Looking for grant money for your research?

Times are tight. It’s tough getting grants from NIH and NSF, but the government has heard your plight and has responded by opening up new avenues to request support: apply for an NCMHD Innovative Faith-Based Approaches to Health Disparities Research grant!

Purpose. The purpose of the NCMHD Innovative Faith-Based Approaches to Health Disparities Research (R21) is to solicit applications that propose translational and transdisciplinary interventions on health disparities, social determinants of health, health behavior and promotion and disease prevention, especially those jointly conducted with faith-based organizations or faith-motivated programs and the research community.  The ultimate goal is to foster empirical, formative, evaluative and intervention research on effective faith-motivated initiatives, concepts and theories that have played an important role in addressing health disparities.  Funding is also intended to provide support for early and conceptual stages of exploratory and developmental research projects.  This focus will allow studies to evaluate the impact of faith-based initiatives and programs in health disparity populations, formulate hypotheses about the role and unique characteristics of faith communities in addressing health disparities, design targeted interventions and track the efficacy of faith-motivated efforts that result from a participatory approach to research in the community. These studies may involve considerable risk but may lead to a breakthrough in addressing health disparities or the development of a model or application that could have a major impact on the field of health disparities research.

It’s not quite as vile as it sounds — they aren’t endorsing the efficacy of faith-based approaches to health, they’re just saying that there are all these churches around and people go to them more easily than they do to clinics, so explore that and see if you can sneak in some science to go with their superstition. Probably. It’s all imbedded in typical murky NIHese, and it does involve forming partnerships with faith-based institutions, so some of your $275,000 direct funds will end up supporting the nonsense we ought to be working against.

Tetrapods are older than we thought!

Some stunning fossil trackways have been discovered in Poland. The remarkable thing about them is that they’re very old, about 395 million years old, and they are clearly the tracks of tetrapods. Just to put that in perspective, Tiktaalik, probably the most famous specimen illustrating an early stage of the transition to land, is younger at 375 million years, but is more primitive in having less developed, more fin-like limbs. So what we’ve got is a set of footprints that tell us the actual age of the transition by vertebrates from water to land had to be much, much earlier than was expected, by tens of millions of years.

Here are the trackways. Note that what they show is distinct footprints from both the front and hind limbs, not drag marks, and all that that implies: these creatures had jointed limbs with knees and elbows and lifted them and swung them forward to plant in the mud. They were real walkers.

i-7c534308c9f00e73cf31353a1066764b-trackway1.jpegi-55a13b4b3a79452858e68be0cd01fa4f-trackway2.jpeg
Trackways. a, Muz. PGI 1728.II.16. (Geological Museum of the Polish Geological Institute). Trackway showing manus and pes prints in diagonal stride pattern, presumed direction of travel from bottom to top. A larger print (vertical hatching) may represent a swimming animal moving from top to bottom. b, On the left is a generic Devonian tetrapod based on Ichthyostega and Acanthostega fitted to the trackway. On the right, Tiktaalik (with tail reconstructed from Panderichthys) is drawn to the same shoulder-hip length. Positions of pectoral fins show approximate maximum ‘stride length’. c, Muz. PGI 1728.II.15. Trackway showing alternating diagonal and parallel stride patterns. In a and c, photographs are on the left, interpretative drawings are on the right. Thin lines linking prints indicate stride pattern. Dotted outlines indicate indistinct margins and wavy lines show the edge of the displacement rim. Scale bars, 10 cm.

