Toccata and fugue in the postapocalyptic wasteland

Baroque is a Playstation 2 / Wii remake of a Playstation remake of a game originally made for the Sega Saturn, and although I am happy, in a way, that it traversed this long chain of production I cannot quite figure out why it did so. Even loose Roguelikes are not usually a winning commercial proposition, the popularity of Persona 3 notwithstanding, and Baroque lacks the concessions that would allow it succeed more broadly. For all its faults, though, there are some interesting ideas in Baroque that deserve some thought.

Spoilers, as usual, after this line of text right here, goddammit.

Baroque takes place in a post-apocalyptic wasteland, and while the fact that it is a wasteland is apparent immediately, that it is post-apocalyptic is something that you must learn, because the main character begins without any memory. Now, Wlad and Michael Abbott and maybe a few others of you know that I usually loathe amnesia as a narrative mechanic, but I did not mind it here. Mostly this is because the majority of games use amnesia as an excuse for long explanatory monologues, while Baroque just tosses you into the story without explaining much of anything. As you stumble forward in the world you encounter an Archangel who gives you a special gun and urges you to make your way to the bottom of a place called the Neuro Tower and use the gun there. "Use it," he urges, "There is meaning in you using it."

The Tower is a dungeon filled with creatures that were once human, but who became twisted into something else by "baroques", twisted ideas that come to dominate their minds. In the world that exists after the apocalypse, ideas that distort the mind can warp the body, and sometimes they remain in the form of a crystal after the body's destruction. By gathering these crystals, and exploring the tower, the nameless main character learns more about the world and its past. The catch, however, is that the Neuro Tower allows travel only in one direction (downwards). To depart the tower and learn from the people in the outer world, the hero must die. He then reappears in the world, having lost all his items (though some can be transferred from the dungeon and found outside) and progress (he is reverted to level 1). To proceed in the story, he usually must enter the dungeon and make his way to the bottom once more.

The hero learns that he, the surviving one of conjoined twins, was fused with God, whom the Archangel had driven mad in part by removing her pain. The Archangel separated the hero from God, causing the apocalypse. The hero must return certain ideas and pain to the multiple personalities that split off from God, and then fuse with her to adapt some kind of livable world from the current existence.

This whole scheme, in my opinion, is practically bursting with potential. One ideal route would be to highlight that the repetitive mechanics make the traversal of the tower a baroque for the main character (or the player). The idea of pain as necessary for sanity, and the parallels between the hero's and God's feelings of incompleteness make for interesting thematic territory. If you wanted to turn the game into a commentary on games and gaming (despite my love of No More Heroes this is not something I generally advise), the subversion of traditional RPG goals would give you a lot to work with. Yet Baroque never really does any of these things; it is simply satisfied to present its minimal story and its Roguelike dungeon and to let you imagine the wonderful things that could have been done with this premise.

Part of the problem here is the repetition; putting repetitive tasks in a game always involves walking a fine line between holding the player's interest and annoying him, and Baroque definitely teeters over into the latter. The real-time combat is dull, and the hero doesn't really become any stronger by leveling up. Instead, the character's strength is almost completely dependent on the items he finds in the tower. Because so many of the items he can find are harmful to him (a common element of Roguelikes), the hero's ability to fight and survive is almost totally random. Because of this, actually fighting through the levels is, after a certain point, a total waste of time. It's better just to run whenever you can. I am still on board for this, but if fighting through the levels is not a worthy end in itself then a game needs to have something else to draw the player along. Baroque doesn't.

Baroque just has too little content to support even the minimal amount of play necessary to finish the game (judging from some FAQs, the game can be completed only entering the Tower 3 times, though it took me 10 attempts to fumble through). Part of the problem here is that the most interesting parts of this tale happened in the past. The backstory of the game is very tense, very emotional, and involves some interesting ideas, but the reality is that you are just playing the epilogue of that epic. Although the pace at which the past is revealed is good, the story doesn't really have an arc, or develop any tension beyond the player's increasing annoyance at going through the Neuro Tower again again. Moreover, the Christian and Kabbalistic imagery is pretty worn at this point, though perhaps it was fresher when the game was first made.

I won't fault Baroque just for being a Roguelike. However, it is fair to complain that the dungeon crawl just isn't entertaining enough to sustain a game on its own, the story isn't strong enough to compensate, and the thematic possibilities never become realities. From its confusing beginning to its end, Baroque repeatedly flashes signs of brilliance, but it never really delivers on the promise. Somebody could make a great game out this.

Sadly, nobody did.

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EGCG disrupts amyloid oligomers

As I mentioned in my post on the recent paper by Kukar et al., the disruption of amyloid plaques has been an ongoing focus in Alzheimer's disease research. However, plaques and inclusions are of concern in many diseases, and as a result there is a great deal of interest in finding molecules that can either dissociate, or prevent the formation of, amyloids of many different kinds of proteins. In the most recent issue of Nature Structural and Molecular Biology there is a paper suggesting that (-)-epigallocatechin gallate (EGCG) may be able to interfere with multiple proteins that form β-rich aggregates.

EGCG is a chemical found in green tea (although it is doubtful you could realistically drink enough green tea to absorb the concentrations used in this study). Previous studies had suggested that it altered the aggregation behavior of α-synuclein (αS) and huntingtin. So, Ehrnhoefer et al. use highly purified EGCG in a number of experiments to determine what effect it had on αS and the amyloid-β peptide (Aβ) (1). What these proteins have in common, besides the fact that their aggregation is associated with disease, is that the single proteins take on a β-strand structure that assembles into fibrils.

Ehrnhoefer et al. find that EGCG interferes with some aspect of this process, reducing the formation of the fibrils while inducing the formation of some alternate oligomeric structure. In the case of αS, the result is a spherical oligomer (Figure 1), although the gel filtration results indicate that a large spectrum of oligomeric states is formed at lower concentrations (the trace suggests that these oligomeric forms are interconverting during elution). NMR and other data show that EGCG associates directly, but non-specifically, with the protein backbone, and that the compound has strongest affinity for the C-terminus of αS, which may play a role in preventing aggregation. The EGCG-treated oligomers had reduced β-strand content (as assayed by CD). Treatment with EGCG appeared to reduce αS toxicity in cultured cells, although this was measured strictly in terms of cell death.

Similar results were seen with Aβ—addition of EGCG reduced the formation of fibrils and the toxicity of amyloids towards cultured cells. Again, the oligomers formed in the presence of EGCG could be quite large, and took a spherical shape.

Based on these data, the authors propose that EGCG binds preferentially to unfolded proteins and interferes with the formation of regular β-strand structure. The EGCG-bound proteins are unable to form fibrils, and therefore EGCG oligomers compete with fibrils for monomers, slowing the formation of the latter. The net effect is to divert these unfolded proteins out of amyloidogenic pathways and into alternate oligomeric structures, which appear to be nontoxic, or at least less toxic.

Can EGCG or a derivative be turned into a drug to treat Alzheimer's disease, or a general treatment for amyloidoses? This is an uncertain proposition. As the authors of a commentary (2) in the same issue of NSMB note, EGCG's nonspecific assault on amyloids may damage some normal structures built on this architecture. Moreover, because EGCG seems to bind unfolded regions nonspecifically, it has the potential to interfere with any of the numerous signaling proteins that possess such regions. The potential for side effects is very high, and the continued viability of cultured cells in the presence of EGCG, while reassuring, is not a particular reason to believe the compound is safe at high concentrations in the human nervous system.

