Jonathan Singer and the Cranky Book

An eminent scientist at the end of his career writes out his thoughts and preoccupations.

Jonathan Singer was a famous scientist at my graduate school. I did not interact with him, but he played a role in attracting me to the program, as I was interested in biological membranes at the time. Singer himself studied with Linus Pauling, and they were the first to identify a human mutation in a specific gene as a cause for a specific disease- sickle cell disease. After further notable work in electron microscopy, he reached a career triumph by developing, in 1972, the fluid mosaic model of biological membranes. This revolutionized and clarified the field, showing that cells are bounded by something incredibly simple- a bilayer of phospholipids that naturally order themselves into a remarkably stable sheet, (a bubble, one might say), all organized by their charged headgroups and hydrophobic fatty tails. This model also showed that proteins would be swimming around freely in this membrane, and could be integrated in various ways, ether lightly attached on one side, or spanning it completely, thereby enabling complex channel and transporter functions. The model implied the typical length of a protein alpha helix that, by virtue of its hydrophobic side chains, would naturally be able to do this spanning function- a prediction that was spot-on. He could have easily won a Nobel for this work.

I was intrigued when I learned recently that Singer had written a book near the end of his career. It is just the kind of thing that a retired professor loves to do in the sunset of his career, sharing the wisdom and staving off the darkness by taking a stab at the book biz. And Singer's is a classic of the form- highly personal, a bit stilted, and ultimately meandering. I will review some of its high points, and then take a stab of my own at knitting together some of the interesting themes he grapples with.

For at base, Singer turns out to be a spiritual compadre of this blog. He claims to be a rationalist, in a world where, as he has it, no more than 9% of people are rational. Definition? It is the poll question of whether one believes that god created man, rather than the other way around. Singer recognizes that the world around him is crazy, and that the communities he has been a part of have been precious oases amid the general indifference and grasping of the world. But changing it? He is rather fatalistic about that, recognizing that reason is up against overwhelming forces.

His specific themes cover a great deal of biology, and then some more mystical reflections on balance and diversity in biology, and later, in capitalism and politics. He points out that the nature/nurture debate has been settled by twin studies. Nature, which is to say, genetics, is the dominant influence on human characteristics, including a wide variety of psychological traits, including intelligence. Environment and nurture is critical for reaching one's highest potential, and for using it in socially constructive ways, but the limits of that potential are largely set by one's genes. Singer does not, however, draw the inevitable conclusion from these observations, which is that some kind of long-term eugenic approach would be beneficial to our collective future, assuming machines do not replace us forthwith. Biologists know that very small selective coefficients can have big effects, so nothing drastic is needed. But what criteria to use- that is the sticky part. Just as success in the capitalist system hardly signals high moral or personal qualities, nor does incarceration by the justice system always show low ones. It is virtually an insoluble problem, so we muddle along, destined probably for continued cycles of Spenglerian civilizational collapse.

Turning to social affairs, Singer settles on "structural chaos" as his description of how the scientific enterprise works, and how capitalism at large works. With a great deal of waste, and misdirected effort, it nevertheless ends up providing good results- better than those that top-down direction can provide. He seems a sigh a little that "scientific" methods of social organization, such as those in Soviet Russia, were so ineffective, and that the best we can do is to muddle along with the spontaneous entrepreneurship and occasional flashes of innovation that push the process along. Not to mention the "monstrous vulgarity" of advertising, etc. Likewise, democracy is a mess, with most people totally incapable of making the reasoned decisions needed to maintain it. Again, the chaos of democracy is sadly the best we can do, and the duty of rational people, in Singer's view, is to keep alive the flame of intellectual freedom while outside pressures constantly threaten.

Art, and science.

What can we do with this? I think that the unifying thread that Singer was groping for was competition. One can frame competition as a universal principle that shapes the physical, biological, and social worlds. Put two children on a teeter-totter, and you can see how physical forces (e.g. gravitation) compete all the time, subtly producing equilibria that characterize the universe. Chemical equilibria are likewise a product of constant competition, even including the perpetual jostling of phospholipids to find their lowest energy configuration amidst the biological membrane bilayer, which has the side-effect of creating such a stable, yet highly flexible, structure. With Darwin, competition reaches its apotheosis- the endless proliferation, diversification, and selection of organisms. Singer marvels at the fragility of individual life, at the same time that life writ large is so incredibly durable and prolific. Well, the mechanism behind that is competition. And naturally, economics of any free kind, including capitalism and grant-making in science, are based on competition as well- the natural principle that selects which products are useful, which employees are productive, and which technologies are helpful. Waste is part of the process, as diversity amidst excess production is the essential ingredient for subsequent selection. 

And yet.. something is missing. The earth's biosphere would still be a mere bacterial soup if competition were the only principle at work. Bacteria (and their viruses) are the most streamlined competition machines- battlebots of the living world. It took cooperation between a bacterial cell and an archaeal cell to make a revolutionary new entity- the eukaryotic cell. It then took some more cooperation for eukaryotic cells to band together into bodies, making plants and animals. And among animals, cooperation in modest amounts provides for reproduction, family structure, flock structures, and even complex insect societies. It is with humans that cooperation and competition reach their most complex heights, for we are able to regulate ourselves, rationally. We make rules. 

Without rules, human society is anarchic mayhem- a trumpian, dystopian and corrupt nightmare. With them, it (ideally) balances competition with cooperation to harness the benefits of each. Our devotion to sports can be seen as a form of rule worship, and explicit management of the competitive landscape. Can there be too many rules? Absolutely, there are dangers on both sides. Take China as an example. In the last half-century, it revamped its system of rules to lower the instability of political competition, harness the power of economic competition, and completely transform its society. 

The most characteristic and powerful human institution may be the legislature, which is our ongoing effort to make rational rules regulating how the incredibly powerful motive force of competition shapes our lives. Our rules, in the US, were authored, at the outset, by the founders, who were- drumroll please- rationalists. To read the Federalist Papers is to see exquisite reasoning drawing on wide historical precedent, and particularly on the inspirations of the rationalist enlightenment, to formulate a new set of rules mediating between cooperation and competition. Not only were they more fair than the old rules, but they were designed for perpetual improvement and adjustment. The founding was, at base, a rationlist moment, when characters like Franklin, Hamilton, Madison, and Jefferson- deists at best and rationalists through and through, led the new country into a hopeful, constitutional future. At the current moment, two hundred and fifty years on, as our institutions are being wantonly destroyed and anything resembling reason, civility, and truth is under particularly vengeful attack, we should appreciate and own that heritage, which informs a true patriotism against the forces of darkness.

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The Fire Inside: Eukaryotic Locomotion

The GTP-based Rho/Rac system of actin regulation runs in unseen waves of activation.