They were also big, approximately 2 meters long. What you see here is a detailed scan of one of the footprints of this beast; no fossils of the animal itself have been found, so it’s being compared to the feet of Ichthyostega and Acanthostega, two later tetrapods. There are definite similarities, with the biggest obvious difference being how much larger the newly-discovered animal is. Per Ahlberg makes an appearance in a video to talk about the size and significance of the mystery tetrapod.

i-7f4600fb6e10f03a446adb1e20fde705-foot.jpeg
Foot morphologies. a, Laser surface scan of Muz. PGI 1728.II.1, left pes. b, Complete articulated left hind limb skeleton of Ichthyostega, MGUH f.n. 1349, with reconstructed soft tissue outline. c, Left hind limb of Acanthostega, reconstructed soft tissue outline based on skeletal reconstruction in ref. 8. We note the large size of the print compared to the limbs of Ichthyostega and Acanthostega, and that the print appears to represent not just the foot but the whole limb as far as the knee. d, digit; fe, femur; ti, tibia; fi, fibula; fib, fibulare. Scale bars, 10 mm.

What’s it all mean? Well, there’s the obvious implication that if you want to find earlier examples of the tetrapod transition, you should look in rocks that are about 400 million years old or older. However, it’s a little more complicated than that, because the mix of existing fossils tells us that there were viable, long-lasting niches for a diversity of fish, fishapods, and tetrapods that temporally coexisted for a long period of time; the evolution of these animals was not about a constant linear churn, replacing the old model with the new model every year. Comparing them to cars, it’s like there was a prolonged window of time in which horse-drawn buggies, Stanley Steamers, Model Ts, Studebakers, Ford Mustangs, and the Honda Civic were all being manufactured simultaneously and were all competitive with each other in specific markets…and that window lasted for 50 million years. Paleontologists are simply sampling bits and pieces of the model line-up and trying to sort out the relationships and timing of their origin.

The other phenomenon here is a demonstration of the spottiness of the fossil record. The Polish animal has left us no direct fossil remains; the rocks where its footprints were found formed in an ancient tide flat or lagoon, which is not a good location for the preservation of bones. This suggests that tetrapods may have first evolved in these kinds of marine environments, and only later expanded their ranges to live in the vegetated margins of rivers, where the flow of sediments is much more conducive to burial and preservation of animal remains. That complicates the story, too; not only do we have diverse stages of the tetrapod transition happily living together in time, but there may be a bit of selective fossilization going on, that only preserves some of the more derived forms living in taphonomically favorable environments.

Niedzwiedzki G, Szrek P, Narkiewicz K, Narkiewicz M, Ahlberg PE (2010) Tetrapod trackways from the early Middle Devonian period of Poland. Nature 463(7277): 43-48.

Crawling pigments

Here’s another of Casey Dunn’s Creature Casts, this time on shifting color spots in marine snails.

CreatureCast – Flamingo Tongue Snails from Casey Dunn on Vimeo.

Pigment cells are always very, very cool. I’ve been intrigued by them for a long time — they show up in my time-lapse recordings of developing zebrafish and are always active. Here’s a quick one, a few hours of time in a roughly 24 hour old zebrafish embryo, compressed to about 30 seconds. You can see one corner of the dark eye at the bottom left of the image, and that oval structure near the middle with two spots in it is the ear and its otoliths. The melanocytes are writhing over the side of the head and down onto the yolk sac; they’re not quite as colorful as the snail, but then, the zebrafish is a mostly black and white animal.

α-actinin evolution in humans

i-e88a953e59c2ce6c5e2ac4568c7f0c36-rb.png

Perhaps your idea of the traditional holiday week involves lounging about with a full belly watching football — not me, though. I think if I did, I’d be eyeing those muscular fellows with thoughts of muscle biopsies and analyses of the frequency of α-actinin variants in their population vs. the population of national recliner inhabitants. I’m sure there’s an interesting story there.

In case you’re wondering what α-actinin is, it’s a cytoskeletal protein that’s important in anchoring and coordinating the thin filaments of actin that criss-cross throughout your cells. It’s very important in muscle, where it’s localized in the Z-disk at the boundaries of sarcomeres, the repeated contractile units of the muscle. This diagram might help you visualize it:

i-60cbd272718c8f724ef1452d79d99d6a-sarcomere.jpeg
Actin (green), myosin (red). Rod-like tropomyosin molecules (black lines). Thin filaments in muscle sarcomeres are anchored at the Z-disk by the cross-linking protein α-actinin (gold) and are capped by CapZ (pink squares). The thin-filament pointed ends terminate within the A band, are capped by tropomodulin (bright red). Myosin-binding-protein C (MyBP-C; yellow transverse lines).

