"There's a great future in plastics..."

There's a deeply disturbing article up at Best Life (hat tip to J. Jacquet of Shifting Baselines, check her page for a nifty link to ORV Alguita's blog) about the pervasive presence of plastics and plastic waste products in the ecosystem, particularly in the ocean. I can't really add anything to the description of the disaster: the article and Alguita blog do a more than sufficient job of that. The problem is daunting, and seemingly insoluble. The particles that cause the most danger are too small to be sifted from the sea, and the use of plastics themselves is pervasive in society. What can we do to fix this mess?

Much like the global-warming problem, two main steps must be taken. Less plastic needs to go into the environment, and we need to be taking plastic out of it as well. Reuse and recycling of plastic are good ideas that will eventually be forced on us anyway by the disappearance of readily-available petroleum. More effective means of recycling will be a must as the supply of raw materials becomes more constrained and expensive. In addition we will likely be forced to complement and then replace the existing petrochemical syntheses and their products with alternate methods and end-products. These should be designed with an eye to biodegradability, either on their own or with the addition of some non-toxic catalyst.

Removing the existing plastic from the environment (and mitigating the damage from future releases) is a significantly greater challenge. Even if we managed to clean up the gyres in some way, degraded plastic appears to be a ubiquitous presence in the water and soil. Supposing that we managed to clean up all that was visible we would still be leaving a substantial fraction behind in the form of highly-degraded particles. It is unlikely that we will ever be able to clean it all up using conventional means. What we really need to do is to enable some creature or set of creatures to digest the stuff.

This would not be a small challenge: even natural polymers are notoriously hard for most creatures to digest. However, as I commented in the Orgel post, we know of bacteria that can digest petrochemical waste products. Given sufficiently masticated plastics, it should in principle be possible, perhaps through directed evolution, to develop proteins appropriate to degrade the polymers and digest the subunits thus produced. The natural solution to the degradation of cellulose as a food source involves perhaps as many as 200 species of bacteria and protozoans; it is unlikely we could produce anything nearly as efficient with a single microbe. However, it may be sufficient to produce just a rudimentary digestive reaction. Once that is good enough for plastic digestion to get started, efficiency is likely to evolve on its own, since the ability to utilize such a food source could constitute a significant selective advantage. Obviously one would want to use an obligate anaerobe or perhaps some kind of plankton for this purpose, to diminish the chances of a "gray goo" problem.

Given a few million years or so, it's likely that microorganisms will evolve that can digest plastic, or help some other organism digest it (as in the case of termites). Regrettably, we do not have millions of years to solve the problem. Of course we should try to come up with some mechanical means of harvesting the larger chunks of plastic from the ocean, but in the absence of significant penalties for dumping, on an international level, such steps will only provide temporary amelioration. Moreover, they will constitute only a partial solution to the problem. In this case, it may be wise for us to try and give evolution a nudge in whatever way we can, lest the prediction of a 'plasticene' geological stratum come true.

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FFXII: Superficially Epic

As I started writing that review of FFXII: Revenant Wings, I realized that I'd never taken out my notes on its predecessor and arranged them into a review. So, I decided I would go ahead and do that. I've played through the game twice now, and I feel it likely that I'll play it again at some point in the future. Final Fantasy XII walks an interesting line: compared to recent entries in the series it seems very much like a new direction. I felt, however, that it actually hearkened back to older entries in many ways. This synthesis had many strengths, but the game did not play to them as well as it could have. For all its epic grandeur, FFXII never really connects emotionally, in the end producing a superficial experience.

The battle system garnered the most attention, because it was the largest departure from Final Fantasy X. A turn-based system with a separate battle screen was replaced by "real-time" encounters in the field. However, the "real-time" system is something of an illusion, and in fact is a throwback, because it is essentially a partially-automated version of the active time battle system that series veterans are familiar with. The real difference here was the elimination of the battle screen, a move that made sense in terms of firmly rooting the action in the world, but which highlighted the oddity of having only half your party involved in a fight. The advantage of the change is most evident in terms of scaling. Giant enemies seem truly enormous in this system, and it also accommodates swarms of enemies that can be a drag in a turn-based system, as anyone who has sat through a Dragon Quest battle with 8 or more slimes can attest.

The removal of discontinuities and transitions between the overworld and battle screen adds a greater sense of immediacy as well, and creates some interesting moments—your characters might end up running through the desert with an enormous dinosaur on their heels, for instance. This engagement is adversely affected by the "Quickenings", rather inexplicable special attacks that seem to take place on some other plane. It would have been better, and achieved more to differentiate the characters, if these had been replaced by in-field special techniques.

The price of all this is the control afforded by a turn-based system. While it is possible to manually direct all your characters, it is more efficient to use the rudimentary AI system (gambits) so that they control themselves. There was some grousing about this, and also the "license" system that forced you, in the early stages of the game at least, to pick and choose what weapons and abilities your character could use. In the end though, these aspects of the system, however controversial, were secondary, because the real selling point of the Final Fantasy games has been presentation and story.

The story was quite a departure. While many of the popular FF entries were highly personal stories intertwined with a traditional fantasy epic, FFXII is much more distant from its characters. In FFX, Tidus repeatedly states "This is my story," but FFXII is emphatically not the (supposedly) main character Vaan's story, nor even Princess Ashe's. The player's characters are all very small relative to the conflict being played out, and for the most part they are unable to affect its course. Only at the very last do the efforts of the characters amount to a thrust against the march of their history. Even then, they affect only the superficial aspects of the moment (i.e. who wins the battle); the great task, the toppling of the Occuria, has already been achieved.

The presentation matched the epic focus in several ways. Full-video cutscenes seemed slightly less textured than they were for FFX—compare Tidus' skin and hair to Vaan's, for instance. Full-video also seems to be used somewhat more extensively for establishing shots than in FFX, and the main characters get rather less screen time in the action shots. Thus, in general, the full-video cinematics feel more remote from the characters. Similarly, the soundtrack is very grand and highly orchestrated. Horn and string ensembles take the lead here, rather than the solo instruments that dominated most tracks of FFX. As a side effect, the music is less intimate, and at times feels almost superficial, like it came out of a fighting game. Although some themes get repeated, the music has nothing like the coherence of FFX. These features work with the nature of the story presented.

So too does the general vastness of the landscapes involved. Entire regions of FFX could fit into single screens of FFXII, and the exterior landscapes are generally quite distinct. Interiors are something of a problem for the game, however; the differences between the various mines in the game mostly amounts to pallette work. Also, in some instances the scale seems very odd—particularly in the corridors of Imperial ships, which are too wide, too purposeless, and have improbably high ceilings. In general, however, the scaling of the environment works to the advantage of the game's approach, emphasizing how large the world is relative to the characters and their story.

This is not to say that there is no personal conflict motivating the story, but it does not play out nearly as intimately as in some other entries. Vaan (the protagonist) and Vayne (the principal antagonist) see each other briefly at the beginning of the game and do not meet again until a few brief moments before the end, but the story tracks their movements and the development of their opposing ideals. Curiously, both wish to be free of oppression: Vaan from the Empire that Vayne represents, and Vayne from the godlike Occuria who try to control history. That Vayne and the Occuria both view their respective rule as benevolent does not impress those who endure it. Despite their key similarity, Vaan and Vayne are presented as physical and emotional opposites, with very different reactions to the resented oppression. Vaan wishes only to be free, and his own master, while Vayne wishes to take the godhead himself, by manufacturing his own version of the magical stones that the Occuria use to distribute power (and with it, control). The problem with this dichotomy is that while Vayne is the unquestioned leader of his side of the argument, Vaan is not a leader nor seemingly even essential to his.

