On decorpsification

Yeah, I made that word up. But you knew that.

On the 12th of January, 2010, the island nation of Haiti suffered a magnitude 7 earthquake that took living conditions in that already poor society into the realm of the unimaginable. Reports at the time hinted at conditions that were positively Boschian.

“There is no dignity in death for most victims of Tuesday’s devastating earthquake, only an anonymous final resting place alongside thousands of others in stench-filled pits carved from the red earth. Bulldozers close the ground back over them when the graves are full.

“Decomposing bodies are first picked from the ground and hurled into trucks operating as makeshift mass hearses that are driving around the city. The vehicles then head for the mass graves, back up to the holes and empty out their contents” (http://www.telegraph.co.uk/news/worldnews/centralamericaandthecaribbean/haiti/7005477/Haiti-earthquake-thousands-of-bodies-are-dumped-in-stench-filled-mass-graves.html).

At the time, public health officials were quick to warn that the bodies did not pose a great risk to the living (at least no greater than the living posed to one another). “The risk is absolutely minimal, unless there is disease in the population. This [hastily dumping bodies in pits prior to ensuring identification] is a mistake and a waste of resources,” according to Sir Nicholas Young, British Red Cross chief executive (http://webcache.googleusercontent.com/search?q=cache:Hq6U0oQjdNUJ:news.bbc.co.uk/2/hi/8465464.stm+haiti+corpses+disease&cd=1&hl=en&ct=clnk&gl=us&source=www.google.com). Similarly, the WHO suggested that “It is important to convey to all parties that corpses do not represent a public health threat. When death is due to the initial impact of the event and not because of disease, dead bodies have not been associated with outbreaks” (http://www.newscientist.com/blogs/shortsharpscience/2010/01/bodies-piling-up-in-haiti.html).

Now, fast forward a year or so. Cholera sweeps Haiti, and the already strained health infrastructure cannot cope. What a difference a year makes. “Stacked with body bags full of corpses of cholera victims, a converted flatbed truck and a colorful tap-tap taxi swerved into the yard of the mayor’s office and their drivers asked where to bury the dead.

“‘Get out of here. Get out of here before they start throwing stones,’ a city hall employee screamed, her voice panicky, her hands flaring.

“A crowd started circling. Three poorly armed police officers showed up and announced more were on the way. Then the city hall employee jumped into a car and motioned the corpse vehicles to follow. The angry crowd shouted and began throwing rocks” (http://www.miamiherald.com/2010/11/19/1934890/living-fear-the-dead-in-cholera.html).

Public health worker Rochefort Saint-Louis warned that “the bodies are very dangerous. ‘People throw them in the street and they have fluid inside them,’ he says. ‘If the fluid comes out and you step on it,’ he adds, you could track it home. You might put your hands in your mouth or your kid could touch your shoes” (http://www.theworld.org/2011/01/dealing-with-haitis-cholera-victims/).

So, which is it? Corpses that need identification and that pose no harm to public health, or corpses that are dripping with infectious material? Well, both, obviously. A natural disaster produces the first, an epidemic the second.

Which brings me to my point: what does a bioterrorism episode produce? Amid the chaos and confusion and terror that would attend such an event, what do we do with the dead bodies? Venerate them? Identify them? Bulldoze them? Burn them? The real problem will be that our options will be limited, and the time will be short, especially if we are dealing with an infectious disease. And it may be that the situation will be far worse than cholera, with diseases like Ebola causing bleeding out and diseases like bubonic plague leading to bursting buboes.

Even handling the corpses, getting them onto a pyre, would be a death sentence, not unlike that visited upon some of the heroes of Fukushima. And the magnitude of the problem would daunt even a well-organized mortuary service. If the body won’t come to the fire, you could take the fire to the body, although in that instance having multiple burning bodies that were randomly sited would not necessarily be conducive to keeping what remained of an urban infrastructure intact.

No, this is a problem for decorpsification. Getting rid of bodies the Green way, without contributing to the ever-present problem of global warming (sardonic alert). A local company (whose name undoubtedly does not need to be mentioned in connection with this grisly post) produces an awesome product known as MELT. From the product brochure: “The MELT System is a hands-free technology for the rapid digestion of fresh or frozen tissue. No homogenization is required. Samples can be lysed easily in a closed-tube format without cross-contamination … using a novel formulation that includes … a cocktail of powerful catabolic enzymes.”

Yessssss. We have the technology. But instead of the milligram scale described in the brochure, how do we scale this up to the megagram level? Can we in fact have big sprayer trucks roaming the streets, hermetically sealed, hosing down the bodies and causing them, and most importantly their attendant diseases to … melt? Who knows. It is unlikely that I’ll be funded to do this in the near future, because, well, like we like to say about Presidential candidate Bachmann: crazy!

But I hope that some careful planner out there is in fact thinking about this, and that somewhere there is a fleet of trucks or planes or hovercraft loaded with enzymes or bacteria. Probably not, since we can’t even have an intelligent conversation about educating the public about bioterrorism without careening into a TMZ-like media feeding frenzy. But still, it would be nice to know that we don’t have to allow the Very Bad situation that is inevitably going to rain down upon us to become even worse. We can stop at Guernica, without going all the way to The Garden of Earthly Delights.

- originally posted on Wednesday, June 29th, 2011

On cannibalism

One of the last, great taboos. And for good biological reasons. In the Eastern Highlands of Papua New Guinea the locals, known as the Fore, practiced what was politely called ‘transumption,’ which led during the late 1950s to the person-to-person transmission of a debilitating disease, kuru, in epidemic proportions. The ‘laughing disease’ led to massive neural degeneration, eventually resulting in death (although sometimes with long latency periods), and was the result of the transmission of prions.

Prions are interesting because they’re sort of the exception that proves the rule of DNA. That is, while DNA replicates sequence, prions replicate conformation. Prions are peptides or proteins that assume a particular conformation. When a prion comes in contact with a similar protein that is not shaped the same the prion forces the protein to assume its conformation, and they aggregate together in a tight knit architecture known as an amyloid. This is the same sort of amyloid that occurs in the very similar prion disease Creutzfeld-Jakob disease (CJD) and in Alzheimer’s. So, you can sort of think of prions as the first domino that initiates a cascade of conformational events that leads to a big, tangled mess in your brain. Not good.