The most prominent elements in the picture are the thin filaments (made of actin) and thick filaments (made of myosin) which slide past each other, driven by motor proteins, to cause contraction and relaxation of the muscle. The α-actinin proteins are the subtle orange lines in the Z disks on the left and right.

The α-actinin proteins are evolutionarily interesting. In vertebrates, there are usually four different kinds: α-actinin 1, 2, 3, and 4. 1 and 4 are ubiquitous in all cells, since all cells have a cytoskeleton, and the α-actinins are important in anchoring the cytoskeleton. α-actinin-2 and -3 are the ones of interest here, because they are specifically muscle actinins. α-actinin-2 is found in all skeletal muscle fibers, cardiac muscle, and also in the brain (no, not muscle in the brain, there isn’t any: in the cytoskeleton of neurons). Just to complicate matters a bit, α-actinin-2 is also differently spliced in different tissues, producing a couple of isoforms from a single gene. α-actinin-3 is not found in the brain or heart, but only in skeletal muscle and specifically in type II fast glycolytic muscle fibers.

Muscle fibers are specialized. Some are small diameter, well vascularized, relatively slow fibers that are optimized for endurance; they can keep contracting over and over again for long periods of time. These are the fibers that make up the dark meat in your Christmas turkey or duck. Other fibers are large diameter, operate effectively anaerobically, and are optimized for generating lots of force rapidly, but they tend to fatigue quickly — and there are more of these in the white meat of your Christmas bird. (There are also intermediate fiber types that we won’t consider here.) Just keep these straight in your head to follow along: the fast type II muscle fibers are the ones that you use to generate explosive bursts of force, and may be enriched in α-actinin-3; the slower fibers are the ones you use to keep going when you run marathons, and contain α-actinin-2. (There are other even more important differences between fast and slow fibers, especially in myosin variants, so differences in α-actinins are not major determinants of muscle type.)

Wait, what about evolution? It turns out that invertebrates only have one kind of α-actinin, and vertebrates made their suite of four in the process of a pair of whole genome duplications. We made α-actinin-2 and -3 in a duplication event roughly 250-300 million years ago, at which time they would have been simple duplicates of each other, but they have diverged since then, producing subtle (and not entirely understood) functional differences from one another, in addition to acquiring different sites of expression. α-actinin-2 and -3 in humans are now about 80% identical in amino acid sequence. What has happened in these two genes is consistent with what we know about patterns of duplication and divergence.

i-75f72fe14fd19d142167119147ebce45-duplication.jpeg
Using sarcomeric α-actinin as an example, after duplication of a gene capable of multiple interactions/functions, there are two possible distinct scenarios besides gene loss. A: Sub-functionalisation, where one interaction site is optimised in each of the copies. B: Neo-functionalisation, where one copy retains the ancestral inter- action sites while the other is free to evolve new interaction sites.

So what we’re seeing in the vertebrate lineage is a conserved pattern of specialization of α-actinin-3 to work with fast muscle fibers — it’s a factor in enhancing performance in the specific task of generating force. The α-actinin-3 gene is an example of a duplicated gene becoming increasingly specialized for a particular role, with both changes in the amino acid sequence that promoted a more specialized activity, and changes in the regulatory region of the gene so that it was only switched on in appropriate muscle fibers.

i-e5ab4d9006f937a9907d72bd7ae1aac0-actn_history.jpeg
Duplication and divergence model proposed by this paper. Before duplication the ancestral sarcomeric α-actinin had the functions of both ACTN2 and ACTN3 in terms of tissue expression and functional isoforms. After duplication, ACTN2 has conserved most of the functions of the preduplicated gene, while ACTN3 has lost many of these functions, which may have allowed it to optimise function in fast fibres.