As the story progresses it sounds two main thematic notes: a suspicion of power and the powerful, and the futility of revenge. Criticism of current events could easily be read into either of these if one wished, though the tale of small nations caught in a conflict between two great empires seems more directly analogous to the Cold War. Intertwined with the theme of vengeance is the resolution of loyalties—familial and political. In general the characters choose to be loyal to the future rather than to memory, which could also be read as a political statement.

One interesting feature of the story is that Vayne and Doctor Cid both have a habit of talking to thin air, seeming to be insane. Yet the "Venat" they speak to, a renegade Occurian god, is actually present (invisibly). The obvious note sounded about the ease of confusing religion and insanity may obscure larger questions about how we can distinguish a good God from an evil one, and whether any God that relates to the world purely through power can be truly benevolent. Is Venat good or evil, or both?

These threads are interesting, but through them we ultimately learn very little about the characters. FFXII spends so much time building up the enormous supporting cast that we never get to connect very completely with or truly understand the putative main players. At the end of the tale, we know that they have done certain things, but we understand very little about why. Nor does the story end with any resolution of tensions or personal transformations. Most of the characters (except Penelo, who is practically a non-character) reconcile with their past, but the significant transformations that Yuna and Tidus underwent in FFX find no echo here. Vaan, Ashe, and Basch are somewhat less bitter at the end of the story, but otherwise not significantly changed. The relationships between the characters of the party never seem to develop or change—at times it's not at all clear why they're even still travelling together. Rather, it is the secondary characters, Larsa especially, who are transformed by events of the story, and their relationships that develop and change. In that respect, FFXII resembled some of the earlier FF games to its detriment.

The only remedy to this weakness would be a change in the narrative. A narrowed focus would have helped—Penelo and Fran could have been excised or relegated to supporting-cast status without much of a loss. Fran and the Viera in general felt like an indulgence of puerile sexuality—the village full of scantily-clad bunny girls was almost fatal to the tone. Vaan should either have been excised or built up significantly—for long stretches of the story he seems like a supporting character who got stuck to the main plot by accident, which is not a problem unless (as here) the events of the story seem as insignficant to him as he is to them. Trimming some of the fat and refocusing the story could draw out Vaan's developing leadership and responsibility, and Ashe's resolution with her past, to a much greater degree. It would also allow more space to play on Ashe's relationships with Vaan and Balthier. At several points the Occuria seem interested in using Vaan somehow, or pushing Ashe towards him. Again, allowing more space for this to play out would also have been wise.

As an epic, FFXII mostly works, and the vastness of the landscape, the massiveness of many of the enemies, and the sweeping score help reinforce this property. However, while the political and military tale is compelling, the personal story is not: the main characters are never developed adequately, their relationships do not seem to evolve, and for all their epic scope the events of the story do not seem to greatly change them. The automation of your allies, the vastness of the world, the remoteness of the score, and the two-dimensionality of the characters creates a fundamentally alienating and impersonal environment only partly ameliorated by the engagement of the continuous-field battle system, and this only weakens the emotional impact further. Ultimately, FFXII is grand but not touching—all glorious gilding, and no lily.

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Revenant Wings doesn't play up to its presentation

Final Fantasy XII : Revenant Wings for the DS opens with some really beautiful video sequences, accompanied by lovely music. Immediately, the top-notch presentation that Final Fantasy games are known for asserts itself. Yet, as is so often the case with FF spinoffs, the actual game doesn't live up to the polish. Despite its production values, Revenant Wings is an uninspired game with merely passable mechanics.

The box promises a version of the FFXII battle system, and Revenant Wings more or less delivers that with its real-time action and skill charging, though the context is quite different. Each of the characters commands a group of monsters in most parts of the game, making it more of an RTS, though there really isn't any resource management going on. This isn't to say that there's no micromanagement—you'll be doing plenty of that, because the AI does not make good decisions and cannot be stopped from making bad ones.

You have no option with the AI other than setting a single "gambit" (automatically used skill). So you cannot, for instance, tell a group of creatures to stand their ground. Ranged creatures especially have a tendency of getting into trouble this way, but any of your creatures might chase after an enemy against your orders. It's also difficult to respond quickly to problems, in part because your creatures move so slowly, but also in part because your units have a hard time moving around one another. In fact, it will not be at all uncommon to see melee and flying units wandering aimlessly through the ranks long after you have ordered them to move to a particular spot, especially when you have lots of relatively stationary ranged units. As you might guess, units have no AI that commands them to stay together, which will occasionally result in a lone member of a platoon charging hopelessly into the mouth of death while his comrades mill stupidly about in your lines.

Other than the traffic jam problem, the touch control works pretty well. Unit selection is straightforward, and never caused a significant problem. Unfortunately, it's not always easy to get units to do what you want—your units and the terrain can obscure opposing units, preventing you from ordering an attack. Also, especially on screens where there is significant variation in altitude, it's not always clear where you are telling your units to go, or even where you can tell them to go. Once you successfully order them, they'll take a while to actually get moving; your commands always seem to take a backseat to whatever the units are doing at that moment.

As a strategy game, Revenant Wings is run-of-the-mill. There's a standard revolving set of weaknesses (flying creatures weak to ranged creatures weak to melee creatures weak to flying creatures) and the requisite elemental strength/weakness setup. It's generally more important to exploit the former than the latter when you choose what creatures and leaders you'll take into battle, but here too you are overly limited. For much of the game you have no real choice about what attack mode your leaders will use. Later on, you have somewhat more flexibility, but in the end you will have only one healing leader (Penelo) and four ranged combat leaders (with two each of flying and melee leaders).

Despite all the limitations, however, the game is not terribly difficult or strategically challenging. Most maps can be solved with a tenacious advance or simple distract and destroy tactics. The exceptions are one ill-advised stealth mission, a few missions involving allies (who apparently long for death) and the final mission, which is somewhat insulting in that you are essentially confronted with a gun that can only be destroyed by attacking the mouth of the barrel. It is doable, but tedious and frustrating, and made more so by the tendency of characters whom you have told to remain in a safe area to wander into the path of the shot (due to the aforementioned AI issues). Naturally, enemy troops are never damaged by the enormous laser blast. In short, difficulty is added in lazy ways—useless allies, massive enemy respawns, over-leveled opponents, and silly terrain effects—rather than more subtle approaches like truly coordinated enemy attacks or realistic competition of strategic goals. This feeling of laziness is accentuated by the interminable recurrence of bosses, especially the Judge of Wings and Ba'Gamnan's crew.

