Is in-cell NMR feasible?

As I mentioned in my last post, it is sometimes difficult to tell if a protein structural model matches the physiologically-active form. One way to address this problem would be to take structural data under physiological conditions, i.e. in the cell. While this obviously isn't a viable approach for crystallography, it seems possible with other techniques such as FRET or NMR. Existing NMR structures are solved under solution conditions, and the cytoplasm is a kind of solution. So we would expect to be able to solve structures, or at least get spectra, in vivo. However, the success of NMR strongly depends on what kind of solution you have, and a forthcoming paper in JACS suggests that the bacterial cytoplasm may not be amenable to NMR.

This paper has a bit of history behind it. A few years ago, Gary Pielak's lab attempted to determine the dynamic behavior of chymotrypsin inhibitor 2 (CI2) and apocytochrome-b5 in living E. coli (1),(2). They found in these experiments that the dynamics of these proteins did not differ significantly between in vivo and dilute solution conditions. A later experiment, however, showed that most or all of the signals that had been observed arose from protein that had leaked out of the cells and into the surrounding medium in which they were suspended. This wasn't true for all proteins: cells expressing α-synuclein (αSN) and FlgM did not leak protein. Subsequently the first two papers were retracted (3), while Gary and his folks tried to figure out just what was going on.

Li et al. encapsulated E. coli cells that were producing either CI2 or αSN in alginate microcapsules to prevent protein leakage and took some basic NMR spectra. Samples of the surrounding fluid proved that neither protein was leaking into the surrounding medium. Nonetheless, although normal spectra of αSN were observed, the cells expressing CI2 did not produce any CI2 spectral signals at all (normal metabolite signals were observed). Experiments showed that these cells did contain CI2 (4). What happened to the signal?

Li et al. hypothesize that this is an effect of the viscosity of the cytoplasm. The intensity of an NMR signal depends on the concentration of the atoms giving rise to the signal, and also the relaxation properties of these atoms, i.e. the rate at which the polarization induced by radio-frequency pulses returns to equilibrium levels. For larger molecules and viscous solutions, the rate at which detectable polarization decays (R₂) is higher, and the rate at which polarization returns to equilibrium (R₁) is lower. The behavior of R₂ is one of the chief obstacles to performing NMR on proteins or complexes that have high molecular weight.

In order to establish that this cytoplasmic viscosity is a reasonable cause of the invisibility of CI2, Li et al. measure the NMR relaxation of αSN and CI2 in a solution doped with poly(vinylpyrrolidone) (PVP), which increases the viscosity significantly. They find that the R₁ of αSN is essentially unchanged, while that of CI2 decreases significantly. The R₂ of αSN resonances increases 2-6 fold relative to dilute solution, but for CI2 this increase is much more significant, up to 40-fold. You can see these features in the graph at right. These results indicate that the relaxation behavior of resonances in a stably folded protein like CI2 is dominated by the global rotational diffusion, while the relaxation of resonances in a disordered protein like αSN is dominated by the significant local fluctuations. Because these latter motions involve less surface area and hence less drag (as compared to the folded protein), the viscosity of the solution has relatively less of an effect. Thus, the vanishing CI2 peaks could conceivably be the result of differential relaxation responses to viscosity.

However, other groups have managed to acquire NMR spectra of folded proteins in E. coli before. These experiments may have experienced the same leakage problem that affected the previous CI2 results, and some control experiments addressing this possibility are likely to be forthcoming. At the same time, the PVP solutions here are supposedly 5 times as viscous as E. coli cytoplasm, and yet CI2 resonances are apparently still visible. This suggests that the complete obliteration of these resonances in cells cannot be chalked up entirely to viscosity. One possibility is that CI2 overexpresses so significantly that it massively increases the effective viscosity of the cell. It may also be possible that the vast quantities of CI2 expressed in these experiments are crammed into a small volume somehow, perhaps as relatively loose inclusions in the cytoplasm or loaded in the periplasmic space, where interactions with peptidoglycan may rotationally lock them. It is also possible that PVP does not capture some important aspect of cellular crowding. For instance, the washing-out of the CI2 signal may be an effect of transient protein-protein interactions. Fluorescence and electron microscopy experiments may be able to address this concern.

If the observations of CI2 are really a result of some unique localization or a consequence of the significant overexpression then it is possible that NMR of different proteins on somewhat less active promoters may yet be viable. In addition, in vivo studies of proteins in systems such as X. laevis oocytes or cultured eukaryotic cells may not be subject to the same difficulties, due to differences in cytoplasmic viscosity. The major implication of this work, however, is that in vivo NMR results cannot be taken at face value. Careful controls will be required in order to ensure that the experiments actually measure protein within the cell, and in the absence of such controls the results should be interpreted cautiously.