One of the amazing capabilities of eukaryotic cells, inherited in part from their archaeal parents, is free movement and phagocytosis. These cells have an internal cytoskeleton, plus methods to anchor to a substrate, (via focal adhesions), which allows them to manipulate their membrane, their shape, and their locomotion. The cytoskeleton is composed of two main types of fibers, actin and microtubules. Microtubules are much larger than actin and organize major trackways of organelle movement around the cell (including the movement of chromosomes in mitosis), and also form the core of cilia and flagella. But it is actin that does most of the work of moving cells around, with dynamic networks that generate the forces behind spiky to ruffly protrusions, that power things like the adventuresome pathfinding of neurons as they extend their axons into distant locations.

Schematic of the actin cytoskeleton of a typical eukaryotic cell.

Actin is an ATPase all by itself. ATP promotes its stability, and also its polymerization into filaments. So, cell edges can grow just by adding actin to filament ends. Actin cross-linking proteins also exist, that create the meshwork that supports extended filopodia. But obviously, actin all by itself is not a regulated solution to cell movement. There is an ornately complex system of control, not nearly understood, that revolves around GTPase and binding proteins. These proteins (mainly RhoA, Rac1, and Cdc42, though there are twenty related family members in humans) have knife-edge regulation, being on when binding GTP, and off after they cleave off the phosphate and are left binding GDP (the typical, default, state). Yet other proteins regulate these regulators- GTPase exchange factors (GEFs) encourage release of GDP and binding of GTP, while GTPase activating proteins (GAPs) encourage the cleavage of GTP to GDP. The GTP binding proteins interact (depending on their GTP status) with a variety of effector proteins. One example is a family of formins, which chaperone the polymerization of actin. At the head of the pathway, signals coming from external or internal conditions regulate the GTPases, creating (in extremely simplified terms) a pathway that gets the cell to respond by moving toward things it wants, and away from things it does not want. 

This is a very brief post, just touching on one experiment done on this system. Exploring its full complexity is way beyond my current expertise, though we may return to aspects of this fascinating biological pathway periodically in the future. An important paper in the field hooked up fluorescent dyes to one of the effector protein domains that binds only GTP/active RhoA. They tethered this to the (inside) membrane of their cultured cells, and took movies of what the cell looked like, using a microscopy method that looks at very thin sections- only the membrane, essentially not the rest of the cell. RhoA, though graced with a small lipid tail, is typically cytoplasmic when inactive, and travels to the membrane when activated. They were shocked to find that in resting cells, without much locomotion going on, there were recurring waves of activation of RhoA that swept hither and yon across the cell membranes. 

Four examples of RhoA getting bound in its active state in a wave-like way, over 7 1/2 minutes in a resting cell. GEF-H1 is ARHGEF2, one of the regulators that can turn RhoA on. The first three panels have ARHGEF2 versions that are operational, but the fourth (bottom right) is of a cell with an anti-RNA to ARHGEF2, turning its expression level down. In this cell, the waves of RhoA activation and recruitment to the membrane are substantially dampened.

These pulses were made even more intense if the cells were treated with nocodazole, which disrupts microtubules, destabilizes the cytoskeleton, and makes the actin regulatory / structure system work harder. They found that myosin (the motor protein that moves cargoes over actin filaments) was also rapidly relocalized, mirroring some of what happened with RhoA. They also found that ARHGEF2 contained two RhoA binding domains, (one binding active RhoA, one binding inactive RhoA), enabling it to feedback-amplify the positive activation of RhoA, thereby explaining some of the extremely dynamic activity seen here. 

And they also found that the arrival of negative regulators such as ARHGAP35 was delayed by a couple of seconds vs the activation of RhoA, providing the time window needed to see wave formation out of a mechanism of positive feedback followed by squelching by a negative regulator. Lastly, they found that these dynamics were significantly different if the cells were grown on stiffer vs softer substrates. Stiffer substrates allowed the formation of stronger surface attachments, concentrating RhoA and myosin at these adhesion locations. 

These researchers are clearly only scratching the surface of this system, as there are endless complexities left to investigate. The upshot of this one set of observations is that neurons are not the only excitable cells. With a bit of molecular / experimental magic, heretofore unseen intracellular dynamics can be visualized to show that eukaryotic cells have an exquisitely regulated internal excitation system that is part of what drives their shape-shifting capabilities, including processes like phagocytosis and neuronal growth / path-finding. 

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3:10 to Yuma

The classic Western, and our moral moment. Spoiler alert!

Americans of my vintage were raised on Westerns. Looking at the Wiki page on TV westerns, it is astounding how many there were, and that isn't even counting their science fiction progeny. I recently watched a classic- 3:10 to Yuma, which is unbearably tense, beautifully shot and acted, and just a crystaline example of the form. It reminded me of the morality that lies at the heart of the American experience. 

Glen Ford (as Ben Wade) heads a gang of outlaws who rob a stagecoach and kill its drivers. Local farmer Dan Evans (played by Van Hefflin) witnesses this with his two sons, and stands clear. Later, the marshal and the owner of the stagecoach line (Mr. Butterfield) capture Wade, who has boldly wandered into town, and get up a posse, which includes farmer Dan Evans, to bundle him off to justice in Yuma. Only, the rest of the gang is still out there, and is ready to shoot whoever stands in the way of getting their leader back. Various plot twists later, Ben is being guarded by Dan in a hotel room close to the Yuma train stop, waiting for the train to come through. 

Glen Ford plays Wade as Satan incarnate- a smooth talking, woman seducing, suave and heartless killer. He keeps up a patter of warnings and blandishments to Dan, telling him that the rest of the posse will desert him (they do) and that Butterfield will also desert him (he does), as Wade's gang closes in around town. 

It is a morality play of power in its most corrupt form, vs civilization and decency. Evans has built a homestead and a family, with salt-of-the-earth wife Alice. Filmed in starkly beautiful black and white, the morality is likewise stark. Yet out here, in the real world, we suddenly are faced with a government that proudly wears the black hats, killing the innocent (and lying about it) and reveling in extortion across the domestic and foreign landscapes. A government that makes a principle of bad policy, selling out our future for donations from the oil industry, and straining every nerve enrich the rich, particularly cronies, and impoverish and denigrate the poor.

It is noteworthy that religion has no role in 3:10 to Yuma. The morality, good and bad, needs no reference to the supposed foundations of morality, sources in higher powers, or advice from clergy. No, morality is, in truth, a secular process, needing only eyes to see and a heart to feel. What is more, our current political depravity has been largely brought about by religious communities, out of their false sense of desperation and victimization. They have hitched their wagon out of grievance over losing the culture wars- to women who can not understand why they should be second class citizens, to minorities, who can not understand why they should be forever trodden upon, to scientists, who won the Dover decision that said that facts, not theology under any guise or subterfuge, should be taught in schools, and to Hollywood, which leads all too often with empathy and inclusion.

For the secret ingredient in all this is truth. 3:10 to Yuma shows how morality is about truth- who has power and how it is used, who is willing to look it in the eye and stand up to it. The first step of predators like our current administration is to gaslight the population, making white black and black, white. The president's every utterance is an exercise in projection, blaming others for his own defects, especially moral ones, to excuse his own depravity. Resistance means sticking to the truth. While predators are only possible in a society that is generally truthful and moral, (since a society of predators would have no one to rob or anything to take), they can only exist if that society is blinded to their true nature, or cowed by their power.