Here’s what’s been making me think. There is a variant CJD (vCJD) that arose a few years ago in England and that could be traced, roughly, to the widespread distribution of what Denny Crane would call “the mad cow disease,” but which is more commonly known as Bovine Spongiform Encephelapathy. Here’s what I find fascinating: you feed cows the remains or waste of other cows, BSE can spread like wildfire (and who can forget the giant bonfires of cows in England circa the late 1980s). But while there were upwards of 180,000 cases of BSE amongst cows, there were only about 153 cases of vCJD (Beghi et al. (2004), Neurol Sci 25:122). The causal link between BSE and vCJD is sort of like the causal link between human activity and global warming: pretty convincing evidence, but hard to definitively prove (and in both cases the appropriate experiments that might directly test the hypotheses are understandably hard to run).

Some folks think that the low transmission rates from cows to humans just hide an epidemic-to-come in which those of us who consume multiple hamburgers per day (what?) will eventually twitch out. But it is more likely that there are huge species barriers in prion replication. This is because at the molecular level a cow prion may only partially recognize and bind to the amino acid sequence in the corresponding normal human protein, limiting kicking over that first domino. “Amino acid mismatches … can dramatically affect the amount of protease resistant prion made and modulate the resistance to cross-species transmission of spongiform encephalopathy infectivity (Priola (1999), Biomed Pharmacother 53:27).” This presumed species barrier is confirmed by experiments with prion transmission in test tubes, cells, and transgenic mice. Thus, cow-on-cow or human-on-human chomping is a much more likely route to disease than humans eating cows (or whatever happens in the future on the Gary Larson-esque “Planet of the Cows”).

So, what are the implications of this understanding? Clearly, don’t eat people, soylent or not. Indeed, the depth of the taboo suggests that our species may have discovered this edict several times over in our past (and there is even a serious-but-unlikely hypothesis that cannibalistic Neanderthals succumbed to spongiform encephalopathies). Keep your factory farms clean. Amusingly, many of the bans on beef that accompanied the outbreak of BSE in England (and more minor outbreaks in the US) were more political than practical.

But I think there’s also a more fundamental issue. While spongiform encephalopathies seem to focus on a relatively small number of neurologically important proteins, a surprisingly huge number of proteins can form amyloids. Indeed, work from the Marcotte lab down the hall (with a small assist from our own lab) has revealed that a surprisingly large number of yeast proteins (like, 30%, of all proteins) seem to self-aggregate. To the extent that those aggregates are amyloids this would mean that there are many dominoes waiting to fall. Why don’t they? Well, most proteins inside of cells are disentangled by energy-burning so-called chaperones (and, indeed, the prion protein that falls down is on the *outside* of cells in the brain).

Indeed, the fact that we’re not cannibals means we don’t know much about these other dominos. Or, to put it another way, from a biodefense point of view prions are a growth industry. How many different human peptides, available orally, just like kuru was, would inevitably take down large fractions of a population after a suitably long and stealthy incubation period? And given the concerns I’ve previously noted about the quality controls in our supply chains (see “On Pepcid,” which incidentally is back on the shelves, woo hoo!), would such prions be relatively easy to introduce (think melamine)? And unlike viral or microbial diseases, where we at least have a basal understanding of how to construct a biodefense, there really is no defense against prions (just as, sadly, there is no real cure for Alzheimer’s). The suggested procedures for prion researchers to decontaminate their workspaces and tools involve essentially treating everything with the most caustic agents you can imagine. That’s not going to work for your brain. And so I am left once again with a conclusion that is an unsatisfying bummer: we’re hosed. Unless the same cultural prohibitions that keep us from eating one another kick in with respect to infecting one another. The thin, nice line. Quaint.

- originally posted on Tuesday, May 31st, 2011

On ribozymes

Hooray, it’s the end of the term. And what better way to relax than to jet off to a conference filled with physicists and historians and philosophers of science. What? Isn’t that what you’d do? Well, being here at the Seven Pines Symposium on the Origins of Life has nonetheless been both relaxing and intellectually stimulating. And it’s been a chance for me to think a bit about ribozymes, which I haven’t done in awhile. For those of you who may have read my previous entry “On Technological Surprise,” ribozymes are of course RNA enzymes.

A case can be made that ribozymes were our first catalysts. Looking forward from origins, it has been hypothesized that the earliest living system was likely a replicating nucleic acid. Looking backwards from modern life, the molecular fossils that live in our cells, such as cofactors and the ribosome, are made of nucleotides or RNA. Therefore, it is reasonable to conclude that there was once a living system (not “life,” see “On Origins”) whose metabolism, including its enzymes, was made largely of RNA. This is the so-called ribo-organism of the so-called RNA world. This organism committed suicide by inventing translation, but that’s another story.

While Crick and Orgel inferred the existence of the RNA world at least as early as 1968, it was indeed Tom Cech’s and Sydney Altman’s technologically surprising discovery of catalytic RNA that gave form to the fantasy. Since the identification of natural ribozymes, there have been multiple attempts to create artificial ribozymes by directed evolution. Many of these attempts have been surprisingly successful, and have yielded RNA molecules with catalytic properties that include the ability to make and break carbon bonds, carry out redox reactions, and of course rearrange bonds to phosphate like nobody’s business.

One of the earliest and best selections of a ribozyme de novo was carried out by David Bartel. Back when he was a lanky graduate student as opposed to a suave member of the Whitehead, David was the only one bold enough to select for ligase activity from a completely random sequence pool. His technical excellence resulted in multiple, different catalysts, one of which has proven to be particularly interesting, the Bartel Class I ligase. This enduring beast is especially interesting in that it is one of the few examples of a scientific miracle. The relative information content of this very fast ligase can be determined, and it turns out that its representation in its original random sequence pool should have been on the order of once in every 10^19 sequences. The problem is that the pool itself was on the order of only about 10^15 sequences. Therefore, either David Bartel was the luckiest graduate student in the world (a distinct possibility), or else … the ribozyme was but one of many other, similarly complex ligases. Should the tape of life (or, well, the tape of graduate student servitude) ever be run again, a different but equally complex ribozyme would emerge from the pool. And this is what you should tell Creationists who insist that histones couldn’t have evolved by accident because they are so highly conserved. Selection, whether natural or artificial, yields up functions, not particular sequences.