1. Bryant, J., Lecomte, J., Lee, A., Young, G., Pielak, G. (2005). Protein Dynamics in Living Cells. Biochemistry, 44(26), 9275-9279. DOI: 10.1021/bi050786j

2. Bryant, J., Lecomte, J., Lee, A., Young, G., Pielak, G. (2006). Cytosol has a small effect on protein dynamics. Biochemistry, 45(33), 10085-10091. DOI: 10.1021/bi060547b

3. Bryant, J., Lecomte, J., Lee, A., Young, G., Pielak, G. (2007). Retraction. Biochemistry, 46(27), 8206-8206. DOI: 10.1021/bi700744h

4. Li, C., Charlton, L.M., Lakkavaram, A., Seagle, C., Wang, G., Young, G.B., Macdonald, J.M., Pielak, G.J. (2008). Differential Dynamical Effects of Macromolecular Crowding on an Intrinsically Disordered Protein and a Globular Protein: Implications for In-Cell NMR Spectroscopy. Journal of the American Chemical Society DOI: 10.1021/ja801020z

Read the rest...

The dimer interface of PhoQ

Relating a protein structure to its function is often not as straightforward as we would like. As any protein crystallographer knows, many biomolecules will only form crystals in specific, limited conditions, with particular buffers, protectants, and co-solutes. Sometimes even the water must be replaced with, say, isopropanol in order to form a crystal. History has shown that despite this, most protein crystals imply models that match with biochemical data. Sometimes, however, crystal structures of a single protein or two highly homologous proteins disagree substantially. In that case, it is up to the biochemists to determine which structure (if any) correctly represents the physiologically relevant ensemble.

That is just what happened in a study soon to be released in the Journal of Molecular Biology from the lab of Bill DeGrado. Goldberg, Soto et al. wish to resolve a question about the dimerization of the periplasmic domain of PhoQ, part of a two-component signaling system in bacteria. These systems sense an external stimulus (in this case divalent cations) and activate a set of genes. PhoQ deals in the first half of the equation, and exists as a dimer (pair of bound proteins) that stretches from the periplasmic space (where it senses the signal) across the inner membrane and into the cytoplasm of the bacterium. Because it is in general very difficult to crystallize membrane proteins in their entirety, the isolated pieces (domains) of this protein were crystallized independently. Unfortunately, the sensor domain does not dimerize strongly in solution, and the crystal structure of the E. coli PhoQ (explore this structure at the PDB):
does not match up particularly well with the structure determined for the homologous protein in S. enterica (explore this structure at the PDB):

Goldberg et al. note that the differences in these structures are unlikely to reflect actual differences between the proteins, because the amino acid sequences are 85% identical. That doesn't guarantee that the structures will be the same (see this post), but as a rule nearly-identical structures are what we would expect. Goldberg et al. set out to determine which structure represents the physiological form, and they decided to do it in vivo, with the full-length protein.

In order to do this, they engineered several mutants of PhoQ, each of which had a cysteine at a particular position in the N-terminal helix of the sensor domain (visible side chains in above figures). Cysteines have the property that they can form disulfide bonds with other cysteines when the chemical environment is right. Consequently, if the cysteine of one dimer is in close proximity to the cysteine from the other, they will stick together or cross-link. Once these proteins are expressed in E. coli and the cells are broken open, the concentration of this cross-linked species will be proportional to the frequency with which the relevant side-chains encounter one another. As you can see, the two structures imply very different predictions about the patterns of cross-linking that will be observed. If the first structure is correct, the successful disulfide bonds should be distributed evenly along the helix. By contrast, if the second structure is correct, the mutations that produce cross-links will all be located at one end.

Goldberg et al. observe the first of these patterns. The side chains that are visible in the figures above are the ones where mutating in a cysteine produced a significant amount of cross-linking. Obviously they are distributed along the entire length of the N-terminal helix. When the researchers ran a simulation that attempted to predict a structure on the basis of the cross-linking data, they also came up with a structure that looked very similar to the first crystal structure above. While not all of the cross-links observed appear to be compatible with the structure, this may just be a result of a few additional conformations in the ensemble.

In this case, the experiment was performed on the E. coli protein, so it is still possible that the structure for the S. enterica protein reflects actual differences arising from the 15% difference in homology. Obviously the next step would be to perform the similar set of experiments on the S. enterica protein; doubtless these experiments are already underway. As the authors note, it is also possible that the two structures represent the active and inactive forms of the protein. Repeating the experiment in minimal media and adjusting the levels of magnesium may address this possibility.

1. Goldberg, S., Soto, C., Waldburger, C., DeGrado, W. (2008). Determination of the physiological dimer interface of the PhoQ sensor domain. Journal of Molecular Biology DOI: 10.1016/j.jmb.2008.04.023

Read the rest...

Rapid evolution of lizards in the Adriatic

Enough about Ben Stein and his lies about evolution. Let's talk some truth about evolution, namely scientific truth. The fine folks at Zooillogix brought my attention to a paper that flew under my radar at a time I may (perhaps) have been more focused on basketball. In it, researchers from Harvard and Amherst studied a population of lizards on a very small islet in the Mediterranean. In less than 40 years, this population of lizards has evolved to have significantly different morphology from its parent population, a new set of endosymbionts, and novel anatomical features rarely in related lizard species. It's an interesting case of evolution in action.