The president may have learned his methods of lying in real estate, but his base learned it in religion. When faith and authority are the standard, truth takes a back seat. Victimization narratives run rampant, when religions are merely up against people who have a higher standard of truth than they do. That includes empathy, which is a form of truth- that of recognizing the true existence of others and extending to them the decency / respect of allowing them their own perspectives and feelings. The evangelical base is so aggrieved by the decline of religion and its (rightful) dismissal by liberal, thinking people, that it is clearly willing to throw all other principles to the wind. They have made a corrupt bargain for power, with a man who hasn't a particle of moral, let alone religious, feeling, and whose greed degrades everything he touches. One would have thought that the base had learned a lesson from their support of George W. Bush, who made tax cuts for the rich and foreign debacles in Iraq and Afghanistan his contribution to American history. No, the evangelical base, triggered by the reign of Barack Obama, doubled down, and is fully on the side of the devil now.

3:10 to Yuma does not deserve its happy ending. But the movie is not about the reality of gunmen and shooting angles. It is about character, and the limit reached by good people when faced with evil people. The settlers of the West were hardly unblemished, trampling as they did in the wake of exterminated and displaced Indians. But in the national mythos that I grew up with, they were the quiet and courageous builders who brought law to wild regions and grappled with the many dimensions of building a new society. They overcame corrupt ranchers, shady miners, gunslingers and gangsters to make the placid Midwest and western farming regions we know today. To think that their descendents would so forsake these principles on the altar of tin-pot fascism is truly hard to fathom.

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Tiny Tunings in a Buzz of Neural Activity

Lots of what neurons do adds up to zero: how the cerebellum controls muscle movement.

Our brains are always active. Meditating, relaxing, sleeping ... whatever we are up to, the brain doesn't take a holiday, except in the deepest levels of sleep, when slow waves help the brain to reset and heal itself. Otherwise, it is always going, with only slight variations based on computational needs and transient coalitions, which are extremely difficult to pick out of the background (fMRI signals are typically under 3% of the noise). That leads to the general principle that action in a productive sense is merely a tuned overlay on top of a baseline of constant activity, through most of the brain.

A recent paper discussed how this property manifests in the cerebellum, the "little" brain attached to our big brain, which is a fine-tuning engine especially for motion control, but also many other functions. The cerebellum has relatively simple and massively parallelized circuitry, a bit like a GPU to the neocortex's CPU. It gets inputs from the places like the the spinal cord and sensory areas of the brain, and relays a tuned signal out to, in the case of motor control, the premotor cortex. The current authors focused on the control of eye movement ("saccades") which is a well characterized system and experimentally tractable, in marmoset monkeys. 

After poking a massive array of electrodes into their poor monkey's brains, they recorded from hundreds of cells, including all the relevant neuron types (of which there are only about six). They found that inside the cerebellum, and inside the region they already knew is devoted to eye movement, neurons form small-world groups that interact closely with each other, revealing a new level of organization for this organ.

More significantly, they were able to figure out the central tendency or vector for Purkinje (P) cells they ran across. These are the core cells of the cerebellar circuit, so their firing should correlate in principle with the eye movements that they were simultaneously tracking in these monkeys. So one cell might be an upward directing cell, while another one might be leftward, and so forth. They also found that P cells come in two types- those (bursters) that fire positively around the preferred direction, and others (pausers) that fire all the other times, but fire less when eyes are heading in the "preferred" direction. These properties are already telling us that the neural system does not much care to save energy. Firing most of the time, but then pausing when some preferred direction is hit is perfectly OK. 

Two representative cells are recorded, around the cardinal/radial directions. The first of each pair is recorded from 90 degrees vs its preferred direction, and adding up the two perpendicular vectors (bottom) gives zero net activity. The second of each pair is recorded from the preferred direction (or potent axis), vs 180 degrees away, and when these are subtracted, a net signal is visible (difference). Theta is the direction the eyes are heading.

Their main finding, though, was that there is a lot of vector arithmetic going on, in which cells fire all over their preferred fields, and only when you carefully net their activity over the radial directions can you discern a preferred direction. The figure above shows a couple of cells, firing away no matter which direction the eyes are going. But if you subtract the forward direction from the backward direction, a small net signal is left over, which these authors claim is the real signal from the cerebellum, which signals a change in direction. When this behavior is summed across a large population, (below), the bursters and pausers cancel out, as do the signals going in stray directions. All you are left with is a relatively clean signal centered on the direction being instructed to the eye muscles. Wasteful? Yes. Effective? Apparently. 

The same analysis as above, but on a population basis. Now, in net terms, after all the canceled activity is eliminated, and adding up the pauser and burster activity, strong signals arise from the cerebellum as a whole, in this anatomical region, directing eye saccades

Why does the system work this way? One idea is that, like for the rest of the brain, default activity is the norm, and learning is, perforce, a matter of tuning ongoing activity, not of turning on cells that are otherwise off. The learning (or error) signal is part of the standard cerebellum circuitry- a signal so strong that it temporarily shuts off the normal chatter of the P cell output and retunes it slightly, rendering the subsequent changes seen in the net vector calculation.

A second issue raised by these authors is the nature of inputs to these calculations. In order to know whether and how to change the direction of eye movement, these cells must be getting information about the visual scene and also about the current state of the eyes and direction of movement. 

"The burst-pause pattern in the P cells implied a computation associated with predicting when to stop the saccade. For this to be possible, this region of the cerebellum should receive two kinds of information—a copy of the motor commands in muscle coordinates and a copy of the goal location in visual coordinates."

These inputs come from two types of mossy fiber neurons, which are the single inputs to the typical cerebellar circuit. One "state" type encodes the state of the motor system and current position of the eye. The other "goal" type encodes, only in case of a reward, where the eye "wants" to move, based on other cortical computations, such as the attention system. The two inputs go through separate individual cerebellar circuits, and then get added up on a population basis. When movement is unmotivated, the goal inputs are silent, and resulting cerebellar-directed saccades are more sporadic, scanning the scene haphazardly.

The end result is that we are delving ever more deeply into the details of mental computation. This paper trumpets itself as having revealed "vector calculus" in the cerebellum. However, to me it looks much more like arithmetic than calculus, and nor is the overall finding of small signals among a welter of noise and default activity novel either. All the same, more detail, and deeper technical means, and greater understanding of exactly how all these billions of dumb cells somehow add up to smart activity continues to be a great, and fascinating, quest.

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Lipid Pumps and Fatty Shields- Asymmetry in the Plasma Membrane

The two faces constituting eukaryotic cell membranes are asymmetric.

Membranes are one of those incredibly elegant things in biology. Simple chemicals forces are harnessed to create a stable envelope for the cell, with no need to encode the structure in complicated ways. Rather, it self-assembles, using the oil-vs-water forces of surface tension to form a huge structure with virtually no instruction. Eukaryotes decided to take membranes to the max, growing huge cells with an army of internal membrane-bound organelles, individually managed- each with its own life cycle and purposes.