The Bartel ligase has since been on many wonderful adventures, ending up for awhile with Gerry Joyce and his group in an experiment that proved that ligase function could be selected continuously. If you squint, and substitute “sugar and minerals” with “nucleotides and enzymes,” the ribozyme ‘grows’ and evolves in the presence of its foodstuffs the same way a bacteria would grow and evolve in a flask.

The Bartel ligase was such a fertile and versatile system that for awhile there were attempts to make it act like a polymerase, not just ligating a single oligonucleotide substrate to itself, but rather serially ligating multiple nucleotides (stop me before I ligate again!). If only it could be made to catalyze a sufficient number of such additions, it could … replicate itself! This would be the primordial Xeroxase, the enzyme that one could point to and proudly say, “That’s life!” (even if there is no such thing). Alas, try as they might, researchers could not make the Bartel ligase catalyze more than a few turns of a helix, reproducing only a shadow of itself.

Until recently. Until now. When Holliger and his co-workers have come along and done the remarkable (Science, 332:209): in a directed evolution experiment that was such a technical tour-de-force that it rivals the selection of the original ribozyme itself, these researchers made the ribozyme replicate … 95 nucleotides. Of a very special template. Still! Almost there! Actually, roughly halfway, based on the length of the parental ribozyme. Given this breakthrough, ribozyme-catalyzed replication (the Xeroxase) may almost be at hand (go Jared!).

Here’s another sort of funny thing about the Bartel ligase, something that is seldom remarked upon: it doesn’t like to be mutated. More properly, it seems to inhabit a very narrow peak on a sequence landscape, and many mutations can knock it from its perch. So, over various selections by various folks, it has only grudgingly changed, and even Wochner et al. (2011) got it to budge by only a few (4) mutations.

This is decidedly not like the sequence landscapes that one observes in natural selection, where biopolymers often move about on large, neutral networks, happily collecting neutral mutations. This difference may be due to the difference in origins: the Bartel ligase emerged from a completely randomized population; natural biopolymers typically evolve one mutation at a time. It’s sort of the difference between betting a gut shot straight in poker, versus having many outs. Most of the time, in trying to hit the straight you go bust, while you may make a lesser hand more easily.

And here’s the payoff, so to speak: because this ribozyme was so very finicky, I wasn’t sure it would ever go anywhere. I’ve often thought it was a beautiful evolutionary dead end. But Holliger and his crew breathed life into it, and revived all of our dreams of making a Xeroxase.

This difference between perception and reality illustrates several important things: (1) I frequently have terrible instincts. We knew that already. (2) Selection can yield amazing products … but only if done with technical excellence. (3) There may be exquisitely narrow fitness peaks with extraordinary properties. It’s this latter that has some biodefense relevance. The role of directed evolution in the production of biosensors and remediation catalysts and whatnot comes and goes, as do most fads. But there may not be natural catalysts that can robustly perform in military applications, and while we continue to wait for the dominance of computational design, directed evolution remains one of the few ways to plumb the nooks and crannies of the world outside biology.

- originally posted on Sunday, May 22nd, 2011.

On zombies

Woo hoo, I put in tags for my posts. To paraphrase Kurt Vonnegut, perhaps from “Breakfast of Champions,” never write your own index (or do your own tags). It’s too revealing. Who knows, maybe opening comments will be next (hint: no, it won’t be).

I am in Atlanta, and my airplane book for the short hop was Garry Wills’ most excellent “Bomb Power.” I don’t give away too much by re-stating the thesis, that the invention of the atomic bomb substantively changed how the nation state approaches national security. Sort of makes sense; indeed, it seems an outgrowth of what I glean from Diamond’s narrative in “Collapse: How Societies Choose to Fail or Succeed.” When we have enough resources, we have the freedom to organize, to get more freedom. Of course, there comes a point at which we war with other societies, for whatever reason. Then we organize to keep the freedom we have, even if we have to give up some freedom in the process (nicely captured by the mantra “Freedom isn’t free,” which I think is a bit more subtle than its adherents often claim). In this light, the establishment of the Manhattan Project, and the subsequent development of an entire apparatus of secrecy and control around its products, makes complete sense. I must admit I haven’t finished Wills’ book yet, so I am curious to see if he proposes an alternative legacy, since the one we have seems sort of inevitable to me.

Which of course brings us to zombies.

We have already engineered society (ours and others’) to take into account the nuclear threat. How do we engineer society to take into account biological threats? There have been some relatively inspired imaginings of this in the science fiction genre, ranging from “World War Z” right on down to teen fiction such as “Hollowland.” I could have equally well used vampire overruns from scifi (that term indelibly removing me from uncomfortable fanboy status), but zombies are much more appropriate, as they better represent the semi- (but not completely) mindless nature of a concerted biological attack, especially via a communicable disease.

And how do we react to large zombie incursions? Quarantine, decapitation, and a police state, although not necessarily in that order. And eventually immunity, depending on the degree of magical zombie-ness that informs a given book.

Now, while keeping zombies firmly in mind (and how can we not?), I careen back towards Wills’ sociopolitical musings. Societies develop a national security apparatus to protect them from other societies (and, in this day and age, that usually means other nation states, since non-state actors are having trouble doing more than flying planes into buildings, which is not quite the same as building a centrifuge to enrich uranium). Ultimately the national security apparatus must take on a life of its own to function efficiently, and its cost / benefit ratio varies over time.

But what would this mean with respect to zombies? Quarantine, a police state, and immunity, check. But decapitation … of who? Well, of the zombie. Not of a nation state. Because zombie uprisings / biological warfare largely differs from other asymmetric warfare in that it does not, for the most part, require something as complex as the enrichment of uranium. I have argued forcefully elsewhere in these droppings that synthetic biology is not De Debbil, and that making a disease is more complex than what the DIYBio kiddies are capable of. But that doesn’t mean that it would be utterly beyond individuals or non-state actors. It would just have to be the right individuals or non-state actors. Like rogue graduate students, for example (again, a recurring meme here).

This therefore raises the question, how do you engineer society to defend against … society?

