Millions of Years Go by in a Day

In vitro evolution has interesting things to say about protein structure, evolution, and even AI.

The advent of DNA sequences has been revolutionary in many ways. It has been technologically transformative, is changing medical practice, has radically validated Darwin's theories of evolution, and has allowed much more accurate phylogenies to be drawn out of the history of life. As Dobzhansky said, nothing in biology makes sense except in the light of evolution. The quest to change those DNA sequences has been another technological frontier, now exemplified by the CRISPR genome editing methods. Geneticists have been inducing mutations forever, (well, for over a century), using insults like mustard gas and X-rays. This long-standing tradition is called a "screen", where, after mutagenesis, one looks for particular effects on the resulting organisms, like changes in color, malformations, defects in development. This is a sort of artificial selection, very highly directed by the experimenter, sometimes resulting in some very weird, if informative, organisms. More recently, biotechnologists have been using directed evolution systems to help develop, through a mix of random and semi-directed mutations, more capable enzymes and other proteins.

But there are many broader questions to ask about the mutational and evolutionary processes. A recent paper demonstrated an interesting mutagenic system hosted in brewer's yeast cells, which can model rapid evolution under a variety of selective constraints. The core of the system is a plasmid, replicated separately from the main genome, by an independent enzyme. This plasmid was found in a distantly related yeast, Kluyveromyces lactis, and encodes its own DNA polymerase that operates independently from the genomic replication system. This opened the way to use the plasmid replication system to host genes of interest and subject them to wildly different (which is to say faster) mutagenic rates than the rest of the organism.

This group has been laboring on this system for several years, and this paper is the culmination, developing a series of plasmid DNA polymerases that have extremely high error rates, while also having high replication activity, and also having a balanced spectrum of error types (that is, G>A as well as G>T, etc.). Indeed, they demonstrate that the error rate (of about 2 errors for every 10,000 bases replicated) is at the threshold of mutational breakdown- the level that is so high that the plasmid's other functions (which are maintained implicitly by purifying selection on activities such as expression of an antibiotic resistance gene/protein and the polymerase itself) are so rapidly impaired that the engineered system can not survive. The error rate of the host cell, in contrast, is about 1 error for every ten billion bases replicated.

What is the point of all this? While, as pointed out above, directed evolution systems and mutation/selection systems have been around for a long time, this is something quite different. This plasmid system creates high rates of mutation all the time, over a very confined target (the plasmid). The experimenters can then decide what kinds of selection pressure to put on their target gene, if any. They can place a positive selection regime on it, to drive the development of, say, a new substrate specificity for an enzyme. They can put it under negative (purifying) selection to maintain its current activity. Or they can let it spin with no selection at all, letting it degrade into a pseudogene unable to code for anything. All of these scenarios are common in nature and of interest to evolutionary biologists.

In this paper, the authors focus on one enzyme, tryptophan synthase, from a thermophilic bacterium. The aim was to see how this enzyme responded to both positive and negative selective forces in the face of high mutation rates. As it converts one nutrient, indole, into another, tryptophan, this is an enzyme whose activity is easy to assay for and to select for. In the main experiment, using many replicate cultures, they started with no selection for fifty generations, then ramped gradually to positive selection over the next hundred generations, and finished with 300 generations of purifying selection. 

Diversity, for one thing, had increased tremendously by the end of this process. At the end, an average of 21 amino acid changes had accumulated, with the most divergent proteins differing by over 60 amino acids, in a protein that started with 398 amino acids total. Secondly, there was a marked migration to net negative charge, which they speculate was due to accommodation of this thermophilic bacterial enzyme to a more temperate environment where it is a bit more difficult to evade agglomeration with other proteins. Third, changes happened more on the outside of the enzyme structure than the interior (image below). This is a very well-known and understood phenomenon, where selective constraints are much higher on interior packing of a protein and on active/catalytic site portions. Several key amino acids that contact the substrate chemicals are colored gray, meaning that they hardly varied at all in this experiment.

Structure of the TrpB enzyme, color coded for change during the evolution experiment. Note how particularly high rates of change happen in one external region (bottom) that interactions with a partner TrpA, which was not present here. Also, gray areas with very low change tend to be in the interior and near the catalytic active site (substrate and cofactor [pyridoxal phosphate] shown in black).

Overall, the rates of mutagenesis created here over a few months in one protein approximate the kind of divergence seen between proteins of humans and mice, which have diverged for about sixty million years. The same studies one can do on such naturally diverged proteins, such as locating selectively important amino acid residues, or comparing activities of highly divergent enzymes, or studying structural constraints, one can do here on artificially evolved enzymes. And this is a general system that could be (with appropriate assays and technology) extended to many other proteins and RNAs of interest. 