Yet, there are complexities. How do proteins get into this membrane? How do they orient themselves? Does it need to be buttressed against rough physical insult, with some kind of outer wall? How do nutrients get across, while the internal chemistry is maintained as different from the outside? How does it choose which other cells to interact with, preventing fusion with some, but pursuing fusion with others? For all the simplicity of the basic structure, the early history of life had to come up with a lot of solutions to tough problems, before membrane management became such a snap that eukaryotes became possible.

The authors present their model (in atomic simulation) of a plasma membrane. Constituents are cholesterol, sphingomyelin (SM), phosphatidyl choline (PC) phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), and phosphotidyl ethanolamine plasmalogen. Note how in this portrayal, there is far more cholesterol in the outer leaflet (top), facing the outside world, than there is in the inner leaflet (bottom).

The major constituents of the lipid bilayer are cholesterol, phospholipids, and sphingomyelin. The latter two have charged head groups and long lipid (fatty) tails. The head groups keep that side of the molecule (and the bilayer) facing water. The tails hate water and like to arrange themselves in the facing sheets that make up the inner part of the bilayer. Cholesterol, on the other hand, has only a mildly polar hydroxyl group at one end, and a very hydrophobic, stiff, and flat multi-ring body, which keeps strictly with the lipid tails. The lack of a charged head group means that cholesterol can easily flip between the bilayer leaflets- something that the other molecules with charged headgroups find very difficult. It has long been known that our genomes code for flippases and floppases: ATP-driven enzymes that can flip the charged phospholipids and sphingomyelin from one leaflet to the other. Why these enzymes exist, however, has been a conundrum.

Pumps that drive phospholipids against their natural equilibrium distribution, into one or the other leaflet.

It is not immediately apparent why it would be helpful to give up the natural symmetry and fluidity of the natural bilayer, and invest a lot of energy in keeping the compositions of each leaflet different. But that is the way it is. The outer leaflet of the plasma membrane tends to have more sphingomyelin and cholesterol, and the inner leaflet has more phospholipids. Additionally, those phospholipids tend to have unsaturated tails- that is, they have double bonds that break up the straight fatty tails that are typical in sphingomyelin. Membrane asymmetry has a variety of biological effects, especially when it is missing. Cells that lose their asymmetry are marked for cell suicide, intervention of the immune system, and also trigger coagulation in the blood. It is a signal that they have broken open or died. But these are doubtless later (maybe convenient) organismal consequences of universal membrane asymmetry. They do not explain its origin. 

A recent paper delved into the question of how and why this asymmetry happens, particularly in regard to cholesterol. Whether cholesterol even is asymmetric is controversial in the field, since measuring their location is very difficult. Yet these authors carefully show that, by direct measurement, and also by computer simulation, cholesterol, which makes up roughly forty percent of the membrane (its most significant single constituent, actually), is highly asymmetric in human erythrocyte membranes- about three fold more abundant in the outer leaflet than in the cytoplasmic leaflet. 

Cholesterol migrates to the more saturated leaflet. B shows a simulation where a poly-unstaturated (DAPC) phospholipid with 4 double bonds (blue) is contrasted with a saturated phospholipid (DPPC) with staight lipid tails (orange). In this simulation, cholesterol naturally migrates to the DPPC side as more DAPC is loaded, relieving the tension (and extra space) on the inner leaflet. Panel D shows that in real cells, scrambling the leaflet composition leads to greater cholesterol migration to the inner leaflet. This is a complex experiment, where the fluorescent signal (on the right-side graph) comes from a dye in an introduced cholesterol analog, which is FRET-quenched by a second dye that the experimenters introduced which is confined to the outer membrane. In the natural case (purple), signal is more quenched, since more cholesterol is in the outer leaflet, while after phospholipid scrambling, less quenching of the cholesterol signal is seen. Scrambling is verified (left side) by fluorescently marking the erythrocytes for Annexin 5, which binds to phosphatidylcholine, which is generally restricted to the inner leaflet. 

But no cholesterol flippase is known. Indeed, such a thing would be futile, since cholesterol equilibrates between the leaflets so rapidly. (The rate is estimated at milliseconds, in very rough terms.) So what is going on? These authors argue via experiment and chemical simulation that it is the pumped phospholipids that drive the other asymmetries. It is the straight lipid tails of sphingomyelin that attract the cholesterol, as a much more congenial environment than the broken/bent tails of the other phospholipids that are concentrated in the cytoplasmic leaflet. In turn, the cholesterol also facilitates the extreme phospholipid asymmetry. The authors show that without the extra cholesterol in the outer leaflet, bilayers of that extreme phospholipid composition break down into lipid globs.

When treated (time course) with a chemical that scrambles the plasma membrane leaflet lipid compositions, a test protein (top series) that normally (0 minutes) attaches to the inner leaflet floats off and distributes all over the cell. The bottom series shows binding of a protein (from outside these cells) that only binds phosphatidylcholine, showing that scrambling is taking place.

This sets up the major compositional asymmetry between the leaflets that creates marked differences in their properties. For example, the outer leaflet, due to the straight sphingomyelin tails and the cholesterol, is much stiffer, and packed much tighter, than the cytoplasmic leaflet. It forms a kind of shield against the outside world, which goes some way to explain the whole phenomenon. It is also almost twice as impermeable to water. Conversely, the cytoplasmic leaflet is more loosely packed, and indeed frequently suffers gaps (or defects) in its lipid integrity. This has significant consequences because many cellular proteins, especially those involved in signaling from the surface into the rest of the cytoplasm, have small lipid tails or similar anchors that direct them (temporarily) to the plasma membrane. The authors show that such proteins localize to the inner leaflet precisely because that leaflet has this loose, accepting structure, and are bound less well if the leaflets are scrambled / homogenized.

When the fluid mosaic model of biological membranes was first conceived, it didn't enter into anyone's head that the two leaflets could be so different, or that cells would have an interest in making them so. Sure, proteins in those membranes are rigorously oriented, so that they point in the right direction. But the lipids themselves? What for? Well, they do and now there are some glimmerings of reasons why. Whether this has implications for human disease and health is unknown, but just as a matter of understanding biology, it is deeply interesting.

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Man is Wolf to Man

The current administration's predatory and corrupt version of capitalism.

What is corruption? Isn't capitalism all about getting as much money as you can? Then doesn't it follow that there can be no such thing as corruption, which is defined as going against the rules? What rules?

We as a country go on a trip with every new president, learning about their nature and values as we accompany them through their brief span of history. Few presidents wear very well after their honeymoon, since the process of getting elected requires some shading of the truth, truth that inevitably comes out later on. The current administration is an odd example, since in his first term, Trump was not allowed (for very good reasons!) to be himself. The second time round has been a different story, and we are getting a deep look at his character. 

The US has always had a double relationship with capitalism, tilting between rampant competition / exploitation and reverence for rules and legal systems. Slavery, obviously, is the foremost example, with slaveholders enshrining in a document dedicated to human freedom their own legal rights to property in comprehensively oppressed people. The founders, on making their constitution, feverishly set to work creating institutions for the common good, such as the treasury, mail system, judicial system, patents, and military. But, at the same time, we have long had an ideology of free enterprise- of land, resource, and human exploitation almost without limit. 