Back in 1971 10 lizards of the species Podarcis sicula were transplanted from one small islet in the South Adriatic to a nearby, somewhat smaller hunk of rock. Over a three year period from 2004-2006, Herrel et al. returned to these small islands to see what became of the lizards (1). What they found is that the transplanted lizards had taken over the second islet. The lizards from the second island were still genetically very similar to those on the originating island (see their supplementary figure 5). However, there were pronounced differences in the diet. Whereas the population on the island of origin ate very little plant matter (<10% of the total diet), the transplanted lizards appeared to subsist mostly on plant matter, with some seasonal variations up and down from 50%.

The change in diet appears to have prompted some substantial changes in morphology as well. The size and mass of the lizards on the second island is significantly greater, and the head dimensions are altered. For the smaller female lizards, this translates directly into an increase in bite force. For the male lizards, however, the differences in head size alone are not sufficient to account for the observed increase in bite force, implying that there are additional adaptations of some kind. Herrel et al. speculate that the increased bite force of the lizards helps them to eat and digest leaves. This is reinforced by the observation that structural features related to the opening of the jaw are largely unchanged.

There are additional adaptations internally. For instance, the lizards on the second island have a structure called a cecal valve (and additional anatomical changes to the cecum) that are believed to aid in the digestion of plant matter such as leaves. This is particularly interesting because this valve structure does not appear in the originating population, and is rare among related species of lizards (the suborder scleroglossa). Moreover, the hindgut of these lizards contained nematodes that are absent from the parent population, suggesting the development of a novel symbiosis. The authors note that the new morphological characteristics are present in juveniles as well as adults, suggesting that the changes involved are genetic, though further experiment is required. The authors also note some interesting changes in population dynamics and behavior that appear to have resulted from the altered eating habits of these lizards.

That these new features appeared within less than 40 years is especially striking. In less than a human lifetime this population of lizards evolved adaptations such as altered jaw morphology, as well as an apparently novel internal feature. While small populations and constrained locations can accelerate the process of evolution, it is still instructive to consider this result when discussing the "likelihood" of evolution. Significant morphological adaptations can evolve very rapidly. Imagine what the power of evolution could do given hundreds of millions of years in which to work.

Oh, wait... you don't have to.

1. Herrel, A., Huyghe, K., Vanhooydonck, B., Backeljau, T., Breugelmans, K., Grbac, I., Van Damme, R., Irschick, D.J. (2008). Rapid large-scale evolutionary divergence in morphology and performance associated with exploitation of a different dietary resource. Proceedings of the National Academy of Sciences, 105(12), 4792-4795. DOI: 10.1073/pnas.0711998105

Read the rest...

Expose Expelled

Maybe you know someone who has been suckered in by the numerous distortions, outright lies, and malevolent accusations of the "documentary" Expelled, starring famous bore and Nixon speechwriter Ben Stein. Perhaps even you have been convinced by its "startling" array of important "facts" about the "controversy" between the scientifically useless design conjecture and staggeringly successful evolutionary theory. If so, I urge you to direct your credulous friend (or yourself) to Expelled Exposed, a website devoted to debunking the lies of this appalling propaganda flick. Even if you favor a creationist viewpoint I think you will find it highly disturbing how freely the creators of this film distort the truth to favor their views.

In addition, for general knowledge on debunking the claims of creationists you should check out The Panda's Thumb, TalkOrigins, TalkDesign, TalkReason, and the National Center for Science Education.

Also, New Scientist has an excellent article up featuring 24 misconceptions about evolution. Read it.

Also check out some other articles about the film and its marketing:
Biologist PZ Myers, interviewed in the film, is expelled from a screening of Expelled. But they let Dawkins in.

An animation in Expelled was ripped off from Harvard University and XVIVO. It should be noted that famous cdesign proponentsist William Dembski was for a long time in the habit of stealing this film to display in paid lectures.

Expelled tries to draw a line between belief in evolution and anti-Semitism. Interestingly, one of the creationist scientists they interview is the anti-Semite Maciej Giertych.

Read the rest...

Creative focus in game and technology development

Tynan Sylvester has a really incisive opinion piece up at Gamasutra illustrating a fundamental disjunction between what developers look at in a game and what players look at. The gist of his piece is that developers often focus on "scaffolding": the mechanics and code that underlay the game world. Meanwhile, the players consider mainly the "masonry", that is, the experience of the game world itself. The not-so-subtle point here is that many of the minds behind games have put their creative focus on the wrong thing.

The developers of Crysis and like games, as well as the graphical powerhouse consoles, succumbed to the tyranny of precision, the mindset dominance of quantifiable fidelity. The simple fact is that the graphical capabilities of hardware and software are easily measured, in terms of resolution and framerates and numbers of polygons on screen at a time, and it's easier to improve something if you know how to measure it. Unfortunately, creation of these super high-definition visuals takes a great deal of effort and expense, and ultimately it doesn't help that much. What players really want is an experience that feels real, not an image that looks real. Experiential fidelity, unfortunately, can't be quantified, and it's difficult to plan hardware (or even software) that creates it.