One thing it can't do, however, is validate machine learning models. The researchers tried to get machine learning models that had been trained on this TrpB enzyme to classify their derived mutants. But this was almost completely unsuccessful, since machine learning (AI) systems only regurgitate what they are trained on, and can not creatively judge novel conditions.

"Although sequences that were predicted to have low fitness did exhibit little or no function in our enrichment assay, we found essentially no correlation between the predicted scores and the real enrichment scores of high-function TrpBs. For example, the highest predicted score was assigned to the nearly nonfunctional TmTriple variant."

It is important to appreciate the significance of this new mutation system, which is far more comprehensive, and a closer model of actual evolution, than are the genetic screens of yore. There, one was hunting for  the "hopeful monster" resulting from one shot of X-rays, that might generate an informative phenotype- maybe by killing a gene needed for red eye color, or amplifying expression of a gene for drug resistance. Here, the levels of negative and positive selection can be subtly adjusted in a background of continuous high mutation pressure simulating millions of years of evolution, and resulting in extensively transformed target molecules. 

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Restraint in Foreign Policy

The restraint school of foreign policy wants the US to do less, and spend less, in foreign affairs.

A significant minority of the foreign policy establishment is trying to turn the tables on half a century of global expansion. Calling for restraint and retrenchment, (though shying away from "retreat"), they argue that we are spending too much and are overextended. What made sense in the hot and cold wars of the last century make less sense now, and indeed generates resentment and antagonism. A recent book by Peter Harris tries to make this case, though it has several defects. First, it uses a lot of loaded language like garrison and occupation, where our overseas bases do not function this way at all. Second, he does not really spend much time actually making the case for restraint, but assumes its logic and spends most of the book whining about why no one- not the foreign policy establishment, not the military-industrial complex, not the US congress, and not even the voters(!) are on board with this new and exciting movement in foreign policy. In despair, Harris veers off into domestic policy, the virtues of ranked choice voting, women's empowerment, and multi-party democracy as the golden keys enabling restraint in foreign policy to finally, some day in the future, to get a proper hearing.

The weird thing is how this community has chosen to frame its movement. Doing less, letting China run things for a change... it is not at all clear why retreat, restraint, and retrenchment would be either attractive or wise policy. We need to take a big step back and consider why we have foreign policy at all. Any nation tries to gain and keep as much power as it can. It tries to shape the international landscape in its interests, hopefully in the most far-seeing way possible. Those are the touchstones of any foreign policy. Claiming to want less power and less reach in the world is simply an intuitive non-starter. The US ended World War 2 as the most powerful nation and remains that at least up to the current administration, in all significant metrics- soft power, military power, and economic power. We need to nurture and preserve these powers for our own sake, and also for that of the system which we are the general sponsors of. As Harris points out, the international institutions that we founded after World War 2 were wonderful, but not very powerful. They were not up to the task of serious policing, and the US took on that role, as the global policeman. With a highly intermittent, sometimes irresponsible, and generally very light touch, we have been the only ones who can knock heads with anyone, any time, while also promoting stability, trade, and the expansion of democratic systems. This environment that we have shaped has been beneficial, for us and for many others around the world. The axis arrayed against us today is significant, but not very large, composed mostly of Russia, China, Iran, and North Korea, plus a few others like Afghanistan, maybe some of the central Asian nations.

An important additional principle of foreign relations is that there are many audiences involved. Other leaders are far from our only target when we show who we are by how we speak and use our power abroad. We seek to promote human rights and democracy to all people, everywhere. We seek to deter aggression from any number of entities, including terrorist organizations of all sizes up to states. The landscape is very complex, so we need to have many tools, and balance those tools carefully. This leads to a totally different framing of the restraint theme in foreign policy.

Take a look at the following diagram. This is a map of the military bases that we have all over the world. Better than all the platitudes those in favor of restraint put out, this one image speaks volumes about how distended one aspect of our foreign policy has become.

This begs belief. At a time when we have technological reach to anywhere and at any time, we have carved out little islands of America in eighty countries. We have over a hundred bases each in Germany and Japan. Maybe in the decade after World War 2 this might have made a little sense. But now, I cannot imagine the point of this gargantuan footprint. There are about 24 bases in sub-Saharan Africa. It is, frankly, unbelievable. None of these are zones of occupation, in the sense that we rule the country they are in. None of them, outside of perhaps South Korea, are garrisons, in the classic sense of guarding that location from harm, particularly from the natives. Our bases are all established on a cooperative basis, in what appears to be a mania for military relations with other countries, to facilitate training, arms sales, a forward footprint for ourselves, and resupply depots. They constitute a sort of international embassy system of the US military.