The promiscuity of EGCG's interactions with unfolded regions poses another problem, in that all these proteins will act to interfere with EGCG's action on its intended target. Fairly high ratios of EGCG were necessary in these assays, and they mostly involved purified proteins. In vivo, all unfolded proteins will act to titrate EGCG out of plasma, meaning that significant quantities of this (or any other non-specific molecule like it) would need to be used in order to achieve the desired effect. This again raises the likelihood of side effects.

Of course, the most severe complication arises from the nature of amyloidoses themselves. Although the obvious presence of plaques and inclusions naturally leads us to suspect that these aggregates are the agents causing the disease, increasing evidence suggests that amyloids are merely the endpoints of some other process that is the actual culprit. In the case of Alzheimer's disease, for instance, a recent article in Nature Medicine has provided very strong evidence that soluble Aβ dimers are the dominant contributors to Alzheimer's pathophysiology (go check out Ashutosh's excellent discussion of this article over at The Curious Wavefunction for more information). Now, given that these dimers do eventually form amyloid, it seems likely that they have β structure in their pathogenic form, which EGCG will probably disrupt, but this is not guaranteed. Small molecules like EGCG that prevent deposition into amyloid may actually exacerbate the problems they are meant to solve. Further research is needed to establish that the EGCG oligomers of αS and Aβ are not toxic in vivo.

Diseases associated with protein aggregation continue to pose a challenge precisely because we have such a poor handle on their pathogenesis. Ehrnhoefer et al. clearly demonstrate that EGCG possesses the ability to alter the behavior of amyloidogenic unfolded proteins. While that may imply that it has promise as a broad-spectrum drug to attack these diseases, the promiscuity of its action is a cause for concern from the perspective of dosage and side effects. And, because soluble oligomers may well be the pathogenic species in many (if not all) of these diseases, our optimism about this approach must be tempered with an awareness that the actual effect of EGCG may be to enhance, rather than diminish, the toxicity of the relevant protein targets.

1. Ehrnhoefer, D.E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., Lurz, R., Engemann, S., Pastore, A., Wanker, E.E. (2008). EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nature structural & molecular biology, 15(6), 558-566. DOI: 10.1038/nsmb.1437

2. Roberts, B.E., Shorter, J. (2008). Escaping amyloid fate. Nature Structural & Molecular Biology, 15(6), 544-546. DOI: 10.1038/nsmb0608-544

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Recent play rundown

Most of the games I've played since finishing The World Ends With You simply haven't done much for me, regrettably. One or two were inspiring, and I'll talk about them at some point when I've had more of an opportunity to digest them, but the majority either disappointed me, or just didn't leave me with much to say about themes or characters. Still, I had some thoughts about these games I wanted to put down. So if you're interested in my opinion of Insecticide, Penny Arcade Adventures Part One, Fire Emblem: Radiant Dawn, or Final Fantasy Crystal Chronicles: Ring of Fates, read on. If you're not, well, screw you; it's my blog.

I didn't finish Insecticide; I may have only barely started it. This was a game I wanted desperately to like, and in fact, except for one incident of what Yahtzee might call "moon logic", I loved the hell out of the adventure portions of it. The ones I worked through weren't anything special in terms of the gameplay, but they were cleverly written and funny, and they successfully evoked happy memories of point-and-click adventure games. However, I loathed the combat portions, to the degree that I ultimately tossed the game aside in frustration. I'm not sure whether the combat segments seemed like a good idea earlier on or what, but at some point someone should have realized that movement was awkward, the controls were sluggish, and they would have a much better game if they skipped all this crap and just put more detective work in. Were the developers really convinced the shooter segments were good? Did they start down a road they couldn't back out of? I can't say. I just wish it had turned out differently.

Continuing with the DS, I followed Insecticide by trying out Final Fantasy Crystal Chronicles: Ring of Fates, a game I finished, but one I also felt fell short of the mark, at least as a single-player experience. The controls weren't so bad, but there were two significant problems. The first was that casting spells was just slow and clumsy. The second was that the only way to get maximum use from the game would be to use your hands for both sets of buttons and the stylus simultaneously, which was awkward and uncomfortable. The AI governing your allies was awful; about all they were good for was wasting your magicite. The platforming was needlessly complicated by your inability to look around the rooms you were passing through. And then there was the story, which worked in a perfunctory way, but strained credulity to the breaking point and ended in an incoherent, unsatisfying mess. If Chelinka really had sacrificed herself, if it had been a bittersweet ending instead of a saccharine one, I would have thought better of it.

Moving from the DS to the Wii, I finally finished Fire Emblem: Radiant Dawn. I loved the previous entry in this series, the Gamecube's Path of Radiance, but I felt let down by the sequel. Part of this was due to the fragmented nature of the narrative. What I liked most about Path of Radiance was taking the Greil Mercenaries from marginal competence to mastery; here there's no continuous arc. Rather, you get thrown back and forth between sides of a very large conflict, leaving some characters behind for several chapters. When you see them again, you've practically forgotten who they are, or why you care if they get killed. This will happen, because when you see them again they are often absurdly under-leveled. Perhaps to compensate for this, you are frequently given a vast army of high-level warriors, most of whom seem to have been lobotomized with a trebuchet. Due to their large numbers the ally turns take quite a while; their extraordinary stupidity makes these phases seem even longer.

A side effect of the construction is that the cast feels far too large, an impression only exacerbated by the replacement of the charming support conversations that took place in camp in Path of Radiance with boring, generic support conversations that take place in the field. This gives you much less of a handle on the characters' personalities, and excises the side plots of the first game. The story of Soren's lineage in that game was one of my favorite discoveries; there's nothing quite like that here. That's a pity, because the main plot of Radiant Dawn is an insipid mess. In Path of Radiance, Ashnard was crazy, but at least he had a goal and took concrete, sensible steps towards it. The villains of Radiant Dawn are apparently evil just to be evil, without any apparent aim or coherent strategy. This is a narrative sin in any game, but it's especially absurd when the gameplay is built around strategic and tactical thinking. The battles where you don't have too many allies are entertaining, but there's no basis for an emotional connection to the characters or the story. You're better off playing chess.

Well, enough being mean. Let's talk about something I really enjoyed: Penny Arcade's On the Rain Slick Precipice of Darkness. As writers of some of the excellent blogs in my sidebar and Blägroll have noted, OtRSPoD doesn't really do anything vastly original in terms of story or gameplay. It's a decent fusion of fairly standard RPG mechanics and point-and-click adventure that has two significant virtues. The first is that it does almost everything right, and the second is that it is great fun to play. The game world is populated by hilarious characters and enemies, and unlike many entries in the genre, OtRSPoD doesn't punish you for trying to see all of it. The concepts are funny, the dialogue is entertaining, and the combat is fun, although I found using items to occasionally be something of a drag. The humor probably isn't for everyone (does a creepy, libidinous juicer sound funny to you?), but if you can see the appeal in fighting a dread mime deity from beyond the edge of reality with a magic rake, it's worth giving Episode One a whirl. I'm pretty sure I'll be grabbing Episode Two.

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A conformational equilibrium controls the Vav DH domain

One emerging view of allostery, and protein behavior generally, describes function in terms of pre-existing equilibria. In this view, proteins are not like switches that get turned on and off, but rather are like dials that are turned "more on" or "more off" depending on the conditions. In this view, regulatory modifications such as phosphorylation do not enforce an active conformation so much as promote it. Because some relaxation measurements are sensitive to conformational exchange, NMR is well-suited to examine systems with this behavior. In the most recent edition of Nature Structural and Molecular Biology, a group from Michael Rosen's lab discover that this kind of equilibrium is governing the behavior of the DH domain from the Vav protein.