The presentation, as I mentioned, is top-notch. The music is especially notable. Although almost all of it is cribbed directly from the original, it was ported to the DS without a significant loss of sound quality. The result is a really enjoyable audio experience, though some of the sound effects are obnoxious. The in-game graphics of course don't match the detail and beauty of the pre-rendered cutscenes. However, they're perfectly serviceable in most instances. The environments are nice, and the map of Lemures is very pretty (this is less true of the Ivalice map). The monster designs have a lot of personality for their size, though I feel that the art department stretched themselves a bit too far on some of the larger espers—these are sometimes too detailed, resulting in an complex yet uninteresting blob.

This description could also apply to the plot, which hits many of the same notes as the original FFXII and adds some Kingdom Hearts-style rambling about hearts (anima). It's interesting to know what happened to some of the minor characters of FFXII, but it's striking that the main characters appear to be totally unimproved by previous events. Penelo is sent to video-game writing purgatory with stock jokes about her bad cooking, and at one point devolves into a sobbing useless wimp. This isn't anything original: I note it only because Penelo's backbone and sass were her only distinguishing characteristics in the previous game and here they are eviscerated for no apparent reason. None of the other characters do much to distinguish themselves—oh wait, one is an emotionally closed-off pretty boy, a novel concept for SquareEnix writers. Cameos by the several of the original FFXII cast amount to just that: cameos, with no additional character development.

Pretty cutscenes and pretty music achieve nothing if they're applied to a run-of-the-mill game with a lame plot. Regrettably, that's exactly what happened here. Revenant Wings' uninspired mechanics, oversimplified strategy, and limp story waste the value applied in the graphical and audio departments. It's not a terrible game, but those who expect a play experience as vivid as the opening videos will come away disappointed.

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What was Leslie Orgel saying?

Well, my first impression on reading Leslie Orgel's recent (posthumous) essay in PLoS Biology was that it was just the sort of rich quote-ore that creationists and cdesign proponentsists cannot resist mining. Indeed, it was not long before famous cdesign proponentsist Casey Luskin began distorting the article in a way that served his personal beliefs. Others have dissected the failures of Mr. Luskin's analysis, and of course volumes were written about his putative misuse of the Research Blogging icon. In all the clamor condemning Mr. Luskin, Orgel's actual paper seems to have gotten lost. I thought I might write about it.

Orgel's article itself doesn't contain any new research, and those who are keeping track might notice similarities to a previous essay on the same topic from 2000. The issue at hand is the origin of life, which owing to our inability to travel back in time is one of the most difficult subjects in biochemical research. It is almost impossible to know exactly what chemicals were present in the prebiotic milieu, what minerals and surfaces were available to perform catalysis, and which of the many possible environments was the one in which life actually arose. As such, the field is highly speculative. Orgel's essay is something of a reaction to this.

It is popular to speak of an "RNA world" of primitive organisms in which most or all of the functions currently performed by proteins were instead performed by RNA. This model has the advantage that RNA can have both informational content and catalytic activity, but it demands the question of where the RNA came from. Some elements of nucleic acids can form nonenzymatically from simple chemicals likely to be present in the prebiotic milieu, but the transition from adenine and ribose to a diverse oligosaccharide with a phosphate backbone is not trivial. If, however, enormous autocatalytic cycles existed on the prebiotic Earth, that would resolve many of these objections.

In his essay, Orgel seemingly meant to quell enthusiasm (I use the term in its pejorative sense) for this idea, not because he believes it is intrinsically false, but because it doesn't really make things easier for us. He takes as an example the reverse citric acid cycle, pointing out that although it is extremely useful, and moreover is autocatalytic, it requires numerous and diverse chemical activities, many of which could go awry if the wrong substrate were used for a step. The assertion that this cycle underpins the existence of life requires appropriate colocal catalysts acting with sufficient efficiency to keep the cycle running. Moreover, it requires that unproductive (or toxic) side-reactions not drain away the reactants at any step. That is, the catalysts present must discriminate between different possible reactants, so as not to break down components inappropriately before they advance in the cycle. It is not immediately apparent that this is possible (certainly it does not appear to be probable), and Orgel was not convinced by several of the attempts to justify prebiotic catalytic cycles. This article summarized his reasons for skepticism.

This attitude has been misrepresented as meaning that Orgel believed (A) that prebiotic autocatalytic cycles were impossible, and (B) that important life cycles are irreducibly complex. Both propositions are false, and it is also false that Orgel believed them.

Part of the rebuttal to proposition A is referenced in Orgel's paper itself, when he mentions Arthur Weber's recent work in which a reaction of various trioses with ammonia gave autocatalytic products. Although the cycle at work in that instance is not yet understood, Orgel points to it as being particularly promising, in part because it requires no additional catalyst, and in part because it yields high-energy carbon compounds of a kind that might be useful substrates for life.

Granted, the reactions of the Weber experiment may not be directly analogous to anything observed in modern organisms, but I feel that this should not be seen as a problem. After all, why would a primitive organism evolve an activity to perform catalysis already occurring naturally? Rather, one would expect that primitive organisms, whatever their informational and structural characteristics, used the products of autocatalytic cycles as raw material for more exotic activities, which were later repurposed to the production of raw materials as a way to gain a competitive edge or to respond to resource scarcity. If, additionally, very complex abiotic cycles similar to the TCA cycle existed to produce more exotic or useful substrates, it ceases to be quite as problematic if they are not highly efficient or specific. Indeed, these sorts of shortcomings would likely serve as a basis for establishing selective advantage for those organisms that evolved compensating protein catalytic activities.

It is not necessary that abiotic cycles intended to serve as a metabolic origin of life work perfectly, or even that they involve products and intermediates similar to those used by contemporary organisms. They need only be efficient and specific enough for life to get started, with intermediates and products that are useful enough for primitive organisms to benefit from them. Orgel's objection is not that this latter situation is impossible; rather he feels that it is sufficiently implausible that actual evidence is needed, rather than hopefulness and modeling.

The sharp-eyed will (hopefully) note the influence of Orgel's rules in the above, especially the First Rule: "Whenever a spontaneous process is too slow or too inefficient a protein will evolve to speed it up or make it more efficient." The rebuttal to proposition B, of course, comes in the form of Orgel's Second Rule: "Evolution is smarter than you are." The belief that any biological systems are irreducibly complex results merely from a failure to understand the enormous problem-solving potential of selection combined with random mutation and millions upon millions of years of time to work. Certainly Orgel would never have accepted that evolution could not produce these complicated cycles once given a chance, or that these cycles could not be broken down in a useful way for primitive organisms. Orgel's point is not that any of this is impossible, it is that it must be shown to be plausible:

The most serious challenge to proponents of metabolic cycle theories—the problems presented by the lack of specificity of most nonenzymatic catalysts—has, in general, not been appreciated. If it has, it has been ignored. Theories of the origin of life based on metabolic cycles cannot be justified by the inadequacy of competing theories: they must stand on their own.

Orgel's paper does not rule out the possibility of autocatalytic cycles, nor does it assert or even imply that some mystical intervention is necessary to explain how life came about. Orgel's paper is a demand for evidence. It is not enough to say that autocatalytic cycles solve the problem of biogenesis, nor even that they are more plausible than other explanations. Negative arguments do not suffice; a positive argument must be made and backed up by simulations, reconstructions, and experimental evidence. Orgel points favorably to several promising avenues of research in this regard.