One option might be “don’t worry, be happy.” You can argue that the very impersonal nature of disease relative to, say, a bomb makes it easier to ignore. The Reagan administration did a bang-up job of ignoring the AIDS pandemic, for example. We’re used to disease, it’s part of our lives, even if it’s not as much a part of our lives as it once was. And, really, given the unbelievable amount of damage caused by that Third (or First, if you’re non-Biblical) Horseman, it doesn’t even really register. 25,000 flu deaths, yawn. Versus: oh my God, there’s a sniper in DC!

It’s not the effect, it’s the cause that seems to cheese people off. The notion of directed disease is so abhorrent as to be one of those few taboos that seems to have some staying power. That said, it seems inevitable that it will be torn asunder at some point, and societal displeasure will follow quickly.

So, I prognosticate that the dystopia we’re in is as nothing compared to the dystopia we’re moving towards (and, again, one person’s dystopia is another person’s Denny’s; I will leave you to figure out where I stand on the matter, although the question of cheeseburgers looms large in that regard). It seems likely that to defend against the fact that we can be thwacked with nuclear fury not by a Los Alamos project that required the combined scientific genius of several continents, but by a wayward dork whose brain chemistry isn’t quite right we must both engineer society to keep track of the dorks, and engineer society to be immune to the dorks. If you quit thinking about biological warfare and go back to thinking about computer hackers, nothing in that last sentence will seem odd. I hope we don’t have to get to the point where we decapitate the dorks.

So, we will become comfortable with genetic engineering, and embrace it. We will become comfortable with organizing society around defense epidemiology and herd-based immunological bulwarks, and will embrace them. We may even countenance more fundamental engineering of the human form, with the same advantages and problems that attended similar changes to plants in the Green Revolution(s).

All so that we don’t have our anonymous undead brethren rising to claw at our throats. Brains!

Tags: Epidemiology, Intelligence

- originally posted on Monday, April 18th, 2011

On designer chemical weapons

I’ve been traveling a fair amount recently, and probably have alot to catch up on here. One of the more interesting meetings I went to was the Gordon Conference on Chemical and Biological Terrorism Defense, in Ventura, CA. In the Gordon Research Seminar for students that preceded the conference I was fortunate to hear one of Clem Furlong’s students, Judith Marsillach-Lopez, speak on “Isolation and characterization of human biomarkers following exposure to organophosphates.” While the talk was great, the background on the compound tri-ortho-cresyl phosphate (TOCP) was really quite interesting. As one of Clem’s (and Oksana Lockridge’s) manuscripts nicely summarizes (Schopfer et al. (2010) Anal Biochem 404:64), this compound was originally associated with so-called ‘ginger jake paralysis,’ which came on because of the presence of this compound in Jamaica ginger, a patent medicine for stomach upsets (and source of alcohol during Prohibition). More recently, though, this compound has made a number of other appearances, most interestingly in association with so-called ‘aerotoxic syndrome.’ Airplanes are one of the last places you can encounter TOCP, since most other commercial uses have been discontinued. Unfortunately, within the complex ecosystem that is an airplane its use as an engine lubricant and in hydraulic fluids sometimes leads to contamination of the recirculated air supply. Passengers have reported ‘dirty sock smells,’ and that exposure has in turn sometimes led to, well, this:

Yes, that’s right, TOCP is essentially a nerve agent. Its mechanism of action is quite fascinating. To borrow again from Clem’s most excellent paper:

I suspect that as usual that’s completely distracting, but I shall eventually figure out Teh Intertubes and thus how to insert pictures and whatnot. Anyway, the basic mechanism first involves hydroxylation of one of the aromatic methyls by ‘liver microsomes,’ followed by activation via cyclic phosphate formation (not unlike what we see for RNA self-cleavage; CH339L students take note!), followed by attack by a serine nucleophile on that cyclic phosphate and covalent attachment of the compound to the protein containing the serum. Unfortunately, such very active serine nucleophiles are commonly found on proteins like … acetyl cholinesterase, one of the primary enzymes that regulates the transmission of chemical signals in our nervous system. Once you modify the enzyme acetyl cholinesterase, you can’t break down the neurotransmitter acetyl choline, and your nerves are more or less stuck in the ‘on’ position.

Acetyl cholinesterase is a common target of nerve agents, which often are phosphate or phosphonate derivatives, and add themselves to this critical enzyme. The science and art of making nerve agents has for the most part involved making phosphates or phosphonates that are reactive, and that have low vapor pressures (and thus get into your body via inhalation quite quickly). More advanced engineered qualities include persistence in the environment and perhaps the ability to stick to virtually everything. Nerve agents are often possessed of a beautiful chemical simplicity, and a diabolical biological outcome. While I often rant on about biological threats, it is really chemical threats that scare the holy hell out of me. They’re far easier to make in many cases, and, like nuclear weapons, we seem to have somehow lost our innocence in terms of their use. Thanks Fritz Haber.

TOPC is not high on the list of things that are really scary. There are many scarier compounds, although mostly not in our immediate environment. What really struck me about TOPC was its mechanism of action. ‘Liver microsomes’ is one of those biochemistry codes that means “transformation by cytochrome P450.” Cytochrome P450 enzymes are usually the good guys in our liver, taking hydrophobic compounds (like TOPC) and adding hydrophilic groups (like hydroxyls) to them, allowing their solubilization and eventual elimination from the body. It is in large measure the wondrous suite of cytochrome P450s (there’s, like, 57 of them!) that have kept us from succumbing to the inventive toxic brews of bacteria, fungi, plants, and insects. Unsurprisingly, since different branches of humanity have encountered different bad things, there are many different cytochrome P450 enzymes. Also unsurprisingly, this makes a bit of a difference in how we handle our own semi-toxic brews (i.e., pharmaceuticals):

“Polymorphisms in the CYP [cytochrome P450] family may have had the most impact on the fate of therapeutic drugs. CYP2D6, 2C19, and 2C9 polymorphisms account for the most frequent variations in phase I metabolism of drugs, since almost 80% of drugs in use today are metabolized by these enzymes. Approximately 5-14% of Caucasians, 0-5% Africans, and 0-1% of Asians lack CYP2D6 activity, and these individuals are known as poor metabolizers.” (from Zhou et al. (2009) Drug Metab Rev 41:89)

We’re entering the age of personalized medicine, where allelic differences between individuals will lead to differences in the way we perform medicine. There are already drugs that initially seemed to have failed in the clinic, only to be revitalized by findings that they work on particular sub-populations with particular enzyme variants. But even though this is true, it wasn’t until I was in the Gordon Research Seminar that I realized that the same principles that apply to modern medicine might apply to modern chemical warfare. Again from Schopfer et al.:

“… some individuals are significantly more sensitive than others to cabin air oil exposure. We hypothesize that this is most likely due to the well-known individual difference in OP (organophosphate) metabolism by cytochrome P450. Cytochrome P450 catalyzes the first step in the converstion of TOCP ….”