Vav activates Rho GTPases by inducing the exchange of GDP for GTP, making it a Guanine nucleotide Exchange Factor (GEF). This activity is performed by the DH domain, and is inhibited by a small neighboring element called the acidic region (Ac). As you can see from the figure of the combined Ac and DH domains (AD) at right, Ac (red) inhibits the DH domain (blue) by forming a helix that binds to the active site (explore this structure at the PDB, noting that the numbering is off by 167). Phosphorylation of Y174 (red side chain) unfolds the helix and exposes the active site, which would be a nice model except for two things. First, as you can see, Y174 is pretty well buried in this structure, which would make it difficult to phosphorylate. Second, mutation of Y174 to phenylalanine, a residue that cannot be phosphorylated, activates DH domain activity. How can Y174 get phosphorylated? What is phosphorylation actually doing?

One possible answer is that the existing structure doesn't tell the whole story. A protein, after all, doesn't have just a single structure, but rather an ensemble of structures across a population or time. While AD may spend most most of its time in this inhibited state, it's possible that sometimes it adopts an alternate conformation that allows Y174 phosphorylation. Li et al. set out to assess this possibility using measurements of R₂ relaxation dispersion. Residues that have a large field-dependence of transverse relaxation (ΔR₂) are undergoing some sort of conformational exchange process that changes their chemical shift between two or more states.

Using CPMG experiments on methyl-bearing side chains, Li et al. identified two groups of residues engaged in conformational exchange processes, shown at left. The first group (orange side chains) have a large ΔR₂ that vanishes once Y174 is phosphorylated. The second group (green side chains) have high ΔR₂ in both the phosphorylated and unphosphorylated states, but the rates are slightly higher in the former. Residues that were observed to have low ΔR₂ are shown with gray side chains. All of this indicates that there are two dynamic processes occuring on the microsecond-millisecond timescale. The first encompasses some change in the chemical shift of the acidic helix, while the second involves some unknown process. However, because the Group 2 residues react to the phosphorylation state, these processes are likely linked in some way. I notice that the Group 2 residues are clustered around loops and joints in the upper half of the domain (in this view), while residues not adjacent to loops or joints do not appear to have significant ΔR₂. It is possible that the observed dispersion represents some flexing of the domain around these loops, and that the rate of this motion increases slightly when the binding site is unoccupied.

That's all very interesting, but it's also bad news for the analysis, because it means the observed relaxation dispersion would have to be fit to a four-state model in order to obtain populations and kinetic parameters. Previous analysis by several groups has shown this to be a dubious proposition, so Li et al. take an alternative approach. Rather than try to fit out populations from the dispersion data, they make a series of mutations to AD to push the populations of the two states in one direction or the other. They find a number of states where the methyl peaks lie on a line between the open (phosphorylated) and bound (unmodified) states. The Y174F mutation lies very close to the phosphorylated state, interestingly enough, implying that the phosphate group itself is not a significant determinant of chemical shifts in the open state. Using a combination of HSQC peak positions and ΔR₂ measurements, Li et al. determine for each mutant or modification what population of the ensemble is in the open state. They find that this NMR-assigned population correlates with the rate constant (k_cat/K_M) for phosphorylation.

This implies a model in which regulation of DH by Ac involves an equilibrium between the bound and open states. In the bound state, Ac forms a helix in the binding site, an effect strongly dependent on a hydrogen bond to the OH of the Y174 (R332 may be the partner here). However, in this state Ac samples the open state about 10% of the time. While in the unbound state, Ac can be phosphorylated, a modification that prevents helix formation or binding; probably by steric interference in the binding site. This stabilization of the open state dramatically increases the chances that the Vav DH domain will be in an active state when it encounters a target. Thus, DH regulation depends on a population shift of an underlying equilibrium, not a singular on/off switch. This model has the advantage of accounting for how Y174 gets phosphorylated and why a mutation that prevents phosphorylation nonetheless leads to a constitutively activated state.

In vivo, Vav consists of many other domains in addition to the AD construct used here. These domains are known to contribute to the inhibition of DH; given these results it is probable that they do so by stabilizing the bound state. Because Ac binding causes the formation of a negatively-charged surface on one side of the helix, charge stabilization is a likely mechanism. Further research will hopefully identify these mechanisms, as well as the origin of the second conformational exchange process revealed by the experiments in this paper. This study is a good example of scientists making the best use of limited data to describe an instance of this important, but difficult to characterize, regulatory mechanism.

1. Li, P., Martins, I.R., Amarasinghe, G.K., Rosen, M.K. (2008). Internal dynamics control activation and activity of the autoinhibited Vav DH domain. Nature Structural & Molecular Biology, 15(6), 613-618. DOI: 10.1038/nsmb.1428

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Structural dynamics of PDZ allostery

If Michele Vendruscolo were trying to get me to blog about one of his papers, he could hardly have assembled a more perfect lure than his upcoming paper in JACS. It brings together all sorts of things I've been talking about on this webpage: NMR dynamics, MD simulations, and dynamics-driven allostery (in the PDZ domain, no less). Previous investigations of this PDZ domain indicated the existence of a network of residues that had a dynamic response to ligand binding. Dhuselia et al. extend this work using molecular dynamics simulations constrained by the existing dynamics results. This leads them to discover not one, but two networks in the PDZ domain, with different properties.

NMR experiments have enormous power to sensitively detect changes in dynamics resulting from a perturbation, but they are also quite limited. Because of the models we use, the parameters we can fit out of relaxation data only give us information about the magnitude and timescale of fluctuations. Chemical shift overlap and interference caused by nearby dipoles limit the number of probes. Moreover, because NMR can only measure an ensemble, it is practically impossible to extract anything other than the most general information about correlated motions. MD has answers to all of these problems, but as a general rule has done poorly at reproducing NMR data about side-chain motions, calling the validity of the conclusions into question. Vendruscolo has taken some interesting strides in this regard by employing the limited experimental dynamics data as a component of the energy function. By constraining the simulation to mimic the known dynamics, we can hopefully learn more about the sites to which we are blind, as well as what kinds of motions the experiment is sensing and how they are linked.

In this instance, the authors make use of the PDZ domain previously studied by Ernesto Fuentes in Drew Lee's lab (there was also some hack working there at the time). Ernie's research followed on previous evolutionary studies indicating a network of communication in PDZ domains (local summary here), and Ernie found, by comparing the dynamics of the free and ligand-bound states, that changes in motions propagated away from the binding site to two distal surfaces. The pathways of communication compared pretty well with the evolutionary results. Dhulesia et al. aim to extend these results by determining which motions are correlated and identifying the mechanisms by which energy is transmitted. They accomplished this by running multiple parallel simulations of the free and ligand-bound states of the PDZ domain constrained by Ernie's dynamics results, as well as NOE and ³J data.

They find that two regions of the protein have correlated motions internally and move in an anticorrelated fashion relative to each other (Figure 3A). One of these regions consists of part of the binding site and all of distal surface 2 (DS2), while the other includes the other half of the binding site and all of distal surface 1 (DS1). When the ligand binds, something interesting happens. The motions of DS2 become more tightly correlated to the motion of an area around V30. The tight correlation between the motions of DS1 and α2 (an element of the binding cleft) switch to a slight anticorrelation.

When a ligand binds to a protein we expect a broad increase in rigidity of the complex so that the proper orientations of bonding pairs are maintained. For the most part, the simulations affirm this expectation, but not for all regions. For the binding site and DS2, the backbone mobility decreases, as expected, but the backbone mobility of DS1 increases (I am going off the text and Table 3 here, rather than Figure 3). The side chains have a similar response. This agrees with other studies indicating that the change in conformational entropy upon binding a ligand need not be homogeneous. What is more interesting is that these results imply that opposite coherent responses can be induced in a small domain by a single stimulus.