Intelligent design is not among those avenues, and it is certain that Orgel would reject it. Having only a (flimsy) negative case in its favor, lacking even the virtue of plausibility, and ignoring his second rule, ID must be seen as weaker than any extant metabolic or genetic theories, which at least are not based on magic. Orgel's overall point is a valuable one. It is disgusting and disgraceful that, with flagrant disregard for his thinking, Orgel's essay should be misrepresented as supporting "if pigs could fly" views of the origin of life.

Primary citation:
Orgel, L.E. (2008). The Implausibility of Metabolic Cycles on the Prebiotic Earth. PLoS Biology, 6(1), e18. DOI: 10.1371/journal.pbio.0060018 OPEN ACCESS

Other peer-reviewed articles referenced:
Orgel, L.E. (2000). Self-organizing biochemical cycles. Proceedings of the National Academy of Sciences, 97(23), 12503-12507. DOI: 10.1073/pnas.220406697 OPEN ACCESS
Roy, D., Najafian, K., von Rague Schleyer, P. (2007). Chemical evolution: The mechanism of the formation of adenine under prebiotic conditions. Proceedings of the National Academy of Sciences, 104(44), 17272-17277. DOI: 10.1073/pnas.0708434104
Weber, A.L. (2007). The Sugar Model: Autocatalytic Activity of the Triose-Ammonia Reaction. Origins of Life and Evolution of Biospheres, 37(2), 105-111. DOI: 10.1007/s11084-006-9059-9

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Dynamics and tunneling in soybean lipoxygenase

Because even the simplest case of enzymatic catalysis involves multiple steps (i.e. association, chemistry, and dissociation), semantic issues have obscured the question of what role protein structural dynamics play. For instance, the opening of the lids in Adenylate kinase appears to be rate-limiting, but there is as yet no evidence that dynamics play any role in the phosphotranfer. Thus, one can argue that dynamics are critical (because they are rate-limiting) or irrelevant (because they contribute nothing to the chemical step). Some authors, Arieh Warshel for example, have argued that the latter finding is general—that the highly successful transition-state stabilization model leaves no place for dynamics in chemical catalysis. Some researchers, however, believe that dynamics may play a significant role in chemistry when catalysis proceeds by other routes, especially hydrogen tunneling. In this vein, Matthew Meyer, Diana Tomchick, and Judith Klinman presented evidence in last week's Proceedings of the National Academy of Sciences that protein dynamics play a significant role in the chemical step of catalysis for the protein soybean lipoxygenase (SLO-1).

Hydrogen tunneling refers to the idea that a hydrogen may in some instances "tunnel through" an energy barrier, going directly from substrate to product without wasting any time or kT in actually surmounting that barrier. A reaction that primarily reflects tunneling should have three properties. The reaction rate should vary only weakly with temperature, because tunneling mostly divorces the rate from kT. Using an Arrhenius plot, this translates to a low calculated energy of activation. Replacing the reactive hydrogen with deuterium should enormously inhibit the reaction—a large kinetic isotope effect (KIE)—because the doubling of mass makes tunneling less likely. Nonetheless, the difference in the energies of activation (ΔE_a) for hydrogen and deuterium calculated from an Arrhenius plot should also be small, if tunneling is the main mechanism. Conceivably an enzyme could enhance the rate of such a reaction by using dynamics to promote favorable vibrational modes for the tunneling to occur, or to temporarily adopt a disfavored structure that puts reactive groups at a better distance for tunneling.

SLO-1 exhibits all three features, and so Klinman's group has used it as model system to try and understand whether and how enzymes promote tunneling reactions. In this case they have performed a series of mutations of isoleucine 553. In addition to existing data on WT and I553A, they analyzed the KIEs, crystal structures, and activation energies of I553L, I553V, and I553G mutants. They find that the protein as a whole is not much distorted by any of these mutations, nor do any of them appear to have significant effects on the binding pocket or substrate dissociation constant—the K_d for each mutant is around 3 μM, while WT is around 10 μM. Yet as the bulk of the mutated residue decreases, the ΔE_a increases significantly. In addition, the kinetic isotope effects decrease much more sharply with temperature than for WT. Again, the magnitude of this increased temperature dependence appears to vary inversely with the bulk of the side chain at I553. From these features, and a decline in the magnitude of the pre-exponential factor, the authors argue that dynamics play an important role in encouraging the tunneling reaction.

Well, you know how I love long-range dynamic effects in proteins. But I don't quite buy it here. An argument based on negatives is never very satisfying in any case, and here we have a lot of questions. For one thing, to say that the structure hasn't changed significantly seems overly simplistic to me. The lowest-energy structure may not have been seriously deformed, but absent crystal packing and with a little extra kT around, the average structure might be different. Additionally, these structures were obtained in the absence of ligand. Even though the energetics of binding do not appear to differ significantly for these mutants, the structure of the enzyme or ligand may have changed in the bound state. Even if I accept that the existing structures argue for a completely identical binding site in the free state, and this could realistically be debated, that's no guarantee that the same is true of the bound state.

I'm not denying that the evidence is strongly suggestive, and getting direct data may be difficult given the size of the protein. However, almost all of these side chains are methylated. That means that perdeuteration of the protein in concert with specific side-chain labeling could be especially fruitful in directly observing the dynamics of the pocket during catalysis. It should also in principle be possible to label other side chains in the region to determine how mutations affect the whole area. It is likely that the substrate can also be labeled, possibly with a nucleus or tag that is poorly relaxed. T₂ is likely to be a major challenge here, but not necessarily an insurmountable one, especially since the enzyme appears to be folded and active up to around 50 °C. The presence of the iron is certainly a complicating factor; however, the dynamics should be the same whether catalysis is occurring or not, so possibly it could be substituted with a non-paramagnetic metal. It wouldn't be the easiest project in the world, but it should be doable.

I think these results are intriguing, and I'd love to hear Warshel's take on them. Nonetheless, I feel I have to reserve judgment at least until dynamic changes in the pocket that seem to correlate with the kinetic observations are directly demonstrated by NMR or some other method.

Meyer, M.P., Tomchick, D.R., Klinman, J.P. (2008). Enzyme structure and dynamics affect hydrogen tunneling: The impact of a remote side chain (I553) in soybean lipoxygenase-1. Proceedings of the National Academy of Sciences, 105(4), 1146-1151. DOI: 10.1073/pnas.0710643105 - OPEN ACCESS ARTICLE

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Marking time on the Phantom Hourglass

Last night, as Hillary accepted the zero delegates she won from Florida, I finished the latest entry in the Legend of Zelda series, Phantom Hourglass. I found it to be a fun, lightweight romp.

Phantom Hourglass is a sequel to the Gamecube game Wind Waker. It inherits cel-shaded rendering, a general character aesthetic, and an oceanic setting from its predecessor, while the terraced look of the islands (once you are on them) is more closely related to Zelda games from the SNES days (see left). Motion and combat are touch-driven, as are all of the tools—boomerang and bombchu routes are drawn on the screen. So too is your steamship's route through the seas (below, right). You can annotate the map (shown in these shots on the upper screen), circling important points or noting safe routes through treacherous areas. The controls generally work well and make most of the tools and weapons very intuitive. There were a couple of hiccups, however.