Charles Ponzi was not working in Italy, after all, but in the US, as was Bernie Madoff. Now crypto is the popular mechanism of picking people's pockets, facilitating mundane crime such as money laundering and ransomware attacks at the same time that it provides flourishing vistas of direct fraud, in rug pulls, hacks, and market manipulation. A recent article reviewed the pathetic world of multilevel marketing, another model of predation where ambitious entrepreneurs are sucked into schemes that are engineered both to fail, and to induce the victims to blame themselves.

The administration has clearly made it its mission to celebrate these forms of business- the predators, the grifters, the destructive businessmen among us who think that taxes are for little people, and rules for someone else. Consumer protection agencies have been shuttered, the IRS eviscerated, investigations cancelled. Pardons have been going out, not only to the January 6th conspirators and their militias, but to the money launderers, the corrupt politicians, and crypto bros. It is a sustained campaign of norm and rule-breaking by a grossly tasteless, shockingly greedy and small-minded president, (and sexual predator), who cannot conceive of rational policy, uncorrupted institutions, or fairness, much less civility, as a principle. A person with deep psychological problems. And thus, is incapable of long-term policy that is the bedrock of durable, functional institutions, either commercial or governmental.

Following gold, like a cat following a laser pointer.

We are all worried about fascism, as that seems to be the aesthetic and the model of power the administration is tending towards. But what they have done so far doesn't even come up to the level of fascism, really. The president is not smart enough to have a coherent policy or ideological platform. The weave does not leave room for a program that would be attractive beyond the nihilistic base. There are inclinations, and moods, and tantrums, love for Putin, and a lot of nostalgia for policies of decades, if not centuries, ago. There is hate. But without a program that binds all these ingredients into even a marginally coherent approach to the future, it will inevitably fall apart. True believers don't do policies or reason- conspiracy theories are enough. Thinking, apparently, is for libtards. 

The fact of the matter is that capitalism is not equivalent to the law of the jungle. A legal system, and rules, are required to prevent capitalists from making military forays into each other's empires, and to prevent the workers from taking up their pitchforks, among much else. It is founded on the limited liability company, itself a legal construct, not to mention all the financial, educational, and physical infrastructure that forms its essential background. There is no going Galt here. Indeed, the whole point of captialism is not to screw everyone and make a few people very rich. Rather, it is to diffuse labor, useful products and productive technologies across society in a way that utilizes everyone's talents and supplies everyone's needs.  The point is general prosperity, not inequality. 

Institutions are built on rules, and they can die two ways- either people disregard and lose faith in the rules, (maybe because they are made by corrupt processes), or the rules become so elaborate and sclerotic that the point of the institution is lost. These ways map roughly onto our political divide, which, when it compromises to the middle produces something akin to a functional mean. But the current administration, and its ideology of thorough-going corruption on personal, business, and governmental levels, is, with the connivance of an equally unhinged supreme court, creating a legacy of cruelty and destruction that is surely a sad way to mark next year's anniversary of our institutional founding.

• How the wingnut evangelicals, rightist Catholics, and their funders have bought into the burn-it-all-down program of predation, with a little help from the Russians.
• Being populist means lying, unfortunately.
• We can and should give real help to Ukraine. How about blockading Russian rather than Venezuelan tankers?
• A hiring hellscape, with AI battling on both sides.
• Destroying science, at the NIH.

Mutations That Make Us Human

The ongoing quest to make biologic sense of genomic regions that differentiate us from other apes.

Some people are still, at this late date, taken aback by the fact that we are animals, biologically hardly more than cousins to fellow apes like the chimpanzee, and descendants through billions of years of other life forms far more humble. It has taken a lot of suffering and drama to get to where we are today. But what are those specific genetic endowments that make us different from the other apes? That, like much of genetics and genetic variation, is a tough question to answer.

At the DNA level, we are roughly one percent different from chimpanzees. A recent sequencing of great apes provided a gross overview of these differences. There are inversions, and larger changes in junk DNA that can look like bigger differences, but these have little biological importance, and are not counted in the sequence difference. A difference of one percent is really quite large. For a three gigabyte genome, that works out to 30 million differences. That is plenty of room for big things to happen.

Gross alignment of one chromosome between the great apes. [HSA- human, PTR- chimpanzee, PPA- bonobo, GGO- gorilla, PPY- orangutan (Borneo), PAB- orangutan (Sumatra)]. Fully aligned regions (not showing smaller single nucleotide differences) are shown in blue. Large inversions of DNA order are shown in yellow. Other junk DNA gains and losses are shown in red, pink, purple. One large-scale jump of a DNA segment is show in green. One can see that there has been significant rearrangement of genomes along the way, even as most of this chromosome (and others as well) are easly alignable and traceable through the evolutionary tree.

But most of those differences are totally unimportant. Mutations happen all the time, and most have no effect, since most positions (particularly the most variable ones) in our DNA are junk, like transposons, heterochromatin, telomeres, centromeres, introns, intergenic space, etc. Even in protein-coding genes, a third of the positions are "synonymous", with no effect on the coded amino acid, and even when an amino acid is changed, that protein's function is frequently unaffected. The next biggest group of mutations have bad effects, and are selected against. These make up the tragic pool of genetic syndromes and diseases, from mild to severe. Only a tiny proportion of mutations will have been beneficial at any point in this story. But those mutations have tremendous power. They can drag along their local DNA regions as they are positively selected, and gain "fixation" in the genome, which is to say, they are sufficiently beneficial to their hosts that they outcompete all others, with the ultimate result that mutation becomes universal in the population- the new standard. This process happens in parallel, across all positions of the genome, all at the same time. So a process that seems painfully slow can actually add up to quite a bit of change over evolutionary time, as we see.

So the hunt was on to find "human accelerated regions" (HAR), which are parts of our genome that were conserved in other apes, but suddenly changed on the way to humans. There roughly three thousand such regions, but figuring out what they might be doing is quite difficult, and there is a long tail from strong to weak effects. There are two general rationales for their occurrence. First, selection was lost over a genomic region, if that function became unimportant. That would allow faster mutation and divergence from the progenitors. Or second, some novel beneficial mutation happened there, bringing it under positive selection and to fixation. Some recent work found, interestingly, that clusters of mutations in HAR segments often have countervailing effects, with one major mutation causing one change, and a few other mutations (vs the ancestral sequence) causing opposite changes, in a process hypothesized to amount to evolutionary fine tuning. 