Although (as far as I know) this PDZ domain has no allosteric behavior in vivo, one can imagine that the binding of a ligand at the cleft could alter the binding of other modules to this domain. The entropic penalty for binding to DS2 would be lower in this case, while the penalty for binding to DS1 would be higher. The opposed nature of the dynamic responses may be related to the broad regional anticorrelation of free-state motions; disruption of this mode (by linking the motion of β2 and α2) may shunt that energy into DS1.

The authors also find, using a series of structural parameters, that a set of residues have clear structural changes. Some of them appear to be associated with coupled changes in rotameric states; the authors map out one pathway in Figure 5. Because it is a rotameric pathway, it should be possible to test whether it is essential to communication experimentally—mutation of the intermediary residues should abolish the linkage. The authors also carry out a network analysis to identify the most connected residues, a prediction that may also be testable by mutagenesis. These "structural network" residues overlap only slightly with the dynamic network, and indeed do not generally intersect with the evolutionary network either. In the absence of identified allosteric behaviors or clear energetic connectivities it's difficult to say what this disjunction means. However, the residues undergoing structural changes surround most of the residues undergoing dynamic changes. It is possible that these changes in structure provide the context that allows the changes in dynamics (or vice-versa); the two properties are inextricably linked.

Although communication between the binding site and distal surfaces is proven in this PDZ domain, and appears to be a general feature of the fold, the absence of a known function for the propagation in this instance makes it tough to assess the quality of these results. However, the findings of Dhulesia et al. make it clear that this approach can produce testable predictions and explanations. Hopefully this approach will be employed in the near future to study PDZ domains known to possess allosteric properties.

1. Dhulesia, A., Gsponer, J., Vendruscolo, M. (2008). Mapping of Two Networks of Residues That Exhibit Structural and Dynamical Changes upon Binding in a PDZ Domain Protein. Journal of the American Chemical Society DOI: 10.1021/ja0752080

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Determinants and evolutionary mechanisms of homosexuality

Debates over the rights of homosexuals in the United States, particularly the right to marry, often get hung up on a thoroughly inane point: whether homosexuality is "chosen" or "innate". While this may seem to be a question of moral import, it is not, and moreover it presents a false dichotomy. Like nearly all human behaviors, sexuality is too complex to be reduced to a choice or a destiny; it is neither, or both, depending on your view. However, the degree to which different factors contribute to sexuality, and the mechanisms by which they do this, are fit subjects for scientific inquiry. Two articles this week present interesting findings, sure to be distorted by all sides of the argument, that may prove enlightening in this regard. I will endeavor, along with others, to be a resource providing an unbiased view.

First, however, a plea for sanity. If science finds, by some transcension of nature, that sexual orientation is entirely chosen, or entirely innate, it does not matter to any debate over the rights of homosexuals. Men may have an innate tendency to try to spread their genes as widely as possible, but we would not forgive adultery on this basis. Toddlers have an innate tendency to become frustrated and throw tantrums, but we still make them sit in the corner. That a behavior is innate is not a basis for withholding moral judgment. And if sexual orientation is a choice? Well, we frequently forbid discrimination on the basis of chosen behaviors—religion, for instance, or political affiliation. What is truly at issue is not choice, but whether it is just to deny rights and protections to one group of citizens for no reason beyond the moral opprobrium of another group.

Thus, the question of rights for homosexuals does not depend, one way or the other, on whether people choose to be gay. I firmly believe one side of this question to be in the right, but this opinion is not informed by my scientific knowledge, because it cannot be. I would urge my readers (all three of you) to view these results strictly as what they are: interesting scientific findings related to a political question that do not support one side of the argument or the other. I would ask advocates for both sides to refrain (for once) from distorting the conclusions of these reports, not only because of the raw immorality of lying, but because by misrepresenting these findings they will have sacrificed their integrity for no gain in the debate.

There, I feel better now. On to the science!

In the first study, a team analyzed the results of a survey of Swedish twins in hopes of parsing out the relative contributions of heredity, shared environment, and unshared environment in shaping sexuality (1). Although the survey was answered by a fairly large number of twins, the authors draw their conclusions using two questions that do not directly ask for sexual orientation. The survey only requested information about actual sexual partners, and did not address homosexual feelings that might not have been acted upon. After excluding twin pairs that were opposite-sex or unclear with respect to zygosity, they had 3826 pairs to work with, of which 5% of men and 8% of women reported at least one same-sex sexual encounter. Because it is suspected that the factors influencing homosexuality may differ between the sexes, males and females were treated separately. By comparing the concordance and discordance of sexual behaviors between monozygotic and dizygotic twins it should be possible to parse out the degree to which genetics and the environment contribute to sexuality.

Despite the limited materials, the authors were able to reach some conclusions, with the caveat that the 95% confidence intervals were quite wide. For instance, for males they found that genetic factors explained 39% of the observations with respect to whether a twin had any same-sex partner in his life. However, the 95% CI on this prediction was 0% to 59%. For men, shared environmental factors appeared to contribute nothing, while unique environmental factors explained 61%. For women, it was determined that genetic factors contributed 19%, shared environment 17%, and unique environment 64%. Similar distributions were seen for comparisons of total numbers of same-sex partners. While the confidence intervals for all factors are quite large, the numbers largely agree with a previous study on Australian twins (less so with a study on American twins).

Obviously, the small sample size and broad confidence intervals on these results suggest that they should be interpreted cautiously. It should also be noted that "unique environmental factors" may run the gamut from hormone exposure in utero to childhood illness to personal experiences. Many unique environmental factors, even for twins, are just as involuntary as genetics, but some result from conscious choices of the individual (which is different from choosing to be gay). Despite their limitations, these results generally support the idea that sexual orientation results from a confluence of genetic and environmental factors.

That genetics play a role in homosexuality may seem curious, because in terms of the classic expression, "survival of the fittest", homosexuality would appear to be a non-starter. After all, a reluctance or outright inability to mate with the opposite sex would seem to result in a substantial reduction in reproductive fitness. However, contrary to what a certain ignoramus would have you believe, the Theory of Evolution has advanced substantially since the days of Darwin, and we are aware of numerous additional evolutionary mechanisms that operate alongside the law of natural selection. In the case of male homosexuality, a new paper by Camperio Ciani et al. argues that sexually antagonistic selection may be at work (2). PLoS ONE is open access, so feel free to open up the article in another window and skim it yourself.

Camperio Ciani et al. begin with the observations that male homosexuality has a matrilineal association, and that the mothers (and maternal aunts) of homosexuals are somewhat more fecund than the population at large. From these pieces of data, and from the fact that homosexuality appears to have been present at low levels in every society that has left written records, the researchers created a set of requirements for some evolutionary simulations, based on different supposed properties of the genetic factors influencing male homosexuality (GFMH). Most of the simulations failed to satisfy the parameters. In many cases (especially with single-locus traits) the GFMH either became extinct or gained too high of a frequency; in others the matrilineal association was not preserved.

Ultimately, the researchers found that the model that best fit the parameters featured two alleles (one of them X-linked), and was sexually antagonistic. What this means is that the trait increases the reproductive fitness of one sex while decreasing that of the other. For instance, a heightened sexual response to men could make women more likely to pass on their genes, while making men possessing the trait less likely to do so. Provided that the effects of this trait are balanced with respect to the population proportion of each gender, it should be possible for it to survive in a population at a relatively constant level.

This result is interesting, and provides some hypotheses that can be tested with genetics. However, it does not prove that homosexuality is genetic, or even that it has a genetic component. Like all simulations, these results merely inform us that a particular possibility is consistent with what we already know. In this case, we now know that the observed aspects of homosexuality are consistent with a 2-locus trait that is sexually antagonistic. However, this model was arrived at simply through process of elimination, and there may be some superior model or more-accurate mechanism that simply hasn't yet been tested. There is always a model we haven't thought of; sometimes that model is the right one. Moreover, as Långström et al. note, some of the data used to determine criteria for successful simulations remain controversial. Camperio Ciani et al. convincingly show how the preservation of homosexuality through evolution could happen, but that is not the same as demonstrating how it did happen. That will require a positive identification of the actual GFMH.