The first was that the touch control is not quite as sensitive as I might like. I had difficulty executing roll attacks consistently, and to be honest I was never quite able to pin down the motion that would produce one regularly. Also, I found stopping time in the final battle to be a bit of a crapshoot—I typically needed to draw five of the required hourglass shapes before one took. The other problem is that there just isn't a very good way to make Link run left (or the right, if you're left-handed). Unless you use an odd grip on the stylus your hand tends to block one screen or the other.

These minor issues aside, the gameplay is not very challenging, especially if you quickly catch on to how the various tools can be used. The eponymous hourglass serves as a time limit on one of the dungeons, in which a modest kind of stealth is required. This, not a serious challenge, is the most difficult part of the game except for one of the boss battles.

The gameplay fits the classic Zelda mold with the same additions as Wind Waker: you travel around the sea, visiting various islands and breaking into their dungeons. The dungeons themselves are pathfinding environmental puzzles—each dungeon contains a tool that is needed to traverse it and a bafmodad you are supposed to retrieve. Though the islands seem a little closer together than they were in Wind Waker you still spend a lot of time in transit, and the occasional appearances by enemies and pursuers are more annoying than entertaining. On the other hand, the addition of a minigame has much improved the dullest task in Wind Waker, i.e. the retrieval of sunken treasure.

Mini-games and sidequests are abundant, as is typical for these games. Seeking out all the various power-ups and perfecting your scores in these accessory games can add plenty of time to the game. However, your actual rewards are somewhat paltry, and mostly come in the form of parts for your ship. You can customize your ship in various ways, but the result is almost entirely aesthetic. Your ship gains greater stamina if all its parts come from the same set, but with one exception there's no reason to choose one set over another. Even a few minor effects would have made this more interesting—if, for instance, the stone hull decreased your speed but added stamina, or the restful cabin healed Link at a slow rate while he was on the ship. Effects of this kind would have added interesting dimensions without taking up much space on the card.

The role of guide and assistant is once more played by a fairy. Fortunately Ciela is much more helpful (and much less annoying) than Navi. She has a story, and it's somewhat interesting, but in general the story of Phantom Hourglass is very lightweight and silly. This was true of Wind Waker as well, but that game did a better job of winking at its own silliness, and also gained a sense of gravitas from the helplessness and regret expressed by the various spirits you encountered. Phantom Hourglass lacks both sensibilities, and as a result no real theme ever emerges.

In the end, the light story and easy gameplay make Phantom Hourglass seem like a slight little breeze of a game. It's easy to pick up and easy to forget once you put it down. The adventure sandwiched in between is fun, and retains much of the charm of its predecessor. Despite a few interesting variations on the way some items are used, however, it just doesn't leave much of an impression.

And now, a definition:

bafmodad (baf mō dad) - In a video game, an object which is not of any intrinsic use but which must be obtained in order to advance the plot. The Medallions from Ocarina of Time are a classic example. The bafmodad is conceptually related to the cinematic MacGuffin. Term invented by Jerry Holkins and Mike Krahulik as a corruption of the name of an actual item from Starfox Adventures.

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The Console Scorecard

It's time to start with a decision-making process. I'm not definitely in the market yet for one of the three next-gen consoles, but I will be, perhaps within the next year. This is an open post that I'll probably keep updating for a while. Keep in mind that my must-have games may not be the same as yours, though I'll welcome your suggestions.

This table looks really screwed up and I don't know why. I have no idea why all these break tags appeared in this post. Sorry for the megascroll. All fixed now. See what effort I go through to bring you my bullshit?

Positives	Negatives	Must-Have Games
Wii
motion control = activity backwards compatible with my GC library awesome downloadable retro games selection system and games more affordable robust controller many WiiWare titles are totally awesome	motion control = activity limited RPG selection (?) shovelware limited original downloadable content DS is enough for casual gaming	Fire Emblem: Radiant Dawn Metroid Prime 3: Corruption No More Heroes Okami Super Mario Galaxy Super Smash Bros. Brawl
XBox 360
robust online service (friends) robust original downloadable games listing great HD graphics very durable controller	robust online service (John Gabriel's GIFT) red ring of doom I don't have an HDTV	Assassin's Creed Blue Dragon BioShock Eternal Sonata Mass Effect
Playstation 3
comes with blu-ray player greater HD graphics Fumito Ueda	expensive... ...or not backwards compatible with my huge PS2 library I don't have an HDTV will cheaper blu-ray cause buyer's remorse? may melt my apartment flimsy controller	Assassin's Creed Final Fantasy XIII LittleBigPlanet Metal Gear Solid 4 Ratchet & Clank Future Uncharted: Drake's Fortune Valkyria Chronicles

Issues to consider:

Where will the RPG market land? Right now the Xbox seems to be leading on the quirky RPG front. Unless the install base widens significantly, it doesn't seem terribly likely that the PS3 will be as successful in that regard as the PS2 was. However, the PS3 is a likely landing place for the main Final Fantasy titles and the Tales series. The low development costs of Wii games may end up turning the 2-D RPG and strategy RPG markets to that console, even though the Gamecube was notoriously weak in the genre. Thus, the Wii may end up catching the narrow-market RPGs I often adore.

Can the HD consoles attract classic adventure games? It can't be a secret that I really like adventure games in the Zelda mold. Assuming that the X360 and PS3 split the RPG market, the clincher for either of these consoles will be success in attracting this generally ignored genre. Games like Beyond Good & Evil suggest that this genre can grow beyond the Zelda clones to include experiences of great emotional depth. Will the HD market go this way, or will the genre languish once again, save for the loving devotion of Nintendo?

Which console will generate the most interesting artistic innovations? I think the knee-jerk response here is the Wii, but it seems to me that developers have so far done much less with motion control than they could have. Instead, the Wii seems to have become a grand repository of casual-gaming shovelware, with a few truly innovative titles here and there. Despite its potential, the Wii has yet to see a game that achieves the true gestalt of emotional communication and interactivity that marks games as an artform. Does No More Heroes fill that void? And Sony Computer Entertainment cannot be ignored in this regard—Ueda's Ico and Shadow of the Colossus were some of the best art made for the last generation of consoles. Also, as the PS3 and Wii have been getting up to speed, Bioware and Irrational have started to take interesting steps on the X360. This is currently an open question, but for me it's a significant one.

And, to track my position by date:
1/30/08 - leaning towards Wii first, HD console (much) later.
2/13/08 - If the PS3 lacks backwards compatibility that is a major minus in my book. Baroque will come out on PS2, so it's no longer in the Wii column. The more I hear about Lost Odyssey the more ambivalent I feel. On the one hand I like the idea that short stories are built into the game, on the other hand I hate games that string cutscenes together using lame gameplay, not to mention that I hate hate hate amnesia as a plot device.
2/15/08 - Okami hops into the Wii category. I was underwhelmed by the gameplay on the PS2, but this is a game that would really benefit from the motion controller.
2/24/08 - Added Insult Swordfighting's controller reviews.
2/25/08 - Valkyria Chronicle looks really interesting. Into the PS3 pro column it goes.