And there you have it: clever chemists of today, armed with the vast armada of information about the distribution and specificities of P450 variants, can now countenance making designer chemical weapons that will work on particular genetic populations. The distribution of P450 variants is such that ethnospecificity is thankfully not within reach, but there are certainly genetic skews that might make your enemy of choice a particularly appealing target for some compounds, while conversely not affecting your own people. This is one of many reasons that I am very happy to be living in a melting pot, protected by a military that is also largely a melting pot.

- originally posted on Thursday, April 14th, 2011

On Pepcid

Unsurprisingly, I have indigestion. My Dad had indigestion, I have indigestion, lots of out-of-shape American males have indigestion. Now, just as antibiotics are one of the things that make modern life better than antiquity, I can point to the presence of drugs like Pepcid Complete as proof positive that civilization is making progress. My Dad would belch for hours, I take a Pepcid and belch for minutes. Progress.

That is, until Fall of last year, when Pepcid Complete unaccountably disappeared. I put it on the shopping list, my wife came back with nada. I went to the drugstore, all the shelves were barren. This phenomenon, the complete and utter disappearance of this product within a relatively short period of time, has been chronicled on many other Blogs (i.e., http://www.totalsuckage.com/2010/11/mysterious-disappearance-of-pepcid.html). What’s sort of weird is that the reasons behind its disappearance have been very sparsely reported. By January, the quality control problems within the McNeil unit of J&J had been brought to light (http://www.nytimes.com/2011/01/16/business/16johnson-and-johnson.html?pagewanted=1&partner=rss&emc=rss), and shortly thereafter the Lancaster, PA plant that made the beloved Pepcid was shut down via a FDA Form 483, a notice of non-compliance. This plant and several others have recently been re-opened essentially under government control (http://www.nytimes.com/2011/03/11/business/11drug.html?src=busln). Whether this results in a return of Pepcid Complete to the shelves remains to be seen.

But here’s the weird thing: why didn’t / don’t we know more about this? You have a recall of E. coli-tainted peanuts or beef, folks go through the roof. But Pepcid disappearing almost overnight? Mostly silence, and ominous murmurs on Blogs. And it must be admitted it doesn’t help that similar quality control problems were dealt with in part by carrying out ‘phantom’ recalls of Tylenol that involved Smurf-like buyers raiding stores and denuding the shelves (http://money.cnn.com/2010/09/16/news/companies/johnson_johnson_recall_hearing/index.htm). Surely such corporate behavior would not carry over into skewing the results of Internet searches. Naw. It’s almost enough to make me want to get out my tinfoil hat and join with Alex Jones’ minions to decry our exploitation by the command-and-control structure created by the Bohemian Grove crowd. Or something like that. It’s actually hard to keep up with the latest in conspiracy theories unless you really work at it.

But back to the point: we have essentially ceded the quality control of our products to watchdog agencies, like the FDA, and cross our fingers and hope that all is well. Now, such an enormous responsibility must overwhelm the FDA even in the best of times, much less these austere times when its budget is being whacked. And the FDA doesn’t regulate all products, anyway, as we saw when we imported melanine-laden pet food from China.

From a biodefense point of view, I find it surprising that we don’t have more independent confirmations of food and drug quality. As commenter ‘j’ on Total Suckage said, you can gain deeper insights into the McNeil quality control problems by reading through FDA reports (http://www.fda.gov/downloads/AboutFDA/CentersOffices/ORA/ORAElectronicReadingRoom/UCM233908.pdf), but really these say very little about what the actual problems are, or how the products may have ultimately been adulterated (or not).

The fact is that there is no profit in monitoring the things we put in our bodies. J&J to this day has been remiss in stepping up to explain to consumers what the heck is going on. The government has been tight-lipped, potentially because hearings, including criminal complaints, continue to waft through the system, but also because what good does it do to destroy J&J? The business of America is business. Thus, we are left with a system that feels unsettlingly non-transparent, even though transparency is a HPLC trace or so away. I suspect that a band of undergraduates with some medium grade lab equipment could probably identify major impurities and perhaps even lot-to-lot variations in many beloved consumer products. But then what? The undergraduates sound the alarm and get sued? Or, worse, the undergraduates get it wrong and the counterculture picks it up and runs with it (we all know about those Satanists at Procter and Gamble, right; http://www.snopes.com/business/alliance/procter.asp?).

Our defense against toxins, whether accidental, intentional, or some corporate combination of both, should be more distributed, less centralized. But this will be damnably hard: while it is likely that biologicals can largely be typed by sequence, accurately determining chemical composition is still non-trivial. There have been calls for developing simple melamine sensors, and such would be useful … for melamine. For the other gazillion compounds of concern, not so much. This is one of those puzzlers where I think I’ve identified an important problem, but right now don’t have much in terms of a solution. Actually, the best solution is J&J’s own Credo (http://www.jnj.com/connect/about-jnj/jnj-credo/), which I really wish they (and many other companies) would live up to. Even though it’s the fox watching the henhouse, confidence in the analytical abilities of our corporate masters is our first, best line of defense against product adulteration. Just consider it an invisible tariff protecting American industry. Oh, wait, in these globalized times there’s no such thing as American industry anymore. Oh well. I can pine for the military-industrial complex along with my pining for just one more bottle of Pepcid Complete. Burp.

- originally posted on Monday, March 28th, 2011

On paleovirology

Like most nerds, I like reading science fiction. It’s not that I think that these fantastical futures will come to pass, it’s more just appreciating the imagination and optimism of those who are unfettered by the realities of experimental failure. Even dystopias seem like Nirvana compared to a day at the bench; the skin jobs actually worked in Blade Runner, didn’t they?