The results of Långström et al. indicate that any GFMH eventually identified, whether or not they materially resemble the predictions of Camperio Ciani et al., will only give rise to a heightened propensity for homosexuality. Environmental factors play a significant, perhaps even dominant, role in determining sexual orientation. Whether genetic or environmental, most factors contributing to homosexuality are involuntary, but some are chosen. If that answer doesn't satisfy you, perhaps you were asking the wrong question.

1. Långström, N., Rahman, Q., Carlström, E., Lichtenstein, P. (2008). Genetic and Environmental Effects on Same-sex Sexual Behavior: A Population Study of Twins in Sweden. Archives of Sexual Behavior DOI: 10.1007/s10508-008-9386-1

2. Camperio Ciani, A., Cermelli, P., Zanzotto, G., Brooks, R. (2008). Sexually Antagonistic Selection in Human Male Homosexuality. PLoS ONE, 3(6), e2282. DOI: 10.1371/journal.pone.0002282 OPEN ACCESS

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NSAIDs bind to amyloid-β

One of the best-known features of Alzheimer's disease pathology is the formation of proteinaceous amyloid plaques in the brain. In Alzheimer's disease these plaques are primarily formed by the amyloid-β peptide (Aβ) derived from the amyloid precursor protein (APP) by the action of β- and γ-secretase. The length of the Aβ peptide varies, but the 42-residue form (Aβ42) is more likely to form plaques and fibrils. Although it remains uncertain whether plaques are a cause of Alzheimer's disease symptoms, or merely an effect of some underlying derangement, finding some way to prevent or reduce plaque formation is a major goal in the field. This week in Nature, a team of researchers from institutions all over the US and Europe show that non-steroidal anti-inflammatory drugs (NSAIDs) may be able to accomplish these goals by binding to APP and Aβ directly.

Previous research from the Koo lab indicated that some NSAIDs specifically reduced the production of the amyloidogenic Aβ42 fragment (1) both in cultured cells and in a mouse model of the disease. APP was still processed into peptides, but these were shorter and less likely to form amyloid plaques than Aβ42. Significantly, the cleavage of other γ-secretase targets was not affected, meaning that side-effects of NSAID treatment might be minimal. Although NSAIDs were expected to ameliorate Alzheimer's symptoms by reducing inflammation, Weggen et al. found that the beneficial effects were not the result of cyclooxygenase inhibition. In a follow-up paper (2), Weggen et al. used experiments on cultured cells to show that the drugs were directly modulating γ-secretase activity. These experiments also showed that mutations to presenilin-1, a core component of the γ-secretase complex, could either increase or decrease the effect of NSAIDs, suggesting that it was the protein directly affected by these drugs.

Kukar et al. set out to test this hypothesis using photaffinity labeling. They took a few compounds known to alter Aβ42 levels and added a functional group that would react with a protein in the presence of UV light. These covalently-labeled proteins could then be detected, and this would serve as a relatively easy way to determine which component of the γ-secretase complex was actually binding NSAIDs. Like many cleverly-designed experiments, this failed in an interesting way: no known components of the γ-secretase complex were labeled. Fortunately, the researchers realized that there was another component to the complex they hadn't tested yet: the substrate.

It turned out that the NSAIDs could label a 99-residue fragment of APP. Moreover, this labeling was reduced by other γ-secretase modulators (GSMs) and unaffected by non-GSM NSAIDs. Using a series of progressively shorter constructs, Kukar et al. localized the binding activity of GSMs to residues 28-36 of amyloid-β.

This on its own is a very useful finding because it provides a target for refinement of these compounds. Knowing where and to what protein a possible drug binds makes it easier to develop assays to test new potential drugs, as well as enabling structure-based design. However, the authors took the next step and asked whether these drugs, because they bind to a region of APP known to be involved in the formation of amyloid plaques, might inhibit plaque formation directly. In cultured cells, they found that treatment with certain substrate-targeting GSMs decreased the formation of Aβ dimers and trimers even under conditions where the overall concentration of Aβ42 was not altered.

This suggests that these GSMs may be able to fight the buildup of amyloid plaques in two ways. By altering where γ-secretase cleaves APP, they reduce the concentration of Aβ42. Moreover, by interfering with Aβ oligomerization they fight the formation of plaques directly. With luck, further work in medicinal chemistry will arrive at compounds that enhance both these activities. The development of compounds that significantly reduce or prevent the formation of amyloid plaques will be a great step forward for Alzheimer's research. Even if such drugs do not prove to be a cure, a clear indication that plaques don't cause Alzheimer's would be a critical insight.

I want to emphasize that although these results are quite promising, they do not prove the efficacy of NSAIDs in ameliorating actual Alzheimer's symptoms. Transforming these findings into a cure or even an effective treatment will require a great deal of additional research, if it is even possible. You should not attempt to treat Alzheimer's with NSAIDs, or begin a regimen of NSAIDs or any other kind of drug or supplement, unless you have first discussed the possible risks and benefits with your doctor. And no, Minnesota, I do not mean a naturopath.

1. Weggen, S., Eriksen, J.L., Das, P., Sagi, S.A., Wang, R., Pietrzik, C.U., Findlay, K.A., Smith, T.E., Murphy, M.P., Bulter, T., Kang, D.E., Marquez-Sterling, N., Golde, T.E., Koo, E.H. (2001). A subset of NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase activity. Nature, 414(6860), 212-216. DOI: 10.1038/35102591

2. Weggen, S. (2003). Evidence That Nonsteroidal Anti-inflammatory Drugs Decrease Amyloid β42 Production by Direct Modulation of γ-Secretase Activity. Journal of Biological Chemistry, 278(34), 31831-31837. DOI: 10.1074/jbc.M303592200 OPEN ACCESS

3. Kukar, T.L., Ladd, T.B., Bann, M.A., Fraering, P.C., Narlawar, R., Maharvi, G.M., Healy, B., Chapman, R., Welzel, A.T., Price, R.W., Moore, B., Rangachari, V., Cusack, B., Eriksen, J., Jansen-West, K., Verbeeck, C., Yager, D., Eckman, C., Ye, W., Sagi, S., Cottrell, B.A., Torpey, J., Rosenberry, T.L., Fauq, A., Wolfe, M.S., Schmidt, B., Walsh, D.M., Koo, E.H., Golde, T.E. (2008). Substrate-targeting γ-secretase modulators. Nature, 453(7197), 925-929. DOI: 10.1038/nature07055

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DegP 24-mers are spacious chaperones

A few weeks back I mentioned a paper in PNAS on allosteric regulation of DegP protease function by its PDZ domains. This week in Nature, the same group (same first author, even) provides some intriguing new insights into the workings of this combined chaperone and protease. Using different arrangements of a base trimer structure, DegP assembles into multimers of 6, 12, and 24 protein units. Krojer et al. determine the structures of the larger complexes using cryo-electron microscopy and X-ray crystallography, and how these structures might achieve the refolding and degradative functions of this HtrA protein family member.

As I mentioned last time, a single DegP protein consists of a protease domain and two PDZ domains, a domain I talk about a lot. This makes for a decently-sized protein, but in fact DegP is rarely encountered in vivo as a monomer. It is known to form trimers and hexamers, and now dodecamers and whatever fancy Latin or Greek word you would use for a 24-mer. In all of these cases what we're really dealing with are higher assemblies of trimers. For instance, the dodecamer is a tetramer of trimers. In addition to its ability to degrade proteins, DegP is known to have a chaperone function, and also to be able to shepherd OMPs (outer membrane proteins) through the periplasm of E. Coli.