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Super Tuesday

Well, today is the day of the Florida semi-primary, yet another chapter of the ridiculous primary leapfrog game that occurred this year. A week from now, almost half the states in the nation will have their own, very early, primaries. The madness will not end there, of course, especially in the case of the Democrats. The long slog of the primary season will continue for some time, and it is entirely possible that the candidates will not be decided until the end of March, leaving only a paltry seven months for the parties to assassinate the character and distort the record of their candidates more than has already been accomplished in the extended primary season. I have decided not to vote in the primary for a couple of reasons that I'll explain. If I were to vote in the primary, however, I would vote for Barack Obama or John McCain, and I'll explain that, too.

Why won't I vote in the primary? Well, it's not because I can't. Massachusetts has a semi-closed primary, so, because I am not a member of either party, I can vote in either primary. I disagree with this policy, because I feel it discourages additional parties and also third-party candidacies. Independents tend to be more moderate than party faithful; open primaries allow these voters to select centrist candidates. Were the electorate confronted with the true extremes of both parties, the enormous repulsion they felt would make a third-party candidate viable and weaken the parties. I'm also unwilling to signal tacit assent to the parties and their corrupt selection system by joining the primary. Finally, both parties only have terrible, horrible, no good, very bad candidates.

If, however, you put a gun to my head and forced me to vote in the Democratic primary, I would vote for Barack Obama, out of negative selection. None of the candidates have any real, relevant experience—Hillary's claim to "35 years" is a bunch of crap, and at the same time, her politics-as-usual behavior in every state, in every primary, belies her claim to "change" as well. She's a rerun of her husband, and frankly, I've had enough of the Clintons and the cynical politics of "triangulation". We didn't need four more years of Bill back in 2000, and we certainly don't need four more years of him now. Moreover, while it may not be fair to Hillary, the fact is that for reasons entirely unrelated to her actual degree of competence she is incredibly divisive. The issues we face right now are simply too difficult for us to have our President and Congress engaged in a four-year partisan wrestling match. Hillary has neither the leadership nor the charisma that the job presently requires.

So that leaves us with Edwards and Obama. I understand that Mr. Edwards is very angry at rich people and corporations, and feels that elites are waging war on the middle class. This is probably true, but Edwards doesn't actually supply any kind of believable answers, except perhaps waving his magic wand to create 1 million jobs. The adversarial position he adopts is not presently advantageous, in my view. We need to partner with corporations to get things done while reining them in gently, not harangue on and on about how bad they are. They are bad, and the present distribution of wealth is dangerously polarized. Edwards, however, doesn't have any better answer for this, his touchstone issue, than the next guy.

Obama is charismatic, has the right priorities, and lacks the baggage. He doesn't have much experience, but that's true of all the candidates. The Republicans hate him less, which means that he has a better chance of getting Congress moving than Hillary does. Moreover, his message of change is legitimate and inspiring. The message itself is trite, of course, but it gives him a chance of getting a true mandate from the electorate, rather than the narrow, confusing results of the last two elections. That will be a much-needed tool to break the intransigence of Congress. So I would vote for him to be a candidate, if I did not hate the parties, the primary system, and the fact that I can't find any candidate with more positives than negatives.

If you put a gun to my head and forced me to vote in the Republican primary, I would probably ask you to pull the trigger. On the other hand, I might hold my nose and vote for McCain.

Giuliani is a joke, and not even a particularly funny one. Huckabee, with his creationism and "fair tax", is an inhabitant of some absurd fairy wonderland. And Ron Paul's only virtue is that he is the only candidate who seems to be aware that "they hate us" more for what we have done than for who we are. I feel sorry for my uncle Sam, who is a fiscal conservative and therefore has no candidate. The whole row of them parrots the line of tax cuts and reduced spending, but nothing in the background of any of them really suggests they'll hold that line. Hell, at the Florida debate Huckabee practically suggested starting the WPA up again.

And Romney... my God, Mitt Romney running on his record of job creation in Massachusetts is laughable. I am not intrinsically concerned by the fact that Romney's position on every issue has changed—a considered change of opinion is every man's right. What bothers me is that all of his new positions just happen to be more expedient for getting a Republican nomination. Changing your opinion so that you will be more popular makes you a jackass, not a philosopher. Moreover, his side-swapping has ruined all credibility with Democrats and Democratic constituencies. He'll be trusted by neither party in Washington—a bad situation to be in with a Democratic Congress.

I disagree with John McCain on almost everything. However, I think he's as strong on the environment as a Republican can get. He has what the Democrats lack, i.e. experience, and has a track record of working with both parties to get things done. If, as a Republican, you want anything that matches your values to emerge from a Democratic congress, then he is probably the best bet. Also, McCain has the best chance out of all the candidates to rebuild the U.S. reputation in the world. Alone of all the Republicans he could believably claim that the United States does not torture, and his word on that would be more credible than any of the Democrats either (trivia time: under which President did "extraordinary rendition" begin?). As an independent, I could tolerate him as a president. He's certainly not a conservative's dream candidate, but since none of these candidates have real conservative credentials, IMO he's the least of the available evils from a Republican standpoint too. At least he would be strong on defense and capable of restoring our standing with our allies.

So those are the least bad in two fields of terrible choices. I'd be least bothered by a race between Obama and McCain, and I wouldn't know what to do in a race between Clinton and Romney, not least because they'd have the same talking points. What the country needs now is an intelligent leader who can work with the other side of the aisle and mobilize public support for necessary changes, not a partisan hack or professional windvane.

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What do we learn from the Protein Ensemble Method?

Anonymous left a comment on my post on Bruschweiller's work, referencing a couple of papers by Amarda Shehu, Cecilia Clementi, and Lydia Kavraki, the cites for which you can find at the bottom of this post. The most fascinating thing about these papers is the remarkable fidelity with which their Protein Ensemble Method (PEM) reproduces NMR-derived order parameters, 3-bond J couplings, and residual dipolar couplings. The authors demonstrate excellent correlations for ubiquitin, eglin c, Fyn SH3, Fnf10, and CI-2, and while all of these are relatively small proteins this is still a major accomplishment. Nonetheless, it is striking how little we learn from the exercise.

Keep in mind that one of the key goals of a structural biology research program is to get a veridical ensemble, i.e. an ensemble of structures that closely resembles those actually sampled by a protein under equilibrium conditions. We can learn important information from other kinds of ensembles, but the one that contains the information we are really after is the veridical ensemble.

The limitation here is intrinsic to the technique, so the technique bears some explanation. PEM utilizes an algorithm derived from robotics to move pieces of the protein. Initially, the approach was designed to map the ensemble of structures available to a loop, and I want to stress that with regards to that task I have no complaints. When given the task of mapping out the range of likely conformations of these regions this seems like an excellent approach, and the second figure of the 2006 paper seems to put this usage on fairly solid footing. The overall idea is that positioning the ends of a loop next to their anchor points is similar to solving a problem for getting a robotic arm with some number of degrees of freedom to adopt a particular pose. The authors' algorithm solves this inverse kinematic problem with a coarse-grained view of the backbone. At this point the backbone is frozen, the side chains are added back and their conformations are sampled randomly. The conformations thus generated are then subjected to energy refinement using a conventional force field. For a loop with no surroundings, this is all well and good.

The problem arises when the whole protein is subjected to the technique. This is done by using a rolling window of residues: the fragment is chosen, an ensemble defined for it while the rest of the protein is held rigid, and then the window moves to the next overlapping fragment. The various structures determined in this phase are all stored; the dynamic properties of a given residue are derived from a weighted average of all snapshots of all fragments that include that residue, with the exception that the first and last few residues of any fragment are out of bounds due to artificial restraints.