There’s a pretty good science fiction book, the Dooms Day Book, by Connie Willis. Won both the Hugo and Nebula Awards (sez so right on the cover). Anyway, one of the neat things in this book is the appearance of a viral epidemic from the past, against which future humanity is defenseless. “I’m afraid Gilchrist was right after all. The virus did come from the past. Out of the knight’s tomb.” (p. 404) Is this art nudging life? There are those who worry greatly about the illicit recovery of either the 1918 pandemic influenza strain or the almost-but-not-really-extinct smallpox from victims frozen in permafrost. Both the fictional and non-fictional scenarios seem equally fantastical to me, not because they’re not possible, but because there’s just so many other good diseases already out there, ready to roll, and because it would be more straightforward (although not necessarily easy) to just engineer a disease.

Still, the notion that you’re a disease jumping the ages, gaining an advantage on a herd that’s long forgotten you, has a certain appeal.

My friend Sara Sawyer and I continue to spar over disease evolution. My position is that we’re bags of meat that attenuated diseases keep around to feed off of. Her position is that our valiant immune systems have fought the vicious pathogens to a stand still. I point out that pathogens evolve thousands of times faster than their hosts, and thus that there is no freaking way that we could keep up. She points out that complex genetic systems may be able to constrain the evolution of simpler genetic systems in a way that boxes the simpler genetic systems into a limited portion of a fitness landscape, where they go through a sort of viral Groundhog Day, waking up and mutating through the same set of genotypes over and over again. Hey, if Chris Rock can be an immune cell (Osmosis Jones), then Bill Murray can damn well be a virus.

It would be pretty awesome if Sara and I were, like, the smartest people ever and we could eventually recollect in our uber-famous recollections those office conversations that set the tone for the revolution in predictive evolution, yada, yada. Nah. We all stand on the shoulders of giants, count on it. Sara (who is also a formidable scholar) pointed out an article from 1973 to me (VanValen, A New Evolutionary Law):

“We can think of the Red Queen Hypothesis in terms of an unorthodox game theory. To a good approximation, each species is part of a zero-sum game against other species. Which adversary is most important for a species may vary from time to time and for some or even most species no one adversary may ever be paramount. Furthermore, no species can ever win, and new adversaries grinningly replace the losers.

From this overlook we see dynamic equilibria on an immense scale, determining much of the course of evolution by their self-perpetuating fluctuations. This is a novel way of looking at the world, one with which I am not yet comfortable. But I have not yet found evidence against it, and it does make visible new paths and it may even approach reality.”

Gives ya shivers, right? It’s like that other famous non-statement, “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” You just know that had to be Crick.

Anyway, again, there is credence to the notion that the past can sneak up on us, that a Lovecraftian virus out of Space and Time could do some damage. Or maybe we would laugh at it, stuck in its rut, one foot of its genome nailed down by our superior immune systems so that it ran in endless circles on its fitness landscape.

Let’s assume the former, just for fun. Besides Ipswich (or perhaps Miskatonic University), where would you look for this virus? Sara also had the great foresight to invite Robert Gifford, from the Aaron Diamond AIDS Research Center, to recently give a seminar on “Paleovirology: investigating the origin and evolution of viruses using genomic fossils.” This was just a kickass seminar, where we got to see all of the dead retroviruses littering our collective genomes. Insertions that swept a population long ago, and that are left in part or in whole, slowly atrophying as they are run over and over and over by DNA polymerases. But that’s the point: they’re very old. Very, very old, in many cases.

And we Know. Their. Sequences. Just like we know the sequences of those youngsters, the 1918 flu and smallpox. But those are just two, and there are literally thousands of retroviral fossils being discovered in the NextGen sequencing revolution that is exploding all around us.

Could it be that we could … recreate … one of these old viruses? And what would it do? I of course immediately suggested the experiment to Sara, and I was pretty excited for about six seconds until she showed me Soll et al. (2010), PNAS 107:19496, “Identification of a receptor for an extinct virus.” But wait, how could researchers actually recreate the virus if, as I’ve asserted, these are dead, atrophied fossils? Ah, the miracles of sequence reconstruction. Based on comparative analysis of many examples of Chimpanzee Endogenous Retrovirus fossils, a consensus, functional envelope gene for CERV was generated. When the protein was expressed on the outside of modern Murine Leukemia Virus particles, it was shown that the so-called psuedoptyped virus could readily enter a variety of primate cells, including human cells.

Wow. Just wow. For all of the depression engendered by bench science, this is the payoff: a story better than any fiction (even yours, Connie Willis). A virus waiting patiently and silently in primate genomes to be reactivated by researchers millions of years later. It wasn’t The Virus Out of Time, The Virus from Another Dimension … it was The Virus from Within!

- originally posted on Wednesday, March 9th, 2011

On aptamers

OK, so it’s been awhile. I busted my arm, which made typing modestly difficult. I’ll gear up here with an easy (but still hopefully meaningful) one:

The word ‘aptamer’ comes from the Latin ‘aptus,’ to fit. Thus, this was meant to be a polymer (incongruously Greek) that ‘fit’ to its target. Of course, the origins of this word are not nearly as rational or pedantic as we like to make it seem. We’d developed in vitro selection methods (as had Larry Gold and Craig Tuerk), and were attempting to do some marketing. I have always liked inventing words (and concepts, although the precedence of one over the other is surprisingly random), but I must say I’m a bit of a piker at it. At the time I had a good friend, Nina Tovish, who regularly thrashed me, badly, in Scrabble. As a Yale Literature major, she is a true wordsmith. And thus it is that I formally credit Nina with the origin of the term ‘aptamer,’ although it is entirely possible that my memory is faulty and either Dave Bartel or Rachel Green did the honors over bridge one evening. Someone should now update Wikipedia, as my Blog undoubtedly counts as a verified ’source’ of information. The Internet looks into the void and sees itself. Anyway, I should point out that Nina’s word is much better than my front-runner, which was “Clingon,” and better still than the replies we got when we first started using it. “Adaptamer?” people would say.

I still like the term much better than “SELEX,” which is what Craig and Larry called the process of in vitro selection (when it wasn’t being called the Tuerk-o-matic). SELEX always sounded to me like something that you’d see on the back of a cereal box. Both words have now enjoyed a long run in the literature, while ‘aptazyme’ has generally faltered and been replaced by ‘riboswitch’ (good job, Ron Breaker).