In the case of the 24-mer, the DegP molecules assemble into a giant, hollow octahedral shape with an interior cavity 110 Å wide, which is larger than the cavity of the well-known chaperone GroEL. The PDZ domains mediate contact between adjacent trimers, and the protease domains form the "faces" of the octahedron. From the first figure in the paper it almost looks like you could cram 2 folded OMP proteins into the cavity formed by this structure. This oligomeric complex is so large it could conceivably span the whole periplasmic space between the inner and outer membranes of an E. Coli. Because the 24-mer has fairly large pores, it seems possible that it could form a tunnel that protects OMPs from aggregation and degradation as they cross the periplasm. In addition, positively-charged residues concentrated on the surfaces of the PDZ domains appear to give the multimer some affinity for membranes; these positive charges are concentrated around the edges of the pores.

To check this idea in vivo, Krojer et al. made a DegP-null strain of bacteria and measured the concentration and location of OMPs. They found that for several OMPs, deleting DegP did not change the concentration of the OMP in a whole cell lysate, but reduced levels of these proteins in the outer membrane. Further experiments indicated that DegP oligomers can protect OMPs from proteases. DegP itself can degrade unfolded OMPs, but stabilizes the folded proteins.

The researchers also managed to catch a glimpse of an OMP inside a DegP oligomer. In the case of the 24-mer this was difficult, probably because the sheer size of the enclosed space allows so many orientations of the OMP that getting a regular structure is impossible. However, they found that the structure of DegP dodecamers bound to OMP was fairly homogeneous, allowing an investigation by cryo-EM. You can see an image of the structure (shamelessly stolen from Figure 5) at left; the DegP molecules are in warm tones, and a molecule of OmpC is visible in blue. Again, the protease domains form the faces of this tetrahedral cage, while the PDZ1 domains (not PDZ2 in this case) mediate trimer-trimer contacts.

But what about DegP's protease function? Chromatography experiments (Figure 2) suggest that at room temperature, the presence of unfolded substrate molecules induces the formation of higher-order oligomers. These experiments cannot tell us whether these 12- and 24-mers have the same conformations as determined in the experiments above, but the fact that the PDZ1 domains mediate trimer-trimer contacts in both complexes suggests a possible mechanism for the allosteric activation noted previously. However, at higher temperatures where protease activity is markedly increased, the dominant species in solution was the trimer itself, even in the presence of substrates.

The authors propose that the hexameric form of DegP is a resting state, and that DegP₁₂ and DegP₂₄ form in response to specific stimuli. This model certainly fits the observations in solution, but it seems possible to me that the presence of membranes could induce formation of DegP₂₄. In vivo studies using fluorescence or TEM may be able to address what form actually predominates in the periplasm. Additionally, these results do not directly address the role of oligomerization in the switch between proteolytic and chaperone function. Particularly crucial in this regard is the question of whether the larger complexes that form during proteolysis are the same as those that form around OMPs. While it's reasonable to think that they are, the demonstrated structural versatility of DegP trimers suggests that these large assemblies may represent alternative conformations. Alternatively, it could be that features of the periplasm make DegP₂₄ and DegP₁₂ poor proteases in bacteria, and that the more rapidly diffusing naked trimer is the only efficient protease in vivo among DegP oligomers.

If, however, the chaperone and protease oligomers are structurally equivalent, a whole new class of questions opens up. Is formation of 12- and 24-mers sufficient to activate proteolysis, or is some additional step required? What protects folded OMPs from degradation by the protease subunits? These are challenging questions, but the new structures will be of great assistance in guiding the design of the genetic and biochemical experiments that answer them.

1. Krojer, T., Sawa, J., Schäfer, E., Saibil, H.R., Ehrmann, M., Clausen, T. (2008). Structural basis for the regulated protease and chaperone function of DegP. Nature, 453(7197), 885-890. DOI: 10.1038/nature07004

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Farah and the Prince

Corvus Elrod's proposed Round Table subject for June is instances of the role of relationships in games. This follows on previous posts of his in which he argued that great relationships, rather than great characters, define a good game. I don't quite agree, because I think the truly great successes occur when great characters and great relationships appear together. That, for me, was something that happened with the Prince and Farah in two Prince of Persia games. Now, I've written about the modern Prince of Persia trilogy before, focusing my treatment on the Prince's transformation and the ways in which the gameplay takes the player along the same path. This time I want to discuss the different ways in which the series' female lead, the Maharajah's daughter Farah, is used in the two games where she appears.

To be honest, Farah is not a fully fleshed-out character in either Sands of Time or The Two Thrones. Her interactions with the Prince show her to be strong-willed and to possess firm convictions about right and wrong, but it's tough to say just from the short time we spend with her what she would be like, were she not trapped in a city crawling with sand monsters. Only in The Two Thrones do we learn the most elementary things about her. Apparently, she likes pomegranates, the color red, and challenges, the last of which certainly serves her well in dealing with the Prince.

Without Farah, we'd know precious little about the Prince, either, and the Prince is a great character (and even more so in The Two Thrones). It's no difficulty to figure out that he's acrobatic and has a thing for revenge, but again this doesn't give us much of an idea what he'd be like in the absence of slavering sand fiends. Yet through the course of the game we also discover that he's a bit spoiled, something of a male chauvinist, fairly bad with women, has a deep sarcastic streak, and is charming despite his flaws. All of this knowledge we owe to Farah—not because of anything she says or does, but because of how the Prince reacts to what she says and does.

Granted, we could have been treated to a limp cutscene in which the Prince is scolded for his insolence by his well-meaning father. This, however, would not have been nearly as illuminating (or amusing) as the lines the Prince mutters while walking across a high beam just after Farah has told him to meet her at the baths:

I'll just ask the first Sand Creature I run into! 'Could you direct me to the baths, please? Well, thank you.' 'Don't mention it, I used to be a bath attendant back when I was alive...'

and later, as he works his way into a cavern full of waterfalls:

Oh, have you been waiting here all this time? I didn't realize you meant these baths! I went to the other baths clear across the other side of the city! I had a lovely wash and a rub with fragrant oils. Too bad you weren't there... Stop talking to yourself!

Without Farah, the Prince is just navigating scenery at these moments. With her around, even if only to push his buttons, we learn a great deal about what kind of person he is, beyond his desire to plunge the Dagger of Time into the Vizier's black heart.

It's important to note that in Sands of Time Farah does really not succeed in inducing any kind of change in the Prince. If nothing else, the end of the story, in which the Prince selfishly uses the dagger to kiss Farah without having her think less of him, should convince you of that. Even the scene in which he obeys her directions to stab the Hourglass plays out ambiguously, making it unclear whether he believes that this action will save his life or end it. Another exemplar in this regard is the entirety of Warrior Within, which neither needs nor deserves further discussion here.

In The Two Thrones Farah's role changes significantly. She's no longer used to illuminate who the Prince is: that task is taken up by the Dark Prince persona. This is a necessary change because of gameplay and story considerations. In this game (as opposed to Sands of Time) Farah is used exclusively in a puzzle-solving capacity, and so she is an occasional, rather than regular, companion. Because the Prince as a person is in flux through much of the game, a constant companion is necessary, and so we have the voice in the head to illuminate his changing character.