The ensemble of structures derived is therefore not veridical. Because of the fragment-replacement approach, only a single part of the protein is ever actually departing from the equilibrium or minimum-energy structure—it is unlikely that motions are actually distributed this way. Moreover, because the endpoints of all snapshots cannot be simultaneously resolved, it is not possible to assemble whole-protein conformational ensembles from the individual fragment ensembles. So, no individual snapshot is likely to reflect a significantly populated member of the ensemble, and also there is no way to collate the snapshots in such a way that the energetics of the real ensemble are accurately sampled. We thus end up with an ensemble of structures that does not reflect the set of structures actually sampled by the protein at equilibrium.

As a result, the structure that is produced can give us only limited information about the protein. For instance, this might be a reasonably reliable way to predict what sorts of deformations are possible or likely in a binding interaction. Also, PEM probably does a good job of reporting at least the lower limit of the range of the structural ensemble. However, because it does not allow for significant compensating deformations outside of the modeled region the conformations obtained probably do not cover the entire solution ensemble even for a particular fragment.

A clear implication of this work is the idea that the data are dominated by local fluctuations. That is, dynamics information derived from NMR relaxation experiments, quantitative J-coupling analysis, and RDCs primarily reflects short-range motions that do not involve major excursions from the overall structure. If this were not the case, it is unlikely that an intrinsically short-range method such as PEM could reproduce the data so well. This is not exactly a surprise, however, and the nature of PEM for the most part prevents us from learning how local motions in one region of the protein affect local motions in a distal region.

In a larger sense, however, this work reinforces the idea that the ideal approach to constructing a veridical ensemble will involve some combination of coarse-grained and all-atom approaches. The key problem here is not the computational method but the windowing. If the inverse kinematics approach used here can be extended to treat the whole protein—or at least multiple regions of the protein—simultaneously, then I think the situation improves. The question is whether this kind of algorithm will be any more efficient than MD if the whole system is in motion; I suspect at least some part of the computational savings (after what comes automatically with the coarse-graining during step one) arises from having rigid context for the fragment motions. However, this approach is also likely to be more amenable to parallelism than standard MD simulations, and because of the coarse-graining it has the ability to sample structures accessible on a timescale longer than MD can treat.

The authors of these studies imply that their future focus will be on extending this approach to larger structures; I would urge them instead to prioritize developing a way to employ PEM or a similar method without relying on fragment replacement.

Shehu, A., Kavraki, L.E., Clementi, C. (2006). On the Characterization of Protein Native State Ensembles. Biophysical Journal, 92(5), 1503-1511. DOI: 10.1529/biophysj.106.094409

Shehu, A., Clementi, C., Kavraki, L.E. (2006). Modeling protein conformational ensembles: From missing loops to equilibrium fluctuations. Proteins: Structure, Function, and Bioinformatics, 65(1), 164-179. DOI: 10.1002/prot.21060

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What will cure your child's cough?

Continuing with the recent mini-theme of pharmaceutical agents going awry, it seems appropriate to revisit last week's FDA advisory that children under the age of 2 should not be given over-the-counter cough medicines. This advisory had been expected for several months, of course, and the companies involved had already pulled most of these products from the shelves voluntarily. The FDA has not yet resolved the question of what to do about cough medicines for children aged 2-11, but because of the risks of accidental medication and the existence of serious questions about the actual effectiveness of these medications, there is a substantial probability that the agency will decide to ban these as well. Of course, this would seem to leave parents in a bit of a bind: what will they turn to now that nasty-tasting cold medicines are gone?

Of course, the situation isn't that dire: after all, cold medicines do not cure colds: they merely treat symptoms. No cold medicine can actually diminish the length of time a child is sick. The main upshot of this decision will be to put more of a burden on parents, who will have to deal with illnesses using more traditional means. At the same time, I am reminded of a recent study indicating that traditional means might work just fine, at least in limited cases. In December, a report in Archives of Pediatrics and Adolescent Medicine stated that honey was more effective in easing cough symptoms in children aged 2-18 than the popular cough suppressant dextromethorphan (DM).

The study did not rely on objective measures of coughing frequency or intensity, but instead depended on the subjective reports of parents to determine the effects of the medicine. Children were treated with either honey-mimic DM (formulated to approximate the taste and consistency of honey), actual buckwheat honey, or nothing at all. While families receiving no treatment were not blind to this fact, the honey and DM treatment groups were blinded. Surveys filled in before and after treatment dictated who entered the trial and assessed the treatment results. The study involved a few more than 100 children from the Hershey PA area; the sample size was limited by the requirement that no cough medicine have been given to the child in the previous 24 hours.

The outcome is shown in the figure at right—higher scores on the survey reflect a worse condition (i.e. 6 = extremely frequent cough). "First night" reflects the pre-treatment condition, while "Second night" comes from the results of a telephone survey carried out after treatment. You'll notice right away that the claim honey is "better" than DM reflects a pretty narrow difference. For the most part, scores are not that different no matter what treatment was used. This is especially surprising in light of the fact that the "no treatment" option was not blind. One might reasonably expect that awareness of null treatment would produce a negative effect on the subjectively reported outcomes; however, this seems not to have happened. Still, there is a clear, if slight, advantage for the honey treatment in this study. At the same time, it does not appear that parents will be much worse off if they do nothing at all. Keeping in mind that cough suppressants only alleviate symptoms, and have no effect on the underlying infection, these results sharply call the benefits of DM treatments for children into question.

This is not actually as crazy as it sounds. As the authors explain, honey has well-known antioxidant and anti-microbial properties. The dark honey used in this study is likely to be even stronger in this regard. Also, the authors note that sweet placebos have often shown a comparable effect to DM, so physiological effects of the sugar may also play a role. Shelley Batts of Retrospectacle, who covered this study when it came out, suggests that the viscosity of honey may also have a role, as it "coats the throat".

There are some complaints to be made about the study: geographically and temporally it is rather limited, and factors such as income and nutrition seem to lack controls. DM might have benefits in children with poor nutrition or additional conditions that would not be picked up by this study. Moreover, the nature of the sample—cases of cough that were severe enough to cause a parent to visit the doctor but not severe enough to induce the parent to use an OTC medication—may be selecting for parents who overreact to a single bad night of an otherwise mild cough. Clearly a larger study with additional data-gathering and controls is desirable. The best situation would be to enroll families at the beginning of cold and flu season in a truly blind trial and track the coughs longitudinally. Also, "no treatment" should be replaced by something, perhaps a colored simple syrup, to preserve blindness, and a fourth group in which DM is mixed directly into honey should be added to assess the possibility that characteristics of the honey-mimic DM broke the blind. Cynically, one would also prefer that such a study not be funded by the honey board.

Despite these limitations, the results are quite striking. In light of this study and its own evolving but clear preference to err on the side of caution it is likely that the FDA will either ban children's cold medications or recommend significantly stricter labeling. This shift in attitude on the part of FDA may mark the beginning of a generalized swing away from medication and back towards traditional treatments for children with mild illnesses. However, the advent of the preference for medication did not arise from medication itself; rather it evolved in concert with social considerations such as the rise in families where both parents work. In the social context of the 21st century a prescription for "plenty of bed rest, lots of fluids, chicken soup, and honey for the cough", may not be as workable as it was 50 or even 20 years ago.