The success of the Wii and DS, however, suggest that good feel can be, if not created by hardware, at least encouraged by it. It's too simplistic to try to explain this success by saying that these platforms "tapped into another market". If that's so, then we must ask why they tapped into that other market. The answer is that these systems create an experience that the user can relate to, despite their shortcomings in terms of graphical excellence. The remote and stylus are very intuitive control devices. As a result, the player can interact with the game worlds easily, and often exactly in the way he expects to, rather than pressing buttons on some bizarre alien boomerang. Although the Wii and DS may not be great scaffolds, they allow for some great masonry.

I don't mean to say that graphics don't mean anything. However, though they are the most easily measured aspect of a game's performance, they do not define what makes a great game. It is the experience that matters. The graphics help create the experience, but even the best graphics in history cannot rescue a game that has lousy audio, an idiotic story, or nonsensical mechanics. Nor am I trying to sell coding short: lousy coding can ruin (or almost ruin) an experience easily. The point is that the creative focus of hardware and software developers should always be on the experience to be created. Everything else must flow from that.

You should check out Sylvester's website, by the way. He's got some really interesting stuff up there. I particularly liked his piece on Hitman and his article on substance and style in game design, although I disagree with some conclusions of the latter.

Read the rest...

Congratulations Doctor Labeikovsky! (Now with photographic evidence)

That cool breeze you felt this morning blew in from the icy recesses of the underworld, the result of a sudden climactic change that occurred because labmate and occasional commenter Wladimir Labeikovsky successfully defended his thesis on the interaction of tau with prolyl isomerase Pin 1 yesterday. He now has his Ph.D., a sure ticket to worldwide fame and fabulous wealth. Congratulations to him on bringing a successful conclusion to a long graduate career, and looking good in a suit. Hit the jump for pictures.

Wlad cleans up nice, doesn't he?

Wlad still laughs at Dorothee's jokes.

Little known fact: as part of your training for grant applications, you must successfully break a piñata in order to receive a Ph.D. in biophysics. The piñata in this case is full of candy and Skyy vodka sample bottles.

Here's a view of the party afterwards. Nothing says "science" like a bunch of folks standing around drinking.

Wlad attempts to murder a committee member with a champagne cork.

Here we see Wlad's adorable godchildren during the brief time that they were not covered in frosting.

Enough said.

By the way, I would appreciate it if whoever received my worldwide fame and fabulous wealth in error would forward them to me promptly.

Read the rest...

Does flexibility increase protein stability?

Since my favorite physicist of the blogosphere put up a great post on the basics of NMR in preparation for a geeky post on the subject, I figure I'll add in a geeky post of my own. As Chad mentions in his post, the dependence of the resonance frequency on local chemical structure allows us to get a great deal of information about covalent bonds and their conformation from NMR spectra. In addition, certain NMR experiments can provide us with detailed information about the motions of atoms with respect to one another. Although the various approaches differ in the level of detail they offer, the sum of NMR dynamics experiments fairly effectively cover the range from picosecond motions to month-long fluctuations. My particular interest in these approaches is in their use to dissect intramolecular signaling in proteins.

The major part of my graduate studies at UNC under Andrew Lee was devoted to understanding the pathways by which changes in dynamics are propagated in eglin c (explore this protein at the PDB). Eglin c is an excellent subject for this kind of study because it has very good spectroscopic properties, and because it has no discernable allosteric properties at all. This means we can do a lot of different experiments on it, and also that our observations about long-range dynamic interactions are likely to be generalizable to any protein that has a hydrophobic core, not just allosteric ones.

My work with eglin c involved making mutations to the protein and determining what dynamic changes resulted. In the course of this I discovered several mutants that caused significant changes in the motions of amino acid side chains on the timescale of picoseconds to nanoseconds (1,2). This is the range covered by the Lipari-Szabo model-free formalism (3). One mutation in particular, V54A (read: valine 54 to alanine), caused almost universal rigidification of the core of eglin c on this timescale. However, the protein was significantly less stable.

This is curious because a lack of stability is known to correlate with an increased frequency of localized unfolding of the backbone. In elements of secondary structure, hydrogens attached to amide nitrogens tend to be caught in hydrogen bonds. When placed in a solution of deuterated water, these hydrogens can only be replaced by deuterium when the hydrogen bond breaks (this is called hydrogen-deuterium exchange or HX). This process can be monitored by NMR (and less specifically by mass spectrometry), and is known to speed up when the structural elements are less stable. So the V54A mutant is more flexible on this slower timescale, and less flexible with respect to some faster processes.

The significant caveat here is that we only looked at some groups in the protein. For reasons that are a bit too complex to discuss in this post, it was only feasible to observe the dynamics of methyl groups. This is a problem because the core of eglin c has a substantial number of aromatic side chains in it. Any picture of dynamic behavior would be incomplete without examining these side chains and quantifying the changes in HX behavior, which I did not do.