Farah's role in The Two Thrones is as a catalyst for the Prince's transformation. The events of Sands of Time have given her emotional power over the Prince, even though the repeated reversals of the story mean she has never actually met him in the timeline of the game. Her attitude towards soldiers and fighting has not changed significantly from the first game, but the Prince's own battle-weariness and longing for her mean that he listens this time. Almost nothing that occurs in the game hurts him more than her rejection on seeing his sand form; absolutely nothing in the game affects him more than her convictions about his duty as Prince.

In the absence of Farah, the main plot of The Two Thrones probably would not change significantly: the Prince would cut his way through the city and destroy the Vizier regardless. However, Farah's presence determines who the Prince is, and why he fights, when he reaches his enemy. In that sense, she is essential to the story. Without her, the Dark Prince is the only Prince—without her, the Prince could never escape the warrior within.

The Prince's relationship with Farah is essential to both the games in which she appears. In Sands of Time, the relationship reflects who he is; in The Two Thrones it defines who he will become.

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E. Coli with a more diverse palate

Today wasn't a good day in the lab for me. There weren't any explosions or failed cultures, just an impossible NOESY spectrum. It's tough to determine your proline isomer when your spectrum doesn't have the characteristic peaks for the cis or trans state (note: there are no other states). At times like this, it helps to be reminded of the value of persistence, which brings me to today's paper, involving an experiment that's been going on for 20 years.

A few weeks back I mentioned Carl Zimmer's excellent book Microcosm: E. coli and the new science of life, which details the study of one of the world's best-understood organisms and the insights it has given us. E. coli has loads of useful properties that make it a great research tool, and one of these is its doubling time, which in the lab typically ranges from a few hours to as few as 20 minutes depending on conditions. This has obvious benefits for researchers who just want to use the bacteria as a means of producing protein or DNA, but it also means that scientists interested in evolution can realistically expect to use E. coli to answer questions that would require tracking a population over tens of thousands of generations. In a recent paper in PNAS, Richard Lenski's long-term evolution experiment (LTEE) has accomplished just that.

One can, for instance, ask whether evolution is random or deterministic. That is, given some specific context, is a particular outcome (or kind of outcome) inevitable, or will the evolutionary history of an organism preclude some possibilities and make others more likely? Stephen Jay Gould maintained the latter—that preceding states would provide such a significant part of the context for future mutations that rewinding time to the beginning and doing the whole thing over again might lead to a completely different world of life. This position has intuitive appeal, but it could also be the case that natural selection is so powerful that certain maximally-adapted states would be achieved regardless of evolutionary history. Obviously, we can't rewind time to test these possibilities directly, but perhaps some model system could help us.

Enter E. coli and the LTEE, an experiment that procedurally sounds very simple. What Lenski did was to found 12 populations of E. coli from two clones. Once the cultures were started, his team took part of each culture every morning and diluted it into some fresh DM25 medium, a minimal medium containing 139 µM glucose and 1.7 mM citrate. Now, E. coli loves glucose, but one of the defining characteristics of the species is that it cannot take up citrate in an aerobic environment. There is citrate inside a bacterium (as an intermediate in a metabolic pathway), and there is citrate outside the bacterium, but what is outside cannot come in. And that is how things stayed for more than 30,000 generations. A number of differences in appearance and other properties have been noted, but for a very long time the ability to eat citrate did not evolve.

Eventually, shortly after the 33,000th generation, random mutations in one of the populations gave its bacteria the ability to transport the more-plentiful citrate across their membranes. Over a very few generations, the maximum density of the cultures exploded as the bacteria gained access to the more-plentiful food source. By itself, that's pretty cool, as it represents laboratory observation of a mutation that changes a defining characteristic of a species. But this new ability also made it possible to test the importance of the previous mutations.

You see, Lenski's lab had taken samples of their E. coli every 500 generations and frozen them in glycerol at -80 °C. This sounds harsh, but this kind of deep freeze allows them to resurrect these populations for future study. And that's just what Lenski's researchers did. They pulled the samples out of deep freeze and replayed evolution for ~3700 generations for each of them, testing to see whether the ability to transport citrate under aerobic conditions evolved again.

If pre-existing context is not particularly important, one would expect that there would be some low probability of evolving citrate transport that was equal for all previous generations. On the other hand, if Gould is right, then there would be essentially no chance of evolving citrate transport for early populations, and then after some potentiating mutation occurred, a higher chance for later populations. Blount et al. show that the latter is the case. In their replays, citrate transport never evolved in populations earlier than the 20,000th generation, and only appeared regularly in replay experiments after the 30,000th generation. This would suggest that some potentiating mutation occurred before generation 20,000 that provided a genetic context in which subsequent mutations could produce citrate transport.

Obviously, one suspect for this mutation would be a change in the DNA reproduction machinery that made subsequent mutations more likely in general. It's true that some of the other populations in the LTEE had enhanced mutation rates, but not this one. A subsequent experiment tracking mutations in another gene showed no difference in mutation rate between the ancestors and the potentiated clones. So the answer is more complex. Unfortunately, the authors do not yet know exactly what mutation occurred in this time frame to potentiate citrate transport. However, our ability to examine the genes of bacteria has increased significantly since the LTEE began. Full-genome sequencing of these bacteria (now underway) should give us some powerful insights into the evolutionary history of this population.

So, would evolution play out the same way if we rewound and started again? These results suggest that it would not, and historical contingency is likely to be the case for many systems. However, it may also be true that some particular characteristics are so constrained, or confer such an enormous selective advantage, that life will take these avenues no matter what. In the case of citrate transport, the existing genetic context appears to be very significant, but generalizing this observation to other systems may not be valid. Nonetheless, Gould's point is proved even if contingency is a property of some systems. The Lenski experiment shows him to be in the right, even if it took 20 years to do it.

Carl Zimmer has a post on this article as well, complete with a few intelligent questions and some crazy rantings.

1. Blount, Z.D., Borland, C.Z., Lenski, R.E. (2008). Inaugural Article: Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proceedings of the National Academy of Sciences 105(23) p. 7899-7906 DOI: 10.1073/pnas.0803151105

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Calling Leon Kennedy

Players of Resident Evil 4 who explored every corner of the sewer filled with hideous insects may remember reading the research Luis left behind describing creatures that changed their behavior due to the presence of parasites. The report about creatures such as Dicrocoelium dendriticum is actually fairly accurate, given its brevity, but doesn't address some important questions. For instance, most of the research on behavioral modifications of this kind comes from laboratory research that may not reflect what actually happens in nature. However, with field research it is sometimes difficult to distinguish aberrant behavior arising due to parasitism from preferential parasitism of aberrantly-behaving hosts. In a recent article in the online open-access journal PLoS ONE, a team of researchers from the University of Amsterdam and Federal University of Viçosa in Brazil set out to get a better grasp on behavioral changes in a caterpillar following parasitism by a wasp.

The wasp in this case is actually a parasitoid species, which differs from a classical parasite in that the death of the host is a certainty. The authors bred both the wasps and the host caterpillars in captivity, and randomly chose the caterpillars that would be hosts. This ensured that observed differences in behavior would be solely the result of parasitism and not some preference on the part of the wasps. Implantation of the eggs was apparently rapid and painless: just by walking over the caterpillars the wasps were able to lay as many as 80 eggs at a go. Perhaps this observation fills you with horror, in which case I say: you are not alone.

After this, the caterpillars were taken out of the lab and allowed to mature on trees on campus at Viçosa. Their movement and behavior were monitored out in the wild and also under controlled conditions in the laboratory. During the incubation of the larvae inside the caterpillar, no significant differences in behavior were observed. Hosts moved and ate at essentially the same rates as their unparasitized brethren. Once the wasp larvae emerged and pupated, however, the hosts' behavior changed dramatically. They ceased to move along tree branches or eat, and instead hovered over the pupae (check out Figure 1 and the supplemental movie 1), swinging their heads at potential predators.