This is the real problem that the restraint crowd is getting at. They suggest also that another function of these foreign bases is as tripwires, to show our seriousness about each alliance and drag us into any war that the host country experiences. This may be true of our core NATO and East Asian bases, but most others are of a much less momentous, and more transactional nature. At any rate, this vast archipelago, as well as the ~500 bases within the US, is much more fertile ground for policy change in the military-industrial complex than efforts to dis-empower our foreign policy more broadly.

The crux is whether we would be more effective with a smaller footprint. While each of these foreign bases is desired at some level by its host country, (with some arm-twisting from the US), the audience is probably quite narrow- the local military, the local support staff and suppliers, some of the political class. It is hard to imagine that most people in most countries are happy to have foreign military in their backyards. Thus, looking at the larger picture of US influence abroad, it is pretty easy to make the case that the benefits of most foreign bases are outweighed by their costs, regardless of their direct price tag. This is where more humility and wisdom are needed. Retrenchment needs to be evaluated, not in the frame of why we should be retreating from the world at large and letting other great powers run their neighborhoods more freely. No, it should be evaluated on how it would benefit our soft power position, beneficially shaping the international environment and attracting more friends to our side.

All these considerations are redoubled when an actual war looms. Has our world policing and forward basing been effective? One would have to give it middling marks at best when it comes to military interventions. We saved South Korea from communism/Juche, and Kuwait from Iraq. but we failed in Vietnam, then in Afghanistan, and should not have even started the war on Iraq. Given the hundreds of thousands of lives lost, it is not a great record of using military means for foreign policy ends. The question is whether we have turned to military tools too frequently, when other options were available. The answer is definitely yes, in the cases of both Vietnam and the second Iraq war. It isn't just hindsight, but foresight at the time could have counseled the US to pass on these misbegotten wars. The Iraq war in particular was a failure on every conceivable level- strategic, humanitarian, political, and tactical. There could not be a starker lesson in how not to use military means to achieve foreign policy objectives.

Looking to the future, the Ukraine war suggests that a fair portion of our military power is also technically obsolete. Our military ability to project power rests in large part on last-century technology like aircraft carriers, tanks, and logistics (those bases!). But the new cyber and drone warfare landscape is starkly different, and may require a significant re-vamping of our overall conception of military power. The restraint school naturally fears that the normal course of the military industrial complex would be to add spending for added capabilities, while keeping all our old machinery and programs as well. The budget is not really the big question, however. Rather, do all capabilities of the government (diplomatic, economic, and military) work productively and in concert to maximize our long-term power and security? Given that better diplomacy and smarter options and thinking at the top could save so many lives and forestall such wide-ranging tragedies as the Iraq war, it makes sense to beef up those areas of the government that provide those goods. Maybe something like a formalized adversarial process of policy development, where red teams and blue teams have independent resources, and develop policy plans, historical interpretations, and forward predictions, which are then evaluated after five and ten-year time periods to gauge who is giving better advice. Maybe a history department, to go with our military, intelligence, and diplomatic departments. One can guess from such exercises that we could use less military, and more policy and cultural expertise, on the whole, in a movement that might be termed rebalancing. And this, in the end, is surely the real point of the restraint caucus.

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• Code red on vaccines.
• A good time had by all.
• Movie of the week: Captains Courageous. I have never seen a movie deal with male culture and male role models as directly and insistently as this (if also melodramatically). It is very topical with all the current talk about men, manosphere, and the problems with boys. Not to mention the evident lack of constructive role models in the life of our current president and his circle. I am extremely fortunate to have had several great role models in my own life.

Sensing a Tiny Crowd

DCP5 uses a curious phase transition to know when things are getting tight inside a cell.

Regulation is the name of the game in life. Once the very bares bones of metabolism and replication were established, the race was on to survive better. And that often means turning on a dime- knowing when to come, when to go, when to live it up, and when to hunker down. Plants can't come and go, so they have rather acute problems of how to deal with the hand (that is, the location) they have been dealt. A big aspect of which is getting water and dealing with lack of water. All life forms have ways to adapt to osmotic stress- that is, any imbalance of water that can be caused by too much salt outside, or drought, or membrane damage. Membranes are somewhat permeable to water, so accommodation to osmotic stress is all about regulating balances of salts and larger ions using active pumps. For example, plant cells usually have pretty high turgor pressure, (often to 30 psi), to pump up their strong cell walls, which helps them stay upright. 