A second property of HARs is that they are overwhelmingly not in coding regions of the genome, but in regulatory areas. They constitute fine tuning adjustments of timing and amount of gene regulation, not so much changes in the proteins produced. That is, our evolution was more about subtle changes in management of processes than of the processes themselves. A recent paper delved in detail into HAR5, one of the strongest such regions, (that is, strongest prior conservation, compared with changes in human sequence), which lies in the regulatory regions upstream of Frizzled8 (FZD8). FZD8 is a cell surface receptor, which receives signals from a class of signaling molecules called WNT (wingless and int). These molecules were originally discovered in flies, where they signal body development programs, allowing cells to know where they are and when they are in the developmental program, in relation to cells next door, and then to grow or migrate as needed. They have central roles in embryonic development, in organ development, and also in cancer, where their function is misused.

For our story, the WNT/FZD8 circuit is important in fetal brain development. Our brains undergo massive cell division and migration during fetal development, and clearly this is one of the most momentous and interesting differences between ourselves and all other animals. The current authors made mutations in mice that reproduce some of the HAR5 sequences, and investigated their effects. 

Two mouse brains at three months of age, one with the human version of the HAR5 region. Hard to see here, but the latter brain is ~7% bigger.

The authors claim that these brains, one with native mouse sequence, and the other with the human sequences from HAR5, have about a seven percent difference in mass. Thus the HAR5 region, all by itself, explains about one fourteenth of the gross difference in brain size between us and chimpanzees. 

HAR5 is a 619 base-pair region with only four sequence differences between ourselves and chimpanzees. It lies 300,000 bases upstream of FZD8, in a vast region of over a million base pairs with no genes. While this region contains many regulatory elements, (generally called enhancers or enhancer modules, only some of which are mapped), it is at the same time an example of junk DNA, where most of the individual positions in this vast sea of DNA are likely of little significance. The multifarious regulation by all these modules is of course important because this receptor participates in so many different developmental programs, and has doubtless been fine-tuned over the millennia not just for brain development, but for every location and time point where it is needed.

Location of the FZD8 gene, in the standard view of the genome at NIH. I have added an arrow that points to the tiny (in relative terms) FZD8 coding region (green), and a star at the location of HAR5, far upstream among a multitude of enhancer sequences. One can see that this upstream region is a vast area (of roughly 1.5 million bases) with no other genes in sight, providing space for extremely complicated and detailed regulation, little of which is as yet characterized.

Diving into the HAR5 functions in more detail, the authors show that it directly increases FZD8 gene expression, (about 2 fold, in very rough terms), while deleting the region from mice strongly decreases expression in mice. Of the four individual base changes in the HAR5 region, two have strong (additive) effects increasing FZD8 expression, while the other two have weaker, but still activating, effects. Thus, no compensatory regulation here.. it is full speed ahead at HAR5 for bigger brain size. Additionally, a variant in human populations that is responsible for autism spectrum disorders also resides in this region, and the authors show that this change decreases FZD8 expression about 20%. Small numbers, sure, but for a process that directs cell division over many cycles in early brain development, this kind of difference can have profound effects.

The HAR5 region causes increased transcription of FZD8, in mice, compared to the native version and a deletion.

The HAR5 region causes increased cell proliferation in embryonic day 14.5 brain areas, stained for neural markers.

"This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. ... Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development."

So there you have it. The nuts and bolts of evolution, from the molecular to the cellular, the organ, and then the organismal, levels. Humans do not just have bigger brains, but better brains, and countless other subtle differences all over the body. Each of these is directed by genetic differences, as the combined inheritance of the last six million years since our divergence versus chimpanzees. Only with the modern molecular tools can we see Darwin's vision come into concrete focus, as particular, even quantum, changes in the code, and thus biology, of humanity. There is a great deal left to decipher, but the answers are all in there, waiting.

• Who's the poodle?
• We are going totally tin-pot.

Fifty Trillion Dollars Are Missing!

Bernie Sanders says that the last fifty years have seen a massive wealth transfer... to the rich.

Bernie Sanders has a new hook for his discussion of inequality. The Rand (AKA Bland) corporation has a study out that shows that the share of income going to workers over the last fifty years has declined to the point that, cumulatively, fifty to eighty trillion dollars have gone missing. Well, they have not gone to workers, but rather to non-workers: capitalists, shareholders, parasites; generally the 1%. 

The basic mechanism here is that capitalism is built on top of a labor market, where workers are paid only what the competition among them renders suitable to sort them into needed jobs. It is not built to fairly share the gains from their work or anyone else's work. In fact, it is built to skim off all the gains / profits and send them to shareholders, owners, managers ... anyone who has power in the capitalist system, which the workers surely do not. Workers get other rewards, but a share of the profits is not one of them. They are regarded as a necessary expense, like feedstocks and machinery, and their cost is to be minimized assiduously, now with robots and AI where possible. I learned this in dramatic fashion when a company I worked for, where the CEO chummily addressed us a team members and collaborators in the grand adventure of bioinformatics, sold the company and took all the winnings for himself.

With that in mind, it becomes obvious where the 50 trillion has gone to. It has been shared out among those who have real power in the capitalist system. First comes management. Managers hold the keys to the finances and the profits. They tell the board what is going on. They have been theoretically upgraded over the last seventy years from employees to entrepreneurs, and have taken shockingly increased amounts from the till during that time. Second come the shareholders, who have been theoretically upgraded from stakeholders in the corporate enterprise to the sine qua non of the corporation- its very soul and purpose, not as a "company" of people, or or a part of a wider culture, or a service rendering organization, but as a money gathering entity. Money that is raked up from whatever the "business model" might be into the pockets of shareholders, with the management as a necessary, if unfortunate, intermediary, and employees as an afterthought.

Ownership is the medium of power in capitalism. The shareholders are part-owners, and their interests are built into the stock market system, which is a perpetual readout of the value of each share. Analysts are constantly sifting through the value of each enterprise in terms of current assets and future potential. Corporations buy each other based on these valuations, and bankruptcy awaits those who lose control of their business model and fail to send money back to the shareholders. What the company produces, or its quality of work life, are completely irrelevant to its value. Whatever money is made over costs is shipped out to shareholders, in the form of dividends, share buybacks, or re-investment towards future growth and greater value. Managers have some power in this system, and have been able to capture quite a bit of the winnings, but workers have essentially none.

Labor markets tend to settle on what employees need to get by, rather than on the worth of what they produce. No compensation review or salary offer makes any reference to productivity or worth of the product. They are always keyed to what the market will bear.. the job market, that is. And job applicants are always in a weak position, since unemployment is such a catastrophic prospect. The choice at most interviews, especially the crucial first one out of school, is some pay or no pay. Employers work very hard to avoid competing for labor, with all kinds of illicit agreements among each other, and a carefully cleansed information landscape where workers don't know what others are being paid. Especially at the low end, this leads to workers competing for crappy jobs that pay little, because the there is always someone who is unemployed who, in light of their dire plight, can make do with even less. 

So, as the US economy has grown, growing things, making things, and gaining productivity year over year, little of that has trickled down to workers. Look at people's lives on the lower end of the economic ladder, and conditions have not visible improved. Indeed, homelessness and hunger are increasingly, instead of decreasingly, common. The middle classes have some increment of technology, like computers, smart phones, and streaming, but the living standard otherwise has not advanced noticeably, even while population pressures increase and the natural world has degraded. This arises simply from the structure of capitalism, when it is taken seriously and grows into the kind of untouchable gestalt it has become over the recent decades. 