Paul IM, Beiler J, McMonagle A, Shaffer ML, Duda L, Berlin CM. "Effect of Honey, Dextromethorphan, and No Treatment on Nocturnal Cough and Sleep Quality for Coughing Children and Their Parents." Arch. Pediatr. Adolesc. Med. 161 (12) 2007 p. 1140-1146.

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Uptake blockers do not improve on statins

It has not been a very good couple of weeks for big pharma in the public eye. They've been taking a pounding in the political arena since the primary season finally got into full swing. Last week, we learned that today's medicines, rather than funding tomorrow's miracles, mostly finance power lunches with your cardiologist. And this week, Merck and Schering-Plough, after much delay, finally announced the results of a major study, called ENHANCE, that did not demonstrate any benefit to using the highly-touted drugs Vytorin and Zetia rather than a generic statin.

Because almost everyone who owns a TV knows that Vytorin blocks both sources of cholesterol, and that "Zetia works differently", the results seem particularly damning. The companies involved did not help this appearance by holding back the results so long, and indeed doing so may have exposed them to lawsuits from shareholders and customers. The study was primarily performed by imaging the carotid artery, and in this regard no statistically significant difference was observed between patients treated with Vytorin (statin + Zetia) and statin alone. Also, there was no statistically significant difference in the number of patients who died from cardiac events or strokes, or suffered non-fatal infarctions in the study. While vytorin lowered cholesterol levels by 56% after 24 months vs. the 41% lowering seen in the statin group, this did not translate into improved outcomes by any measure.

So, is this a case of evil pharmaceutical companies trying to gouge consumers over worthless medications? Not exactly. Like every campaign promoting prescription drugs, commercials for Zetia and Vytorin oversold the benefits and did not sufficiently emphasize the risks of these medications. Their failure to improve outcomes in this study is troubling, but because adverse outcomes were so rare overall, it would be difficult to establish an effect one way or another. Also, this study was not geared towards measuring plaques and clots directly; rather it (indirectly) measured the effect of these drugs on atherosclerosis. The effect of uptake blockers on infarction rates was an incidental measurement.

One important consideration when interpreting these results is that the study group was not constructed to resemble the general population. Rather, the study was performed on individuals who had familial hypercholesteremia, a genetic condition that causes greatly elevated levels of LDL in the bloodstream. Without the data in hand I of course cannot make a solid judgment, but one possibility that immediately suggests itself is that in this population even a significant diminution of LDL levels is not sufficient to improve outcomes. There's just too much cholesterol for these uptake blockers to affect the outcome. In a normal individual with significantly lower levels of LDL Zetia and Vytorin might have a much greater effect.

Moreover, all the individuals in this study were already taking statins—which contributed to the low rate of adverse outcomes. It may be that the marginal improvement from adding an LDL blocker on top of a statin simply isn't that great. However, for individuals who cannot take a statin due to side effects, taking an uptake blocker might be a significant improvement over doing nothing. The study does not, as far as I can tell, speak to this possibility.

There is always the possibility that this study points to completely unsuspected aspects of arterial disease. For instance, these results might indicate that once lesions form they attract cholesterol very strongly, and therefore only extreme reductions in circulating LDL can affect their growth. Alternately, this outcome may indicate that mechanisms unrelated to circulating lipids play a more significant role in determining plaque thickness than previously suspected. In light of the highly unusual population used as study subjects, any grand pronouncements in this regard are premature. If these findings are replicated in upcoming studies of greater duration on more representative samples, however, a substantial re-examination may be in order.

The findings of the ENHANCE study don't really indicate that you should burn your Vytorin prescription and go back to just a generic statin, but it does create doubt as to whether this approach will prove efficacious in the general population. Further study, promptly published, is called for, and it would be wise for Merck and Schering-Plough to pull their advertising campaigns for the time being. Doctors should also be less eager to prescribe Vytorin for patients who are responding well to statins alone, but for patients who are not improving greatly with statins (or cannot take them at all), then a prescription for Vytorin (or Zetia) would still seem to be justified. As always, the best approach is to exercise and eat healthy foods, though that's much easier said than done.

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A Chilling Dilemma

Back when I was at Chapel Hill, a fellow student asked my help in figuring out a strange phenomenon he observed with one of his proteins. It seemed that the protein was perfectly soluble at room temperature, but precipitated when it was cooled. Because proteins typically denature at higher, rather than lower, temperatures, he was mystified. Mostly out of luck, the first question I asked him proved to be the right one: "What buffer are you using?" As it happened, he was using Tris buffer, a very common buffer in the biosciences, but also one that changes pH significantly upon cooling. It was not temperature that was denaturing his protein, but rather a change in the alkalinity of the solution it was dissolved in. The change of buffer pH with temperature is one of the most subtle and least-discussed issues in biochemistry, but as in my friend's case, can often produce confusing effects that significantly complicate research. An article by scientists from the University of Illinois online now at Chemical Communications (click link for open access), however, aims to resolve this problem by producing a temperature-independent pH (TIP) buffer for biological applications.

The approach they employed was actually very simple: rather than search the ends of the earth for a buffering agent that would maintain its pH across a huge temperature spread, they just found two that had opposite reactions to chilling and mixed them together. The buffer system they ended up with contained 60% HEPES and 40% potassium phosphate and experienced a pH change of less than 0.07 ± 0.1 over a temperature range from 25 °C to -180 °C. Readers who have encountered this subject before will recognize that HEPES and K₂HPO₄ are already fairly pH-stable over most of the laboratory temperature range, but as the paper's first figure shows, the TIP maintains these benefits down to very low temperature. A colorimetric assay was used to track the pH to very low temperatures.

So, problem solved, right? Well, not exactly. This finding is very nice as far as it goes, and has some promise for labs that do a lot of work that involves freezing proteins—structural biology labs come to mind. But while it would be nice to be able to select buffers on the basis of pH characteristics alone, that's rarely feasible. Not all buffers work with all proteins. In some cases, this can be rationalized: proteins that bind ATP or nucleic acids often bind or interact with phosphate buffers, resulting in poor solubility or aberrant structural dynamics. In other cases the observations are harder to understand—it seems that some proteins just "don't like" some buffers. So long as you're dealing with very dilute proteins, this is not usually an issue, though the biochemistry is sometimes deranged as a result. However, when high protein concentrations are employed—again, this is characteristic of structural biology—incompatible buffer-protein combinations tend to result in crashed protein.

The good news is that, with the exception of proteins that normally interact with phosphate moieties, both of these buffers are fairly well tolerated by a wide spectrum of systems. So long as a nearly-neutral pH is desired, this buffer combination should be useful. Researchers wishing to safely put pH-sensitive small molecules into long-term cold storage are also likely to find this buffer a boon. Moreover, because the buffer components are common and inexpensive almost any lab will be able to use this approach.

Sieracki, N. A., Hwang, H-J., Lee, M.K., Garner, D.K. and Lu, Y. "A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures." Chem. Commun. 2008 DOI 10.1039/b714446f

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