In an upcoming article in Biochemistry, Josh Boyer and Andrew Lee address these deficiencies (4). In order to look at the aromatic side chains, they employ a clever biosynthetic labeling scheme devised by Akke's group (5). While this only allowed them to look directly at δ-carbons of these residues, the general rigidity of aromatic rings meant that they could generalize the observations to essentially the whole side chain. They found using this approach that the dynamic response of aromatic side chains to the V54A mutation was more heterogeneous than that of the methyl side chains. As you can see in the figure I have shamelessly stolen from their paper, the central portion of the core appears to have rigidified across all residue types, while the edges of the core and the outward-facing residues of the protein all appear to have become somewhat more flexible. The location of V54 is shown here in black.

This heterogeneity of the dynamic response was also observed when the HX results were taken into account. The distribution of responses, however, is very unexpected. The top portion of the figure at left shows rigidified side chains as a transparent surface, and destabilized backbone amides (as measured by HX) as solid surfaces. Note that the two regions of destabilized amides are linked by contiguous side chains. The lower figure shows side chains that have become more flexible as transparent pink surfaces. The solid backbone surface indicates a region of the protein that has become more stable. You will notice that this stabilized region lies a significant distance from the mutation site (though as the figure above shows it is connected to it by a network of contiguous side chains), and that it corresponds with a bundle of aromatic sidechains that have become more flexible. A significant portion of the protein is clearly destabilized, as was expected from the previous results on V54A. However, the co-localization of side-chain rigidity and backbone instability (and vice-versa) doesn't jive with our expectations.

Our physical intuitions suggest that rigid things are stable and flexible things are not, and in many cases these intuitions have been borne out—in studies of backbone dynamics of proteins from thermophilic organisms, for example. However, the present results can be rationalized if one supposes that conformational entropy makes a significant contribution to stability. Indeed, because the structure of V54A is known to be essentially unchanged from wild-type (2), the enthalpic contributions (hydrogen bonds, charge-charge interactions, etc.) will not be altered. Entropic contributions will therefore dominated the observed changes in stability.

Rigid regions of a protein may have no place to disperse thermal energy other than localized unfolding, while flexible regions may have the option to dump some of that energy into side-chain fluctuations. When mutations increase the available space for those fluctuations, therefore, stability may be expected to increase, and vice versa. Obviously, the benefits of flexibility to ordered backbone structure will diminish as the fluctuations approach a magnitude that permits solvent to permeate the site.

Boyer and Lee's results, though probably expected by those who have followed previous research in eglin c, do not mesh with our macroscopic understanding of the relationship between rigidity and stability. However, they are not so outlandish that they can't be rationalized in terms of what we already know about proteins. An investigation of this anticorrelation in other eglin c mutants (promised in the paper) would be welcome. In addition, it will be interesting to see if this phenomenon is observed in other proteins (especially thermophilic proteins). As new labeling techniques and experimental approaches expand the range of molecules that can be effectively investigated using NMR, we may learn significantly more about the role of conformational entropy in the stabilization of protein folds.

1. Clarkson, M.W., Lee, A. (2004). Long-Range Dynamic Effects of Mutations Propagate Through Side Chains in the Serine Protease Inhibitor Eglin C. Biochemistry, 43(39), 12448-12458. DOI: 10.1021/bi0494424

2. Clarkson, M., Gilmore, S., Edgell, M., Lee, A. (2006). Dynamic Coupling and Allosteric Behavior in a Non-Allosteric Protein. Biochemistry, 45(25), 7693-7699. DOI: 10.1021/bi060652l

3. Lipari, G., Szabo, A. (1982). Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. Journal of the American Chemical Society, 104(17), 4546-4559. DOI: 10.1021/ja00381a009

4. Boyer, J.A., Lee, A.L. (2008). Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via 13C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c. Biochemistry DOI: 10.1021/bi702330t

5. Teilum, K., Brath, U., Lundstrom, P., Akke, M. (2006). Biosynthetic ¹³C Labeling of Aromatic Side Chains in Proteins for NMR Relaxation Measurements. Journal of the American Chemical Society, 128(8), 2506-2507. DOI: 10.1021/ja055660o

Read the rest...

Odin Sphere gameplay notes

So I came to a compromise on the review issue. I'm not going to do much discussion of the aspects of a video game that don't relate directly to the theme or the atmosphere, which are the subjects I want to focus on in the critiques. At the same time, there are going to be some instances, like this one, where a game really deserves some additional criticism or discussion for some of the design aspects. In some cases this will be because of exceptionally good choices that were made in the design process, but in others it will be because of the flaws in the process. And the simple fact is that Odin Sphere had several flaws, some of which nearly made me toss the controller down and take the disc out.

Odin Sphere is an example of a rare case in which the coding, rather than the artistic direction or the writing, was the weakest link in the game. Music, graphics, story, and design create the experience for the player. Coding can't create a good experience if these elements are trash. It can, however, work against them by pushing a player's mind out of the world of the game. It's a thankless task to be a programmer, I'm sure. The designers, artists, and musicians get all the print. But it's the people in the trenches writing the game engine that make all the other aspects work together to create art. In this case, unfortunately, the programming (and perhaps the hardware) wasn't equal to the task that the experiential elements demanded.