And it is obvious that this is bad. Bernie Sanders is completely correct to point out that Americans across the spectrum could be much better off if the 1% got less of the income and wealth, and the rest of the citizenry got more. Inequality is obviously corrosive socially and politically, quite aside from the misery it causes at the lower end of the economic ladder. The capitalist system as we practice it is a relic of feudalism, when capital was scarce, sword and blood trumped merit, and serfs knew their place.

There are many ways to approach this problem, which is incredibly systemic and entrenched. Obviously we need to start with a bit of a cultural reboot that reconceives corporations as cultural entities with important roles and stakeholders that go beyond just making a dollar. We need a minimum wage that pays not just subsistance, but a normal, civilized living. Unions are another, if quite fraught, way for workers to retake power in the capitalist ecosystem. We need a tax system that values labor income over passive, unproductive income, and which taxes accumulated wealth as well. Employees should have a seat at the table in all companies- the corporate board table. We need serious regulation and enforcement that levels the playing field for companies that play by the rules, and sets rules that not only sustain, but build, society and the environment over the long haul.

Another idea that is quietly gaining revolutionary steam is employee ownership. There is an excellent book that discusses this, as it is currently practiced by hundreds of companies in the US. One example is the Publix supermarket chain, which has been wholly employee owned through five decades of growth, and is by far the largest such company. While the ESOP (employee stock ownership plan) model may start with a company stock buying plan, the serious work comes with majority ownership by employees. There are about 14 million employees in the US with some kind of company stock ownership plan, and 650,000 who majority-own their companies. This is usually arranged by a loan-financed buyout from a founder or other owners. Then the proceeds of the business pay off the loan as the employees accumulate distributed stock. This mode of buyout is an excellent way to transition from the early entrepreneurial phase of a company to a mature business, as it keeps the company culture intact and avoids the many problems of private equity, which may debt-strip a target company and leave it in bankruptcy, or of public stock ownership, which leads to disinterested owners that demand short-term financial performance over long-term health. 

This book is a bit thin on how such companies are managed and how the stock allocations and valuations are made. The stock is partially restricted against outside sale, (though some ESOP companies are publicly traded in part), so companies typically engage experts to value themselves each year. Employees gain stock with time, so it becomes a seniority system, a bit like waiting for a pension or union seniority. That is again not entirely fair to employees who may contribute more despite being young. At any rate, these companies still have professional management, but since it is all employees on the board, and with open financials, there is a track record of fewer layoffs, better morale, more cooperation, and ultimately, better financial performance. And critically, profits are not dispersed to faceless and uninterested "investors", but to the employees.

Imagine if every mature company was not buffeted by stock market analysts and fads, not bled dry by stock buy-backs to fund rich investors, nor haunted by the specter of a private equity strip-and-dump, but was owned wholly by its own employees, who reaped the profits of their collective work and controlled its conditions as well. As increasing numbers of companies become employee-owned, one can imagine a phase transition (analogous to the universality of certain benefits like health insurance) where people just would not want to work anymore for other kinds of companies. Small and startup companies would have to share equity, (as they used to do in Silicon Valley), and would naturally progress as they grew to broad ownership by every employee. That would be amazing! 

• Predation, chaos, and crime are the policy.
• Unitary, schmoonitary.
• Half an hour on how terrible medicare advantage is.
• A new way to heat, together.

Students Deserve Mentors

The best form of education is personal mentoring. More of our educational and work system should get back to that model.

We learned some important things from the Covid pandemic. One is that fiscal stimulus really works. Another is that mRNA vaccines are highly effective, and their rapid and flexible development cycle makes them a superior platform for future vaccines. And another is that social interaction is deeply important, especially for young people. We all got used to Zoom, but for school children, that was a poor substitute, when it was even possible. Children were left significantly behind both academically and socially.

A recent segment on the PBS NewsHour touched on this in a discussion of adolescent development. Its message was that learning requires challenging opportunities and human relationships. Adolescents are going on a heroic quest to become adults. They thrive on active engagement with the world and need models of successful adulthood to learn from. How to provide these key functions in an optimal way? We know how to do this- by apprenticeship and mentorship. This model has been understood forever, from the schools of Athens to the medieval trade guilds to the graduate schools of contemporary academia. My grandfather was a baker in Germany, and in his turn trained many apprentices and journeymen to be bakers. I went to graduate school, which turned out to be a glorified apprenticeship under a renowned researcher, then went on to a journeyman position (aka post-doc) with another mentor. This model is an education in many dimensions- the technical ingredients of a craft, the management practices that make a successful organization, how to participate in a larger community that pursues socially important goals, and the discipline and moral integrity it takes to be a competent adult, capable of leadership.

Example of a certificate of attainment of mastery, 1927, for a bricklayer, attested by his mentors and examiners.

However, as a society, we are reluctant to make these kinds of investments in children and adolescents. Efficiency demands that class sizes be large, colleges impersonal, and money squeezed out of the system. Companies clamor for fully trained job candidates, expecting students to go into debt in trade schools before being hired into a paying job. Few young people get the kind of lengthy, personal training that they would most benefit from. Mentorship becomes a hazard of chance, if a boss in an early job takes an interest, or a teacher decides to make extra time.

Principally, I fault the corporate system, which has sloughed off its civic responsibilities to train people and propagate cultural knowledge. The economy is full of interesting and important jobs representing exquisite technical knowledge and other expertise. As a culture and economy, we are not going to maintain a high standard if we keep losing these skills and knowledge with every generation. Just look what has happened to the industries we have ceded to China. Innovation hubs like Silicon Valley are successful in part because training becomes a shared enterprise. New companies benefit from a large pool of experienced workers, who can switch between organizations with ease. No individual company carries the whole burden of training, but as companies become larger and more specialized, they have to take on the costs of training a larger proportion of their incoming employees. Yet they still benefit from the cross-fertilization of being in a highly skilled employment ecosystem.

To better serve young people, we need to make integration into corporate skills training more accessible and normal. The idea that students should be battling for unpaid internships is absurd and insulting- all internships should be paid, and they should be longer as well. The German trades system is an example, where companies and government cooperate in providing training to young people. The companies get a much better familiarity with future hires, who are also better trained. Many trades/sectors have a communal "training tax", which all companies pay, and which funds salaries to trainees and other training costs. This is one accomplishment of the union system in Germany, which is much stronger and better integrated into their industries than that in the US.

This model could be made more general in the US as a federal program, crossing all organizations in the public and private sector, funding internships and training for more students than is now done, setting up a more lengthy and regular apprenticeship system. The training/salary costs would grade over the first few years of employment from tax-supported to company-supported. Lowering the burden of a young first hire, both in financial terms and terms of knowing the candidates better, should encourage more hiring and more training by employers. 

Companies are often citadels of hermetic wisdom, when they are not going off the rails as predatory enterprises. Integrating more young people and an additional purpose of training into US corporate culture would counteract both of these problems, while helping the youth and preserving / propagating cultural knowledge more effectively.

