Saturday, September 30, 2006

The Zen of Cloning

Ugh. Loooong week. Ive been trying to get that last construct (the uninfectious bastard from Tuesday). I make these guys by cloning—not the half man/half cow clones George W has nightmares about at night, but molecular cloning.

I cut and paste lots of bits of DNA together into a plasmid, pop the plasmid into some bacteria, and give the bacteria lots of food so they will cheerfully make billions of copies of my plasmid. Just when the bacteria are getting comfy in their warm soup… I blow them up and harvest their innards!!! BWAHAHAHAHAHAHAHAHA!

Anyway, I cant get a good plasmid for that last construct. By *good* I mean one that doesnt have any weird random mutations, one that will make all the proteins I want when I put it in mammalian cells, one that actually makes infectious viruses, etc. But all of these procedures take forever, so I don’t know Ive got a bad one until Ive wasted three days. After ranting to my boss a bit yesterday—he said something very Zen:
“You are trying to make three pieces of DNA that might be 'bad' come together and make a 'good' construct. Why don’t you take parts from constructs you know are good and put them together? Use the good fragments to make a good whole.”

Of course, this is just common sense, and I don’t know why I didn’t think of it—but it just sounded very Zen to me.

Thursday, September 28, 2006

More Syncytia Sweetness

Syncytia formation is a fun technique I use in the lab to see if the viruses Ive made are infectious. But theyre important in vivo too— you wouldn’t be alive without retroviruses and their syncytia trick.

During embryological development, your first cells made a viral protein. No, you cant use ‘early embryological viral infection’ as an excuse for why youre so messed up. A functional viral env gene is in all of our genomes, and its still there right now. But this endogenous retrovirus is only actively transcribed and translated in embryos—Its env is expressed on the surface of embryo cells, and they use the syncytia trick to build you a placenta! When experimenters used siRNA ** to knock down this endogenous retrovirus, the embryo freaks out and wont make a placenta!

Its not exactly *ethical* to try this on a real baby—so the same group that found this ERV looked in mice to see if they could find something similar. They did! Another group found an ERV with the same function in sheep! The sheep guys went so far as to knockdown the expression of the sheep embryo ERV in actual embryos in a mommy sheep… and the embryos died. So that pretty much clenches the fact we need this endogenous retrovirus to survive.

Okay, so that makes sense. All mammals need placentas, I bet this retrovirus helped us ‘kingdomate.’ Not quite—this gets weirder. So far we know humans, mice, and sheep have ERVs that perform the same function… but all of them are different! Syncytin-1 and Syncytin-2 in humans are different from Syncytin-A and Syncytin-B in mice, which are different from enJSRV in sheep! Each of these species independently acquired a retrovirus that became endogenous and performs the same function! Kinda like how ‘eyes’ all perform similar functions, but eyes independently evolved at least 40-60 times! Or how bats and birds and insects have wings, but they all evolved them a different way! Convergent evolution!!

We don’t have the whole story on the origin of these ERVs, or whether there was a common mammalian embryo ERV that got replaced by ‘better’ embryo ERVs in each species over the course of time… I love it when we find tips of icebergs!






**(Instead of finding out what a gene does by irreversibly screwing it up and seeing what happens to the cell, siRNA lets you just make the gene shut up for a minute, you can see what happens when the gene isn’t translated. When the siRNA gets degraded, the gene gets expressed again and you can watch what happens then.)

Human paper

Mouse paper

Sheep paper

Wednesday, September 27, 2006

I suppose ‘Manbearpig’ should have been a clue.

South Park’ is starting their tenth season this fall. There have been a few stinkers over the years (Jackovasaurus, Crab People, Cancelled), but it has mainly been a fun show that Ive enjoyed since The Spirit of Christmas. That’s why I was disappointed to read this:

But Parker says atheism is more ludicrous to him than anything else.

"Out of all the ridiculous religion stories — which are greatly, wonderfully ridiculous — the silliest one I've ever heard is, 'Yeah, there's this big, giant universe and it's expanding and it's all going to collapse on itself and we're all just here, just 'cuz. Just 'cuz. That to me, is the most ridiculous explanation ever," he says. "So I think we have a big atheism show coming."


Great. South Park’ is taking the Poo-Choo-Train straight to Shitsville. A ‘big atheism show’? 22.5 minutes of ‘I don’t believe in a god. Do you believe in a god?’ ‘No. You wanna go get nachos?’

Look, Im all for making fun of atheists. I think I could write an awesome episode making fun of atheists—like the Jews did a better job making fun of the Holocaust than the Muslims. But considering SPs unempathetic attitude towards the Problem of Evil in Red Hot Catholic Love, and the stupid comment quoted above, Im not hopeful that this episode will be an accurate portrayal of atheism in America.