Although it is graphically beautiful, Odin Sphere doesn't seem to run on very efficient code. Anytime a large number of moving elements are present on screen the framerate drops, sometimes significantly. This isn't just a problem with too many enemies—the frequent slowdown in the Netherworld stages is associated with background elements. Even stages with relatively few enemies can suffer significant drops if too many phozons are left floating in the air. Because several of the boss battles are very complex, with lots of moving elements, they can be especially afflicted. The battles against Odette and Belial get to be very frustrating because of the framerate failures. The feeling that you could punch in a combo and go make a sandwich before the animation stopped really removes you from a fight.

Speaking of going to make a sandwich, Odin Sphere features some truly amazing load times, especially when going between the Pooka Village and the restaurants. No number of charming little animations will hide the time it takes to move between screens and stages. Often there is substantial loading after you enter a stage as well. Slowdown and load screens kill a player's engagement in a game.

Aside from the technical base, a few design elements were notably off. The controls were a bit muddy, and made to feel more so by the fact that cancels were impossible. Once you start a combo your only option is to see it through or stop acting entirely, which was something of a problem in the more complex boss fights. The inventory system also could have been much improved—it seemed like half the time you weren't fighting was spent managing the tightly constrained inventory. Shrinking that down so much didn't feel like it served much of a purpose; there's no need to be "realistic" about the inventory size in a game where each level ends with a gigantic treasure chest falling from the sky. The inventory management was a needless distraction from the game's better elements.

Another flaw was that the revelation of recipes for alchemy and the restaurants didn't match up with the modular nature of the story. You find a recipe for a moderate healing potion in the first book of the story, but the stronger healing potion isn't found until the fourth book. In the meantime, however, you have brought several characters to a point where the "H" potion isn't of any benefit. There was no gameplay or story reason to hold the Elixir back, and doing so forces the players to replay books in order to give their characters a meaningful healing potion for the final stages. Revealing the recipes piecemeal would have been more acceptable if the player was free to experiment and find things on his own. However, you aren't allowed to make any alchemy mix for which you don't have the recipe.

As for the restaurant, the fact that the story is told very much out of order makes its recipe system seem extremely strange. Why can Velvet buy "egg on toast" right away, when in the linear timeline that recipe won't be found until much time has passed? This complaint can also be leveled at the alchemy recipes. In short, the recipe system was illogical and needlessly forced a replay of previous books.

The reason the replay of previous books was a problem was that you literally had to replay them. That is, once you finished all five main books and wanted to buff your characters for the final battle, you had to start each of those books from the beginning, rather than entering each one at a point later on where you had access to all or most of its levels. As a result, you had to play rather a long time to reach a point where you could buy the bags you needed for inventory or get the raw materials necessary to make elixirs. It seems like this could have been avoided.

For the most part, however, Odin Sphere features reasonable design choices. The diversity of fighting styles of the main characters means that the repetition of levels and bosses never gets boring. The character development system is intuitive and still manages to feature an intelligent trade-off. And for the most part, the game is very forgiving about providing you with materials to produce the potions you need in the areas where they're needed (the notable exception being Titania). While most battles allow you to get by just by whacking at your enemies and using special powers, you can usually find additional strategies or tricks for each particular fight that makes it easier. The game is balanced, has a relatively even difficulty progression, and only one really unreasonable enemy (the slimes). The choices are solid but not spectacular; for the most part this is a game where the mechanics try to make themselves as unobtrusive as possible. That's why the smaller failings seemed so glaring.

Read the rest...

Eleventy-one!

Discount Thoughts turned eleventy-one with that last post, which I think is an excellent time to shake up the structure of this annoying little site. I've decided to kiss the keywords list goodbye and revamp the links. I've replaced it with a non-exhaustive list of keywords and the most popular posts (relative to my other posts) from the last month or so. The list of papers (formerly headed as "Shameless Self Promotion") has been removed and will now exist only in this post. Let me know what you think of the changes.

"Solution Structure of Polymerase μ's BRCT Domain Reveals an Element Essential for Its Role in Nonhomologous End Joining"

"Dynamic Coupling and Allosteric Behavior in a Non-Allosteric Protein"

"Role of Structural Plasticity in Signal Transduction by the Cryptochrome Blue-Light Photoreceptor"

"Long-Range Dynamic Effects of Point Mutations Propagate through Side Chains in the Serine Protease Inhibitor Eglin c"

"Increased Rigidity of Eglin c at Acidic pH: Evidence from NMR Spin Relaxation and MD Simulations"

And some contact information, rendered in my native language:
Email : clarkson dawt emm double yew at geemail dawt cawm
Speak it to learn my address.

Also in this post: the Discount Thoughts lexicon!