That’s cool, but what we need is an experiment: we need to knock out the gene and see what happens. Mutations in α-actinin-2 are bad—they cause a cardiomyopathy. Losing α-actinin-4 leads to serious kidney defects (that gene is expressed in kidney tissue). What happens if we lose α-actinin-3?

It turns out you may be a guinea pig in that great experiment. Humans acquired a mutation in the α-actinin-3 gene, called R577X, approximately 40-60,000 years ago, and this mutation is incredibly common: about 50% of individuals of European and Asian descent carry it, and about 10% of individuals from African populations. Furthermore, an analysis of the flanking DNA shows relatively little recombination or polymorphism — which implies that the allele has reached this high frequency relatively recently and rapidly, which in turn implies that there has been positive selection for a nonsense mutation that destroys α-actinin-3 in us. The data suggests that a selective sweep for this variant began in Asia about 33,000 years ago, and in Europe about 15,000 years ago.

There is no disease associated with the loss of α-actinin-3. It seems that α-actinin-2 steps up to the plate and fills the role in type II fast muscle fibers, so everything functions just fine. Except…well, there is an interesting statistical effect.

The presence of a functional α-actinin-3 gene is correlated with athletic performance. A study of the frequency of the R577X mutation in athletes and controls found that there is a significant reduction in the frequency of the mutation among sprinters and power-lifters. At the Olympic level, none of the sprinters in the sample (32 individuals) carried the α-actinin-3 deficiency. Among Olympic power lifters, all had at least one functional copy of α-actinin-3.

Awesome. Now I’m wondering about my α-actinin-3 genotype, and whether I have a good biological excuse for why I always got picked last for team sports in high school gym class. This is also why I’m interested in taking biopsies of football players…both for satisfying a scientific curiosity, and for revenge.

You may be wondering at this point about something: α-actinin-3 has a clear beneficial effect in enhancing athletic performance, and its conservation in other animal species suggests that it’s almost certainly a good and useful protein. So why has there been positive selection (probably) for a knock-out mutation in the human lineage?

There is a weak correlation in that study of athletic performance that high-ranking athletes in endurance sports have an increased frequency of the R577X genotype; it was only seen in female long-distance runners, though. More persuasive is the observation that α-actinin-3 knockouts in mice also produced a shift in metabolic enzyme markers that are indicative of increased endurance capacity. The positive advantage of losing α-actinin-3 may be more efficient aerobic metabolism in muscles, at the expense of sacrificing some strength at the high end of athletic performance.

This is yet another example of human evolution in progress—we’re seeing a shift in human muscle function over the course of a few tens of thousands of years.

Lek M, Quinlan KG, North KN (2009) The evolution of skeletal muscle performance: gene duplication and divergence of human sarcomeric alpha-actinins. Bioessays 32(1):17-25. [Epub ahead of print]

MacArthur DG, Seto JT, Raftery JM, Quinlan KG, Huttley GA, Hook JW, Lemckert FA, Kee AJ, Edwards MR, Berman Y, Hardeman EC, Gunning PW, Easteal S, Yang N, North KN (2007) Loss of ACTN3 gene function alters mouse muscle metabolism and shows evidence of positive selection in humans. Nat Genet.39(10):1261-5.

Yang N, MacArthur DG, Gulbin JP, Hahn AG, Beggs AH, Easteal S, North K (2003) ACTN3 genotype is associated with human elite athletic performance. Am J Hum Genet 73(3):627-31.

Evolving fish of the lower Congo

The lower Congo river is deep and complex, and there are a surprising number of hydrologic features that act as barriers separating populations of fish — this very nice video explains the diversity of species and the ongoing evolution of the fish in this environment.

They too briefly showed a blind depigmented cichlid that apparently lives in very deep troughs in the river — I wanted to see more about that. It’s probably out of the question to send divers down into that maelstrom, but cameras? Someday? Please?