I also think its funny that Parker thinks scientific explanations of the universe are ‘the silliest’ things hes ever heard. Heres the deal, Parker—the leaders of the scientific community, the members of the National Academy of Science, are nearly exclusively atheists/agnostics/nontheists. Though Im still very young, Ive never worked with a True-Believer. Everyone is atheist/agnostic/apathetic/secular Jewish/etc. Why? Because reality is soooooo much cooler than any of those ‘wonderfully ridiculous’ myths people like. ‘Wonderfully ridiculous’ explanations of the universe cease to be 'wonderful' when you understand physics, chemistry, and biology. How can you compare this to ‘yeah, theres this big, giant universe and its expanding blah blah.’ Ugh. Ill say it again—Poo-Choo Train.

Tuesday, September 26, 2006

At Least Theyve Got Booze in Casinos...

My job is so damn addicting. Its like playing the slots—you can plug in quarters forever thinking “Maybe this time Ill win! I can feel it! One more spin!” And just when you’re starting to give up—BAM! Jackpot!!! Today was almost a jackpot:
Syncytia…

Syncytia…

Syncytia…!

AWWWWWWWWW!! Non-infectious construct! God dammit!

Ugh. 6 months of work. One more week and Ill get that last construct. I can do it.

What youre looking at:
My job is to make various laboratory strains of HIV to help us understand exactly how HIV attaches to our cells, so we can design vaccines in a more logical manner. I take those viruses and infect TZM-bl cells.

TZM-bls are ‘indicator cells.’ That means that some genes have been introduced into their genome-- luciferase and B-galactosidase, attached to the HIV promoter region. If the viruses are infectious, they will infect the TZMs, and bring along with them a gene called ‘tat’, which helps transcribe the infectious viruses genome, and also the artificially inserted genes. You can either do a ‘luciferase assay’, or a ‘beta-gal staining assay’ to see if you got any infected indicator cells. Those blue globs you see in the pics are infected cells.

Another cool thing about these indicator cells is that when they are infected with HIV, they will form ‘syncytia.’ The envelope gene is expressed on the surface of the cells. HIV will normally steal the cells membranes, and their env, when they bud off from the infected cell. When the env on the surface of the cells comes into contact with receptors on uninfected cells, they will fuse together, forming multinucleated blobs!

Sweeeet.


Monday, September 25, 2006

Irritating the CDC

Hehehehehehe Okay, Creationists always like to complain about how those meany scientists are peeeeeeeeeersecuting them. They have all this awesome evidence that not only PROVES their creationism myth is true, but ergo PROVES their god exists! But those EVILutionists wont let them publish their research! Well, ignoring the fact that they don’t, you know, do any research—how does the scientific community deal with ideas that go against the grain?

There is only one question we ask when hearing an ‘alternative’ idea—Where’s your research? Bad news for the Creationists, but good news for people who have discovered something interesting. You can use your research to convince others you are right, and they can review your work—maybe even go on to use your research as a foundation for more novel research of their own. Eventually, your ‘alternative’ idea can become mainstream! Hell, you may even win a Nobel!

But as we learn in Mooney’s ‘Republican War on Science’—the scientific community and governmental ‘scientific’ organizations don’t always see eye to eye. That brings us to todays paper:


Factors Associated with HIV Prevalence in a Pre-Partum Cohort of Zambian Women

The cohort Im working with is in Zambia too. What Ive learned over the past 3 years is that most women are infected through heterosexual contact. Their husbands run off to whores, get infected, come home, infect their wives. That’s even been the basis of some research, trying to explain why HIV Subtype C has outcompeted other subtypes, despite its lack of virulence in a Petri dish. Somehow, Subtype C has evolved to be the most efficient transmitter through heterosexual contact (example)

But this paper found something… different:

Even after accounting for the proportion of HIV infections associated with the domains listed above ( Demographics, Sexual behaviour, Alcohol and substance use, Cultural practices, STDs), medical injections emerged as the single most powerful predictor of HIV seropositivity.

Their idea isn’t totally new, which they mention in the intro. Gisselquist published several papers that reached a similar conclusion, but his analyses were all theoretical mathematical models. The authors of this paper simply did a retrospective study with data they collected for other reasons (KSHV transmission patterns). And they got evidence to support the claim that HIV transmission through contaminated needles in hospitals is gravely underestimated in Zambia, and probably all of Africa.