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.

cameralepsy — The repeated recrossing of a boundary between two camera fields of view. Generally occurs in games which have fixed cameras and camera-relative controls. If the smallest angle between the cameras is > 120 degrees, there is a high probability of a cameraleptic seizure, which can only be cured by ceasing to move or (in games like Grim Fandango) changing the control scheme.

disingenue — A person, often famous, who falsely claims that his or her bad deeds are due to the influence of poorly chosen friends.

magibabble — The fantasy equivalent of Treknobabble, magibabble is pseudoscientific, jargon-filled dialogue concerning technicalities of magical physics that will either cause the destruction of the world, allow the heroes to save the world, or both. Excessive use of magibabble is a particularly common sin of fantasy RPGs. The emphasis on jargon is a critical feature of magibabble. Ordinary myths and legends often hinge on some technicality of magic, but they are not examples of magibabble because the rules are explained in plain language. Magibabble is typically used to disguise plot holes or laziness.

tatropmr (tä trōp mər) — The Abstract Transcendental Realm Of Perfect Mathematical Relationships, a nonexistent place which is frightfully dull, rather difficult to comprehend, and not inhabited by natural laws. All those who fundamentally misunderstand the process and meaning of science are eternally condemned there. First identified by Paul Davies, also the first to be publicly damned to it.

the turtles — A key problem with many arguments for creationism or intelligent design is the turtles: specifically, the fact that they go all the way down.

Read the rest...

The ring of death and life

The beginning of Odin Sphere seems to have little to do with the operatic introductory sequence or the characters shown in it. A young girl, exploring with her cat, finds a library of old books in a musty attic. She begins to read, and as the storybooks come (gloriously) to life, she finds herself lost in an earlier world, one that is tied to her own by a cycle of death and birth.

Spoilers follow below the fold.

One need look no further than the title to recognize that Odin Sphere is aiming to evoke the feeling of Nordic sagas and the Ring of the Nibelung. There are some direct parallels to be found, and persistent references to these stories help establish the mythic atmosphere. Like the sagas, the story emphasizes destiny and prophecy; the more the characters struggle to avoid their fate, the closer they bring it. The player controls five characters (in different "books"), but the story is not presented linearly. Instead, each chapter opens a new angle into events that have already occurred.

The image of the ring lies at the heart of Odin Sphere. Much of the story is concerned with the struggle for a literal ring, called Titrel, that controls an enormous spherical Cauldron with the power to create immensely powerful weapons called Psyphers. Odin captures the Cauldron, but his illegitimate daughter Velvet, fearing the Cauldron's destructive power, feeds Titrel to an enormous dragon named Wagner. Odin's legitimate daughter Gwendolyn saves Velvet from the vengeance of Odin's armies, but in doing so defies her father and so she must be put to sleep. She is told that she will love the first man she sees on waking.

In the meantime, the Shadow Knight Oswald (left), one of Odin's enemies, has fallen in love with Gwendolyn. In order to win her hand, he defeats Wagner, giving the ring to Gwendolyn as a symbol of his love. Seeking her father's approval, Gwendolyn gives Titrel to Odin. The remainder of her story consists of her efforts to win back the ring and rescue Oswald from death, as she has fallen in love with him. In the end she refuses to yield up the ring to her father.

Events spill off from this core narrative in all directions, and the player takes up the part of Velvet, Oswald, the fairy princess Mercedes, and the hapless prince Cornelius as they struggle futilely against fate. Cornelius, cursed by Velvet's brother Ingway, accidentally releases Velvet's mad grandfather from the Netherworld, who wreaks havoc and brings a world-ending dragon to life with the aid of the Cauldron. Mercedes, trying to save her kingdom from Odin, unwittingly destroys the only weapon that had a chance of restraining the cataclysm. Oswald learns that Gwendolyn was lied to, that when she wakes her heart will still be her own, yet decides to awaken her anyway in hopes that he can make her happy. Each of them comes into contact with the ring, as well as confronting many of the same enemies (and each other) in combat. The repetition of areas and bosses (especially the dragon Belial, seen at right) is itself a kind of cycle within the game. It should be noted that this never actually gets dull, because each of the characters plays in a different and interesting way.

The game contributes another kind of cycle in that each of its two-dimensional stages is actually a circle. If you continue far enough in any direction you will come back to the spot that you left. From a gameplay perspective this allows the construction of rather complex level maps in the context of what amounts to an old-school side-scroller. But the circular level structure also emphasizes what the player should expect from the game.

Similarly, the results of combat foreshadow the end of the game. In death each enemy releases a number of brightly glowing "phozons". The player can choose to absorb these into the character's weapon, enabling special attacks and increasing the character's combat strength. If, however, the player has planted certain seeds in the stage, those plants will draw in the phozons as they grow and bear fruit. The character can eat these to heal and increase HP. In this regard, deaths are made to literally produce life. Even the phozons drawn into the psyphers have the same ultimate effect, because at the end of the game these weapons are used to power the Cauldron so that it can cover the world with new life after Armageddon. Why not Ragnarok or Gotterdammerung? I have no idea.

This outcome of the game's final cataclysm again echoes the Nordic myths. As the great serpent falls from the sky, Velvet uses Titrel to command the Cauldron, and a lush new world is born from the death of the old. Only Gwendolyn and Oswald, who were bound together by the ring, have survived the apocalypse to repopulate the world. Again we are treated to the circle, the emergence of death from life, reaching the end of all things only to find yourself at the beginning of all things.