Naming aside, and for those who did not know at the outset of this drabble, aptamers are selected nucleic acid binding species, the nucleic acid equivalent of antibodies. You take a large pool of random sequence information (often upwards of 10^15 species, dwarfing the diversity of the human immune system, at least in terms of numbers) You can select for one or more of these diverse species to bind to a thing, where the thing has been as small as zinc and as large as a rat tail. Having bound, the species can be eluted and amplified by traditional molecular biology trickery: reverse transcription, PCR, in vitro transcription. Multiple cycles of selection and amplification lead to the purification of those binding species with the highest affinities and specificities.

It is a cool technology, if I do say so myself. And all kudos go to Jack Szostak, who believed in it even when I didn’t. But in the end, all technologies must be brought to account, and one has to ask: what are aptamers good for? The answer is: so far, not much. They have an awesome research track record, and an absolutely shitty commercialization track record. One can argue over the scientific and / or business reasons for this, but to my mind the bottom line is this: aptamers aren’t as good as antibodies. My lab now works on both, often side-by-side, and I must say that antibodies kick ass. They bind roughly 10- to 100-fold better than aptamers in many cases, and work consistently under a variety of conditions. You can tout advantages for each (aptamers are smaller and more penetrable; aptamers can be reversibly denatured; aptamers are synthetically accessible, aptamers can be programmed to undergo conformational changes. In contrast, antibodies often have less background binding; antibodies frequently recognize denatured epitopes; antibodies can be easily adapted to standard analytical formats, like ELISA; antibodies can be raised against a greater range of targets). For different applications you can choose your poison. But in the end, antibodies bind better, and mostly that’s what makes the difference.

Until now.

I sometimes think that I’ve had a long-running feud with Larry Gold, but really it’s just a long-running conversation that sometimes gets testy. And I must say that Larry has now done something quite remarkable that gives aptamers a new life. You have to ask, why is it that antibodies are so much better than aptamers? The answer would likely be: they have more chemistry available to them. Cysteine has a sulfur; the branched chain amino acids provide many opportunities for specific hydrophobic interactions; lysine, arginine, an histidine bear positive charges that no self-respecting nucleic acid would countenance. Recognizing this, Bruce Eaton and others started years ago to develop amino acid-like nucleotides, where the nucleobases had side chains somewhat like the amino acids. This has finally reached an apotheosis in which Somalogic, the latest incarnation of an aptamer company, has developed literally thousands of aptamers with modified nucleotides that bind to their targets in the low nM to pM range. (see, for example, Gold et al. (2010), PLoS One, 5:e15004). And a shout-out to my pal Marty Stanton for the pipeline; awesome!

Thus beginneth the Second Coming of Aptamers. I would argue that the new side chains put aptamers on par with antibodies, finally, and that it is now a horse race. Now, business or other considerations may still handicap that race in various interesting ways, but at least the fundamental chemical properties of a replicating polyanion are not the limiting factor. From a biodefense point of view, this Second Coming should have an interesting impact. Like everything else in the defense community, aptamers fall in and out of favor as various generations of Program Managers come and go (the question of why there is no corporate memory in defense-related research can wait for another day). We’re probably on, what, the third cycle of favor now. But in part because the United States is so very good at turning its research apparatus on a dime, we’re bizarrely behind other countries that are still bullish on aptamers … because they paradoxically never really had the chance to be disappointed in them. In particular, I’m going to retire to Korea, not just because it is an amazingly beautiful country, but because I could probably live off of giving testimonials to the various institutes that work on aptamers. The increase in publications on aptamer biosensors from China is also stunning / staggering, depending on my mood.

It’s not clear who will win the horserace (either between biopoymers or countries), but it should be fun to watch and participate. Proteins have alot of tricks up their sleeves still, some of which I hope we’re going to help invent. But in the end, the Second Coming may not go to the swift, but to the greedy: the old aptamer patents have about reached their limits, and the new frontier will be the composition of matter surrounding the side chains. Somalogic has its favorites, but there’s still alot of chemical territory to be covered. And as I previously speculated regarding novel base-pairs, it’s possible that there will be patents that have national security implications, where the composition-of-matter of coveted side chains are owned by a single company or entity that excludes practice by others.

- originally posted on Sunday, March 6th, 2011

On debilitation

Mostly when we think about biothreats we think about critical rather than chronic attacks. This is because our adversaries are most likely to use these weapons against us to sow destruction and chaos, rather than to attempt a long-term modification of our society (although, as we have seen with 9/11, critical attacks can lead to chronic changes).

While we undoubtedly face adversaries who think over decades or even centuries, in the absence of a Moriarty-like planner who can manage to coordinate untold numbers of skeins in a conspiracy tapestry it seems unlikely that we will ever have to worry about biothreats as part of long-term takedown plots. That said, it is relatively easy to think of chronic bioweapons that at first glance would seem to at least be technically feasible. Ethno-specific viruses is an easy one. Many viruses already show some specificity in terms of what portion of a population they’ll attack (often the old, the young, and the immunologically infirm), and engineering ethno-specificity can be readily imagined. You carry the gene for Tay-Sachs? Fine, a virus (or a transposable element on a virus) could be engineered to seek out and insert into this allele. For a slightly more complex science fiction scenario, there is the possibility of binary weapons: two agents that spread inexorably through a population, but cause disease only when they meet up. Again, such a scenario has a basis in reality: mortality in the recent H1N1 influenza epidemic was apparently greatly abetted by Streptococcus co-infection, and there is still some question whether the great killer of the last century, the 1918 flu, may have had help from a commensal micro-organism.

Such fictions can be multiplied almost indefinitely, and indeed they already have. One way you know this stuff is bunk is its repetition by conspiracy theorists. HIV=1 was a man-made virus designed to kill blacks, and introduced via an innoculation program (despite the clear historical and phylogenetic antecedents for the zoonotic transfer of the virus). Genetically modified food is designed to make your immune systems weak and to give you allergies (despite the many studies to the contrary, and in contradiction of the common sense notion that every protein we eat is already a ‘xenobiotic’ that is broken down by the same digestive system). Vaccinations are not the greatest public health discovery of the last Millenium, they’re really meant to make your kids autistic.