The hypothesis that the parasitoids control host behavior to gain some sort of advantage would suggest a couple of predictions. One is that the behavioral changes are exclusively associated with parasitized caterpillars. Another is that the behavioral changes increase the probability that the parasitoids survive to adulthood. In addition, one might expect that the behavior has no benefit for the host. All of these predictions turn out to be accurate.

Unparasitized caterpillars from the same batches of eggs as the hosts did not show any of the host behaviors, strongly supporting the contention that the behavioral difference is solely the result of the parasitoids. In addition, when unparasitized caterpillars were placed on branches with pupae, they ignored both the pupae themselves and potential predators. The modification clearly has benefits for the wasps, as Janssen et al. found that the presence of a zombie caterpillar doubles the survival chances of the pupae. As for the caterpillar hosts, they die around the time that the adult wasps emerge, without themselves reaching maturity. That means that there's a great deal of benefit for the wasps, and none for the poor caterpillars.

What actually causes the change in behavior? Because it persists even after the pupae themselves have been removed from the vicinity, because the pupae alone do not induce behavioral changes in unparasitized caterpillars, and because the time between parasitism and larval emergence is about a fortnight, the authors conclude that neither the pupae or adult wasps are directly responsible. Dissection of the hosts after egression indicated that one or two active larvae were usually left behind. Similar to the case of D. dendriticum, these lingering parasitoids may be sacrificed to protect their brethren by manipulating their host.

There's no need to go grab your shotgun: these wasps are pretty small and aren't about to go parasitizing humans. In addition, the behavioral effect, though striking, seems to involve very general manipulations. Las Plagas these ain't. Still the mindlessly aggressive protection of the pupae calls to mind a boss monster, helplessly guarding the hideous young of an alien species. Best keep Leon on speed dial, just in case.

Carl Zimmer has also covered this over at The Loom. He's a great writer, so check it out... and also consider grabbing a copy of his new book Microcosm. Ed Yong has his own excellent article on this research.

1. Grosman, A.H., Janssen, A., de Brito, E.F., Cordeiro, E.G., Colares, F., Fonseca, J.O., Lima, E.R., Pallini, A., Sabelis, M.W., Raine, N.E. (2008). Parasitoid Increases Survival of Its Pupae by Inducing Hosts to Fight Predators. PLoS ONE, 3(6), e2276. DOI: 10.1371/journal.pone.0002276 OPEN ACCESS

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I was a teenage reaper

A boy comes to his senses in a crowded intersection, surrounded by people who seem not to notice his existence. It's fine with Neku: he doesn't like dealing with people anyway. He uses headphones and a harsh attitude to shut others out, and he might take The World Ends With You as a mission statement. If Neku's going to escape the nightmare Shibuya he's trapped in, though, he'll need to learn to trust others, to let them into his world, so that it begins, rather than ends, with him.

Spoilers follow. I am not kidding.

Role-playing games usually work best with a story of personal growth. This doesn't necessarily mean a coming-of-age tale, although the heroic fantasy that forms the cultural context for most RPGs is thick with that plot. What's important is that the story of the hero's growth as a person is matched with the growth-based mechanics. Even though the player is not always in control of the main character's personal development, the act of developing the character in gameplay provides a parallel that the player does control. The World Ends With You takes this idea and runs with it.

It's fairly easy to identify the game as a coming-of-age story: all the characters are teenagers, and much of the gameplay involves acting like a teen. After all, what you do in the game is run around a hip section of Tokyo, hang out in malls, keep up with the latest trends, and eat junk food all day. Neku has some familiar adolescent characteristics, in that he is a moody, selfish, sarcastic prick. Many of the secondary characters are facing normal teenage problems, too: Shiki is so jealous of a friend she takes on her shape, and Beat has arguments with his parents. In agreement with the impressions of most teens, adults come across as hopeless squares—one advertising executive (Makoto) wanders around town trying to sell pins, using lame, out-of-date catchphrases.

Pins play an important part in the game because they contain essentially all of Neku's combat abilities. Shibuya is inhabited by demons called 'Noise' that seem to be related to people's negative emotions. In order to defeat them, Neku has to use 'Psychs' associated with his pins, but his attacks alone are not enough. In order to fight through the Noise he'll need the help of a partner (who acts in the DS upper screen). Neku and his partner—a different one each week of the game—share their HP, so there's no resurrection spell that can be cast. Once you die, it's game over. The strength of a pin is determined by the PP it has accumulated (separate from the EXP that increases Neku's HP), and also by the popularity of the pin's brand in the particular district of Shibuya where Neku is fighting.

Much of the story is also concerned with popularity. One of the early tasks in the game is to popularize Makoto's pin, and several incidents in the first two weeks involve the Prince of Ennui, who defines what's popular in Shibuya with his "F Everything" (F for Fabulous) blog. Yet, even the prince has to fight to be himself. Shiki, as mentioned, longs to be popular, but finds she doesn't enjoy being the person she thought she wanted to be. The mechanics force players to acknowledge popularity by strengthening and weakening the pins, but they also allow them to take control of the trends. Succeeding in battle with unpopular pins or clothing increases the popularity of their brands in that section of Shibuya. In this sense, the gameplay offers the player the same choice that the characters face: follow the trends, or blaze your own path. The former is easier, the latter more rewarding.

Some pins can level up and evolve into more powerful forms using only the PP that you obtain from battles. However, there are additional mechanisms for gaining PP. For instance, turning off the DS grants you "Shutdown PP" relative to how long you leave it off. If you carry it around with you and encounter another DS (or play a minigame against a friend), you can also gain "Mingle PP", and earn new items in shops. Shutting off the DS grants you another benefit, related to the food that your characters consume. Each item of food that they eat grants them a temporary bonus for a certain number of battles, and then a permanent stat increase once it is digested. However, the characters' stomachs can only take a certain amount of food per day—not per game day, per your day. So shutting The World Ends With You off overnight allows you to eat more food and gain more stat bonuses.

All of this benefit to turning off the game seems very strange at first blush. The difficulty of combat, the tunability of the game system, and the intricacy of its rules makes it very clear that this will mostly appeal to core gamers. Yet the beneficial aspects of shutting off the DS enforce a schedule more familiar to casual players. The point of the game's story, however, is that keeping to yourself is the wrong way to live. By rewarding the player for turning the game off and hanging out with his (DS-owning) friends, The World Ends With You encourages the same behavior in the player's life that it is suggesting for Neku's. The mechanics are constructed to make the player's actions fit the theme.

Adolescence is not a peaceful period for anyone. Transforming from a passive child who is controlled by the world into an adult who can shape it involves a lot of pushing against friends and parents and authority figures. Neku's reaction to this is to close himself off from everyone else; he wears headphones to shut out other people's noise. When he is eventually forced to cooperate with others it doesn't go very smoothly: the friendships he forms during the game have plenty of friction. The difficulty of the combat system may be intended to evoke the frustration (and occasional awesome rewards) of trying to get along with others. But, as Neku himself eventually realizes, it is conflict that makes people grow (a sentiment that invokes the mechanics of nearly all RPGs). By confronting the Noise he has been shutting out, Neku's world expands.

The story of The World Ends With You is interesting, and in spots it's cleverly written (not when it's using amnesia, though), but by itself it's hardly remarkable. In a way, the plot falls apart at the end, never really explaining why the city was saved, but the story isn't really about saving Shibuya... what's at stake is Neku himself. Still, what makes The World Ends With You great is not the story, but the way in which the mechanics reinforce the story's message. The genius of the design is that the theme is developed not only when the game is being played, but also when it isn't. The World Ends With You doesn't just aim to expand Neku's world—it aims to expand yours.

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