The characters of Odin Sphere struggle against fate, striving to avoid their own deaths, the death of their loved ones, and the death of the world. But it is the nature of all things to die. And it is the nature of the world for new life to arise from death. In the epilogue, even Velvet, who struggled hardest to avoid the death fated to her, comes to accept this.

One way or another, every world ends, and then, another begins.

As of 4/2008 Odin Sphere is still available new from its publisher, Atlus.

Read the rest...

Zetia and Vytorin update

A few months back I mentioned a study called ENHANCE from the makers of Vytorin and Zetia that showed these drugs had no effect. That study has again entered the spotlight thanks to the release of the results and a pair of commentaries in the New England Journal of Medicine. The published results are essentially as was previously reported: addition of ezetimibe to an existing simvastatin regimen was of no benefit to patients, either in terms of reducing a measure of atherosclerosis or in the frequency of adverse events. The article (citation below) is worth checking out if you are interested.

As I mentioned in the previous post, these results are not definitive. The group under study was a high-risk group, but the fact that most of the participants were taking statins already significantly complicates the interpretation of the results. Is the lack of improvement with the addition of ezetimibe simply a case of diminishing marginal returns?

The really curious thing here is that ezetimibe did what it was supposed to do. Patients who took it had lower LDL levels than patients who didn't. Yet this reduction in LDL did not improve the measured outcomes. Does this mean that the measurement (intima-media thickness) was not appropriate? Or do these results imply that there are deficits in our understanding of the interplay between LDL, statins, and arterial disease? Additional studies using other observables are underway, but will not be completed until 2011. Until those results come in, the American College of Cardiology recommends that ezetimibe be used only when other approaches (notably statins) have failed to control LDL. I want to reinforce that these results do not mean that ezetimibe is dangerous, or even that it does not help prevent cardiovascular disease. The present situation is one of uncertainty. If you are presently taking ezetimibe as Vytorin or Zetia, continue to do so until you have a chance to discuss your medications with your doctor.

You can read the study here:
Kastelein, J.J., Akdim, F., Stroes, E.S., Zwinderman, A.H., Bots, M.L., Stalenhoef, A.F., Visseren, F.L., Sijbrands, E.J., Trip, M.D., Stein, E.A., Gaudet, D., Duivenvoorden, R., Veltri, E.P., Marais, A.D., de Groot, E. (2008). Simvastatin with or without Ezetimibe in Familial Hypercholesterolemia. New England Journal of Medicine, 358(14), 1431-1443. DOI: 10.1056/NEJMoa0800742 OPEN ACCESS

The commentaries have DOIs 10.1056/NEJMe0801608 and 10.1056/NEJMe0801842. Both are open access.

PalMD of the Denialism Blog has another commentary on this issue.

Read the rest...

NCAA finally picks a tournament right

Well, my bracket is officially toast, even though the two teams I picked for the final are still in it. The reason? For the first time in forever all four #1 seeds made it to the Final Four. Obviously, I have mixed feelings on this. I'm happy UNC and UCLA made it, though I would have preferred to see Davidson in the semifinal. Playing Kansas will be hard on coach Williams, though I expect Ray and his family will be hoping Williams gets a karma dosage. And as far as I'm concerned, Memphis can go right to hell.

I genuinely felt bad for Davidson, and I really respect what they did this year. They scheduled some tough teams during the season (including UNC), played hard throughout the tournament, and made it a long way. This wasn't a fluke—they are legitimately a great team, and they didn't make it all this way just because of Stephen Curry (as Barr's performance today showed). They made it to the tournament the right way, playing tough all year, and they deserved their Elite Eight berth. For beating them, the way they're playing right now, Kansas deserves a lot of respect. The close score in today's game is a mark of Davidson's quality, not Kansas' weakness.

UNC-Kansas will be a great game, perhaps the game of the year. I'm not sure that either team can take the championship after winning, though. The game will definitely be emotionally taxing, no matter what the outcome. I think Kansas can bounce back from it more easily than UNC, but either way it will be really draining. As for the other semifinal, I hope UCLA mops the floor with Memphis. Did I mention that I don't like Memphis?

Conference-wise, I think the tournament played out about right. The SEC and Big Ten had down years, so the absence of their teams is reasonable. Obviously, C-USA has no right fielding a Final Four team; most of the conference is trash. The glaring omission here is the Big East, which had a pretty strong slate of teams this year. I really thought Georgetown would make the semifinals (I had Texas representing the Big XII in my bracket), and that Pitt and UConn both had a chance. But we know how that turned out.

On a related note, I really like Grant Wahl:

We've been asked to discuss the NCAA tournament on Lou Dobbs' national radio show on Monday. Part of me wants to see how Lou responds when I tell him about the impressive play of Brook and Robin Lopez, Lorenzo Mata-Real, Edgar Sosa, Juan Palacios and Orlando Mendez-Valdez. Then I think I'll invite him to join me at a U.S.-Mexico soccer match.

Read the rest...