While there may be less nonsensical opportunities for debilitation, they seem to be caught almost immediately. Low levels of melanine poisoning would seem to be a great way to slowly debilitate the American populace, but our pets served as early warning systems. Farm animals are also our friends where biosecurity is concerned. Prions in animal offal lead to mad-cow disease, and there is a tiny glint of statistical evidence that these infective conformations can cross into humans, possibly leading to all of us carnivores going ga-ga in 20 years or so. That said, the huge outcry has led to mad-cow seemingly being locked down, at least for the moment. I continue to worry about the ready supply of illicit drugs in the US, not because of the social costs (which are nonetheless serious, though you can argue that they’re running neck-and-neck with the social costs of eradication), but because of the opportunities for sabotage. Contaminants in street Demerol, and perhaps Ecstasy itself, have led to Parkinson’s-like diseases. These were quickly observed, and perhaps we should therefore think of drug abusers as our self-appointed, front-line guinea pigs. The realities of addiction make this a grotesque statement, but doesn’t make it less true. The wizards at Monsanto have made Roundup-ready seeds that are not only economically addictive, but that can contaminate adjacent crops. In a different world in which Monsanto was geared for world-domination rather than just monumental profit we might have to worry about what else was in those seeds … although recent breakthroughs in sequencing technology might make that more than a little bit difficult to hide.

What got me thinking about all of this was not all of these false alarms, but the opportunities afforded by new technologies, in particular in the burgeoning fields of epigenetics and metagenomics. I’ve recently had a chance to catch up a bit on epigenetics, and its Lamarckian reach is truly remarkable. Links between history, methylation, and suicide. Links to immune function. If it proves possible to engineer the pattern of epigenetic markers (and it will), then there may be awesome opportunities for changing physiological state in a way that does not necessarily involve genome modification in the conventional sense. Even more accessible will be changing the metagenome of the gut, which is proving increasingly tied to disease state and even mental health. Now, it would of course be easy to come up with diabolical plans worthy of Fleming’s foils. But that’s not the point: there may really be, in the near term, ways to improve the health of entire populations through engineering how we’re methylated (or how our histones are marked), and through what we lay down in our gut. Inevitably, there will be those (especially on the conspiracy theory side, which is sadly quite vocal, and which tends to stray into the great middle more often than not — see the election of Rand Paul for details) who will decry the possibilities. And thus we will sow the seeds of our own debilitation. For when you cannot advance, you of necessity decline, if only in comparison.

- originally posted on Sunday, February 6th, 2011

On miscegenesis and grappling

Over many posts now we have discussed the definition and impact of synthetic biology, and also where and how technological surprise in biology might arise. I now want to bring together and summarize a few strings of thought in one place. First, the genome is the unit of engineering. Not parts, not circuits, but the genome as a whole. In this regard, systems biology eats synthetic biology. Similarly, we’ve also seen that technology surprise may arise not from assemblies of known parts, but from the unknown: for example, from random proteins interfacing with systems, or perhaps from the largely unplumbed depths of information and function that are being revealed by metagenomic analyses. Again, the real threat is not from so-called synthetic biology, but from systems biology.

I think that at root this is a biomimetic analysis. Where does new function arise in biology? Sometimes it arises from novel assemblies of pre-existing parts, and from the construction of interesting new circuits. It can be argued that yeast lysine biosynthesis was a great achievement in synthetic biology, in its day. Almost every other organism makes lysine via the diaminopimelate pathway, but not yeast. In some ancient catastrophe or adaptation, yeast lost the diaminopimelate pathway, and instead devised a new route to lysine by duplicating parts lying around the genome: citrate synthase and isocitrate dehydrogenase became homocitrate synthase and homoisocitrate dehydrogenase, ending up at keto-adipate. From there it was just a reduction and a few simple transaminations to lysine.

But I would assert this is the exception rather than the rule. In general, when micro-organisms acquire new function they acquire it from other micro-organisms, by horizontal transfer. Indeed, bacteria seem to have whole swaths of their genomes that serve as landing pads for new function, whether it be pathogenicity islands or CRISPR loci.

If we accept this analysis, then the question becomes: how do we engineer via horizontal transfer? Now, that’s sort of a silly statement, because what are plasmids, if not horizontal transfer? We’ve been engineering by horizontal transfer for a good long time now. But even with facile plasmid construction methods, like Gateway vectors, this engineering has been decidedly low throughput. This has led me to think about how we might come up with a new approach, a new method, which I am nominally (and in a spirit of political uncorrectness) calling miscegenesis: the breeding of everything with everything. Such a method would go well beyond genome shuffling, which to date has involved a relatively limited number of genomes. It would be horizontal transfer on steroids, so to speak, with organisms freely and continuously exchanging information.

Clearly biology does this over long evolutionary timescales, but how could we do this quickly? Homology-based methods are almost certainly out; site-directed recombination presents a fairly steep barrier to organisms even a few steps away in the tree of life.

I have some ideas along these lines. Indeed, I woke up a few nights ago not from a dream of a snake eating its own tail, but rather from a dream about pirates. You know those great old (and continuing) cartoons in TIBS, that sort of showed how scientists anthropomorphized molecules and processes? It was like that, a dream of molecular piracy. What kind of vector could I make that would steal the genes from another organism? Viruses do this, of course, but again in a sort of inefficient way. Viral particles will often misload random mRNAs into themselves, and these escape capsules may reach another organism and be reverse transcribed into the genome, but again, slow, slow, slow. No, I want a sleek vector vessel that sails into its host and grapples the genome, grabbing a chunk and chortling “Yo ho ho!” as it sails out again. Indeed, in our lab conversations we have begun to call this vector “The Grapple.”

We haven’t done it yet, but the blueprints are laid in, and the minions are conjuring. A first order, single-step Grapple is within our grasp, I believe. The more ambitious continuous Grapple, that truly does sail between organisms, continuously grabbing and dropping off chunks of genetic information is still just a bit beyond the horizons of my imagination. But I am encouraged by biology, which does the oddest things in the oddest ways. I am encouraged by old literature, from a time before restriction enzymes, when microbiologists mouth-pipetted their samples and liked it, by God. I am encouraged by a Fata Morgana that in pure evolutionary terms should probably not exist, a selfish element that has learned to do unselfish things. I am encouraged by the F’.

- originally posted on Sunday, January 30th, 2011 at 2:24