Well, great! Now we know another place to focus our efforts to prevent new infections, right? Hehehe Nope! *Word has it* the CDC is pissed at this paper because it makes them look stupid. The CDC hadn’t focused on this issue, and they should have. It’s not exactly a shocking conclusion. But, whether the CDC liked it or not, the paper got published. This was just a little battle in the War on Science, but it’s a victory none the less.

Sunday, September 24, 2006

Mobile Elements: Drivers of Genome Evolution

This spring I sat in on a course in Viral Evolution. I quickly fell madly in love with Patrick Forterre hypothesis: that everything on Earth now evolved from and because of—viruses.

Of course, one of the booby-traps that scientists need to watch out for is… falling madly in love with a hypothesis. You have to look at the data objectively—regardless of how much you might like or dislike an idea.

So even though I cant get into a time machine, go back a few billion years, and watch what happened myself, we can look at our (and other organisms’ genomes) to see if we can find any clues. And that’s the topic of this first paper: Mobile Elements: Drivers of Genome Evolution.

Approximately half of the mammalian genome is composed of mobile elements, cousins of retroviruses. Mobile elements can be in the form of:

DNA transposons: Cut and paste themselves into DNA, normally staying close to the original insertion (‘Local Hopping’), but can move to a distant site in the genome

LTR retrotransposons: Duplicate themselves, rather than cut/paste. They do this by being transcribed into RNA, reverse transcribed into DNA, then inserted into DNA. They are very similar to retroviruses (have gag and pol) but lack a functional env. Endogenous retroviruses fall into this category.

Non-LTR retrotransposons: LINE-1s, or L1s. Only encode a nucleic acid binding protein, endonuclease, and RT. They are reverse transcribed right on the genomic DNA, not in a viral-like particle in the cytoplasm like LTR retrotransposons.

Oddballs: Look like bits from the other groups, but lack all of the genes necessary to be autonomous. They might be missing a protease/integrase/RT/etc.


Okay, well, that’s neat—but what do these bits of DNA have to do with the evolution of genomes? Well, these little guys can really screw around with your chromosomes. Humans have about 80-100 L1s, and about 1 in 50 people have a novel L1. These L1s can plop themselves in the middle of a gene. They can repair breaks in your DNA. Increase/Decrease the transcription of genes. And they (potentially) control X-chromosome inactivation in women! Trust me, that’s a good thing.

L1s also act in trans to activate Alu sequences, or SINES. Alus are only ~300 base pairs long, but they make up 11% of our genome! 1 in 30 people has a *new* Alu. Alus really have the ability to screw around with genomes—they can cause unequal crossovers (instead of two identical chromosomes splitting into two eggs/sperm, one is chromosome is larger, one is smaller), rearrange coding regions, and duplicating portions of chromosomes. And, L1s can do some rearranging of their own, as well.

So what are the effects of these chromosomal rearrangements? Obviously, screwing around with genes can lead to diseases. Alu insertions are the cause of over 20 diseases, and L1s cause some as well. Obviously, there can also be problems with chromosomal deletions, and L1s changing the transcription levels of genes can also have negative consequences.

But, changing transcription levels can also have evolutionarily beneficial consequences. After comparing our genome to the recently finished chimpanzee genome, we noticed that it wasn’t so much our genes that make us different from our chimpanzee cousins, but the transcription levels of the genes we share. Chromosomal duplications can also have a benefit—if you have two copies of a gene, then one of those genes is no longer under any evolutionary pressure. Its free to mutate, potentially creating a new useful gene, or mutate into a pseudogene.

So maybe I cant completely agree with Forterre yet, but obviously, viral elements have played a huge role in the evolution of our genome, and every other organism on this planet.

Saturday, September 23, 2006

DI: A Day Late and a Dollar Short

I dont suppose *news* that the Discovery Institute is a little slow on the uptake, but a couple weeks ago, Casey Luskin finally got around to 'criticising' Chris Mooneys take on ID Creationism in The Republican War on Science. A book that came out over a year ago. While Im sure Casey was a little more concerned about controlling the damage from the Dover Trial last fall/winter, this still seems a little silly to me, but lets see what he has to say:

In its entirety

#1-- Mr. Mooney overpraises Darwin.
#2-- Mr. Mooney claims ID traces itself to the theological arguments of William Paley.
#3-- Mr. Mooney critiques a blatantly false, straw-man version of intelligent design.
#4-- Mr. Mooney implies there are no peer-reviewed scientific publications supporting ID.
#5-- Mr. Mooney alleges that the controversy over evolution is "manufactured".
#6-- Mr. Mooney insinuates that Discovery Institute opposed Dover's ID Policy because Discovery Institute allegedly believes ID is unconstitutional.
#7-- Mr. Mooney implies it is inappropriate to "teach the controversy" over evolution.
#8-- Mr. Mooney insinuates the Santorum Amendment inappropriately "singles out" evolution.
#9-- Mr. Mooney argues that intelligent design is not science because some
of its proponents have Christian religious beliefs and motives.
#10-- Mr. Mooney argues that Discovery Institute is "disingenuously pretending that modern science basically amounts to institutionalized atheism".
#11-- Mr. Mooney appeals to authority as a valid argument against ID.
#12-- Mr. Mooney's misrepresents Stephen Meyer's peer-reviewed pro-ID science article.
#13-- Mr. Mooney claims the Kitzmiller v. Dover case is the "death knell" of ID.
#14-- An Error of Omission--Mr. Mooney ignores the real "war"--the attack upon the academic freedom of scientists who support intelligent design in science and the media.
See? Its just the usual DI dreck! (If you dont know why these 'arguments' are retarded, Chris gives this garbage the attention it deserves here) It took Casey a year to pull this together? Ugh.

Evidently Casey is actually pissed off that all those darn 'scientists' are listening to Chris:
"Why do so many people eagerly listen to a journalist with neither scientific nor legal training discuss a complex scientific and legalissue like intelligent design?" asks Casey Luskin, an attorney with a science background, working with Discovery Institute's Center for Science & Culture (CSC).
Gee Casey, dont ya think its weird that a lot of people with credentials agree with Chris, and virtually everyone with credentials doesnt buy your Creationism bullshit? If you want to play the 'credentials' card, youre still neck-in-neck with the astrologers and faith healers. But fine, want me to disagree with Chris on something? Sure!

I dont mind Chris's lack of scientific credentials. He cited his sources and consulted appropriate experts and documents of the topics he addressed. What I have a problem with is his naive (hopefully not pretentious) attitude about how it is to be a scientist in the real world. There was one sentence in the new epilogue that pissed me off:
"But scientists have too often failed to counter creationist efforts ar a local level, preferring to remain in their ivory towers."

Bull. Shit.

I am still just a 'kid', yet I try to be as active as possible on the local Anti-Creationism front. Right after I moved down to OKC, I attended a local church to hear a Creationist speak (just a bottom feeder-- Brad Harrub . No one 'cool'). After sitting through a 2 hour hate speech - and I do mean hate speech - against science, reason, atheists, etc, the audience was invited to ask questions of Mr. Harrub. Questions written on a piece of paper, filtered through the ministers/elders, and finally to Harrub to answer or not answer. However I was allowed to ask as many questions of Harrub as I liked... as I was shepherded away from the rest of the church, so they wouldnt be tainted with my evil questions. I returned to the church a few days later and asked to give a presentation about evolution to anyone was interested. The ministers humored me for a bit, but then they suddenly were unable to return my emails and messages.

This is what you come up against being a science advocate in the real world. These people have absolutely no interest in 'teaching both sides.' They want to teach their side, their creation myth, nothing else. They blockade themselves in their churches and their religious schools and religious camps and nobody gets through with an outside opinion. Filtering questions?? Common!! I would ask Chris, why hasnt he promoted his book on 'The 700 Club'? He says we need to plead with the Religious Right to come back to reality, so why doesnt he go on 'The 700 Club' and do just that? Well, the same reason why I cant leave my 'ivory tower' to speak at local anti-evolution churches. I might want to go, but theyarent letting me in.

The answer is not to further condemn frustrated science activists, but to properly condemn the evil individuals that would rather keep their flocks ignorant than lose their tithes. Evil little twits like Casey who continue the religious mental abuse of teenagers and young adults with their IDEA clubs. Dont bitch to me about staying in my 'ivory tower' when youve never been in the dark trenches of the Bible Belt.

There, Casey. Feel better?

All the cool kids have blogs.

Thats it. After being a long time fan of ScienceBlogs, I want to join the cool-kids-club too.

Im going to (attempt) to keep the personal stuff to a minimum, and focus on science. While there are lots of great microbiologists around (Aetiology is great, Respectful Insolence gets into immunizations and such a bit), Ive yet to find a virology blog, so I want to fill that gap. Taking papersIm reading and transferring them into blog posts will hopefully help me understand the material better myself.

Of course, seeing as I live in Oklahoma, and seeing as my research relies on the validity of evolution, Im probably going to jump into that fray as well (even though everybody at ScienceBlogs is probably better qualified to address that issue).

Thanks for humoring me as I awkwardly start. I shouldnt be that bad. I did go to a liberal arts university :P