• Mentoring, Shanghai style.
• Surrender Dorothy!
• Mini-me and the future of Ukraine.
• Free buses work.
• Crypto, on the other hand, remains a criminal enterprise.
• Biology is not so easy. Really.

Ground Truth for Genetic Mutations

Saturation mutagenasis shows that our estimates of the functional effect of uncharacterized mutations are not so great.

Human genomes can now be sequenced for less than $1,000. This technological revolution has enabled a large expansion of genetic testing, used for cancer tissue diagnosis and tracking, and for genetic syndrome analysis both of embryos before birth and affected people after birth. But just because a base among the 3 billion of the genome is different from the "reference" genome, that does not mean it is bad. Judging whether a variant (the modern, more neutral term for mutation) is bad takes a lot of educated guesswork.

A recent paper described a deep dive into one gene, where the authors created and characterized the functional consequence of every possible coding variant. Then they evaluated how well our current rules of thumb and prediction programs for variant analysis compare with what they found. It was a mediocre performance. The gene is CDKN2A, one of our more curious oddities. This is an important tumor suppressor gene that inhibits cell cycle progression and promotes DNA repair- it is often mutated in cancers. But it encodes not one, but two entirely different proteins, by virtue of a complex mRNA splicing pattern that uses distinct exons in some coding portions, and parts of one sequence in two different frames, to encode these two proteins, called p16 and p14. 

One gene, two proteins. CDKN2A has a splicing pattern (mRNA exons shown as boxes at top, with pink segments leading to the p14 product, and the blue segments leading the p16 product) that generates two entirely different proteins from one gene. Each product has tumor suppressing effects, though via distinct mechanisms.

Regardless of the complex splicing and protein coding characteristics, the authors generated all possible variants in every possible coded amino acid (156 amino acids in all, as both produced proteins are relatively short). Since the primary roles of these proteins are in cell cycle and proliferation control, it was possible to assay function by their effect when expressed in cultured pancreatic cells. A deleterious effect on the protein was revealed as, paradoxically, increased growth of these cells. They found that about 600 of the 3,000 different variants in their catalog had such an effect, or 20%.

This is an expected rate of effect, on the whole. Most positions in proteins are not that important, and can be substituted by several similar amino acids. For a typical enzyme, for instance, the active site may be made up of a few amino acids in a particular orientation, and the rest of the protein is there to fold into the required shape to form that active site. Similar folding can be facilitated by numerous amino acids at most positions, as has been richly documented in evolutionary studies of closely-related proteins. These p16 and p14 proteins interact with a few partners, so they need to maintain those key interfacial surfaces to be fully functional. Additionally, the assay these researchers ran, of a few generations of growth, is far less sensitive than a long-term true evolutionary setting, which can sift out very small effects on a protein, so they were setting a relatively high bar for seeing a deleterious effect. They did a selective replication of their own study, and found a reproducibility rate of about 80%, which is not great, frankly.

"Of variants identified in patients with cancer and previously reported to be functionally deleterious in published literature and/or reported in ClinVar as pathogenic or likely pathogenic (benchmark pathogenic variants), 27 of 32 (84.4%) were functionally deleterious in our assay"

"Of 156 synonymous variants and six missense variants previously reported to be functionally neutral in published literature and/or reported in ClinVar as benign or likely benign (benchmark benign variants), all were characterized as functionally neutral in our assay "

"Of 31 VUSs previously reported to be functionally deleterious, 28 (90.3%) were functionally deleterious and 3 (9.7%) were of indeterminate function in our assay."

"Similarly, of 18 VUSs previously reported to be functionally neutral, 16 (88.9%) were functionally neutral and 2 (11.1%) were of indeterminate function in our assay"

Here we get to the key issues. Variants are generally classified as benign, pathogenic/deleterious, or "variant of unknown/uncertain significance". The latter are particularly vexing to clinical geneticists. The whole point of sequencing a patient's tumor or genomic DNA is to find causal variants that can illuminate their condition, and possibly direct treatment. Seeing lots of "VUS" in the report leaves everyone in the dark. The authors pulled in all the common prediction programs that are officially sanctioned by the ACMG- Americal College of Medical Genetics, which is the foremost guide to clinical genetics, including the functional prediction of otherwise uncharacterized sequence variants. There are seven such programs, including one driven by AI, AlphaMissense that is related to the Nobel prize-winning AlphaFold. 

These programs strain to classify uncharacterized mutations as "likely pathogenic", "likely benign", or, if unable to make a conclusion, VUS/indeterminate. They rely on many kinds of data, like amino acid similarity, protein structure, evolutionary conservation, and known effects in proteins of related structure. They can be extensively validated against known mutations, and against new experimental work as it comes out, so we have a pretty good idea of how they perform. Thus they are trusted to some extent to provide clinical judgements, in the absence of better data. 

Each of seven programs (on bottom) gives estimations of variant effect over the same pool of mutations generated in this paper. This was a weird way to present simple data, but each bar contains the functional results the authors developed in their own data (numbers at the bottom, in parentheses, vertical). The bars were then colored with the rate of deleterious (black) vs benign (white) prediction from the program. The ideal case would be total black for the first bar in each set of three (deleterious) and total white in the third bar in each set (benign). The overall lineup/accuracy of all program predictions vs the author data was then overlaid by a red bar (right axis). The PrimateAI program was specially derived from comparison of homologous genes from primates only, yielding a high-quality dataset about the importance of each coded amino acid. However, it only gave estimates for 906 out of the whole set of 2964 variants. On the other hand, cruder programs like PolyPhen-2 gave less than 40% accuracy, which is quite disappointing for clinical use.

As shown above, the algorithms gave highly variable results, from under 40% accurate to over 80%. It is pretty clear that some of the lesser programs should be phased out. Of programs that fielded all the variants, the best were AlphaMissense and VEST, which each achieved about 70% accuracy. This is still not great. The issue is that, if a whole genome sequence is run for a patient with an obscure disease or syndrome, and variants vs the reference sequence are seen in several hundred genes, then a gene like CDKN2A could easily be pulled into the list of pathogenic (and possibly causal) variants, or be left out, on very shaky evidence. That is why even small increments in accuracy are critically important in this field. Genetic testing is a classic needle-in-a-haystack problem- a quest to find the one mutation (out of millions) that is driving a patient's cancer, or a child's inherited syndrome.

Still outstanding is the issue of non-coding variants. Genes are not just affected by mutations in their protein coding regions (indeed many important genes do not code for proteins at all), but by regulatory regions nearby and far. This is a huge area of mutation effects that are not really algorithmically accessible yet. As a prediction problem, it is far more difficult than predicting effects on a coded protein. It will requiring modeling of the entire gene expression apparatus, much of which remains shrouded in mystery.

• Oh, no! Not the shopping cart!
• Jevons and Malthus will have their say, if we do not get our act together.
• It is time to scrap GDP, and the mania for growth.
• Blaming the victim.
• Everything crypto will be just perfect.
• Can I play piano with my mind?
• We are killers.