Since cells are filled with large, impermeable molecules like proteins, nucleic acids, and metabolites, the default setting is inward water migration, and thus some turgor pressure. But if excess salts build up outside, or a drought develops, the situation changes. A smart plant cell needs to know what is going on, so it can pump salts inwards (or build up other osmolyte balances) to restore the overall water balance. While osmosensing in other kinds of cells like yeast is understood to some extent, what does so in plants has been somewhat mysterious. (Lots of temperature sensors are known, however.) Yet a recent article laid out an interesting mechanism, based on protein aggregation.

Some cells have direct sensors of membrane tension. Others have complex signaling systems with GPCR proteins that sense osmotic stress. But this new plant mechanism is rather simple, if also trendy. The protein DCP5 is a protein that looks like an RNA-binding protein that participates in stress responses and RNA processing / storage. But in plants, it appears to have an additional power- that of rapid hyper-aggregation when crowded by loss of turgor pressure and cell volume. These aggregates are now understood to be a unique phase of matter, a macromolecular condensate, that is somewhere between liquid and solid. And importantly, they segregate from their surroundings, like oil droplets do from water. The authors did not really discuss what got them interested in this, but this is a known stress-related protein, so once they labeled and visualized it, they must have been struck by its dynamic behavior.

"This condensation was highly dynamic; newly assembled condensates became apparent within 2 min of stress exposure, and the condensation extent is positively correlated with stress severity. ... In cells subjected to continuous hyperosmotic-isosmotic cycles, DCP5 repeatedly switched between condensed and dispersed states."

Titration of an external large molecule, (polyethylene glycol of weight 8000 Daltons, or PEG), which draws water out of a plant cell. The green molecule is DCP5, labeled with GFP, and shows its dramatic condensation with water loss.

Well, great. So the researchers have found a protein in plants that dramatically aggregates under osmotic stress, due to an interesting folding / bistable structure that it has, including a disordered region. Does that make it a water status sensor? And if so, how does it then regulate anything else? 

"In test tube, recombinant DCP5 formed droplets under various artificial crowding conditions generated by polymeric or proteinic crowders. DCP5 droplets emerged even at very low concentrations of PEG8000 (0.01 to 1%), indicating an unusual crowding sensitivity of DCP5."

It turns out that DCP5, aside from binding with its own kind, also drags other proteins into its aggregates, some of which in turn bind to key mRNAs. The ultimate effect is to shield key proteins such as transcription regulators from getting into the nucleus, and shield various mRNAs from translation. In this way, the aggregation system rapidly reprograms the cell to adapt to the new stress.

As one often says in biology and evolution ... whatever it takes! Whether the regulation is by phosphorylation, or proteolysis, or sequestration, or building an enormous brain, any and all roads to regulation are used somewhere to get to where we want to go, which is exquisite sensitivity and responsiveness to our environment, so that we can adapt to it ever faster- far faster than the evolutionary process does.

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Party Of The Future

Not the past ... what the Democrats can do to succeed.

The Democratic party has gotten a little lost, perhaps led a bit too well by Joe Biden, who wandered out of a more civilized and decent time. But focusing on personalities would be wrong- there are much deeper currents at work. While it remains hard to believe that billionaires have successfully hijacked the US government on the back of demogogic appeals to the uneducated, resentful, and bigoted, that is pretty much where we are. Those billionaires are dismantling the US government as fast as humanly possible, so it is imperative for the Democratic party to regain its grip on reality, and on a winning coalition.

One of the more remarkable aspects of the new administration is just how retrograde it is. Its obsession with tariffs comes out of economics at least a century out of date. There was the weird fetishization of William McKinley, and of a replay of our colonial interlude from the late 1800's vs Greenland, maybe even Canada. There is the rollback of the regulatory state, the literal gilding of the oval office, and blithe dismissal of the last hundred years of public health advances. Rather than making America great again, it is making America backwards again, waging a bitter war against the universities and any elites not bending the knee. At this rate, we are heading backwards by the week, to perhaps before the Revolution and onwards to the depths of the royal prerogatives of Henry VIII. Who everyone says was a very great king, by the way!

It is fine to complain, (while we still can), but the more important task is to come up with a better message and coalition, so that the US can get back to looking forward, instead of backward. One way to look at the electorate is in very broad thirds- one elite third, rich and business-centric, another elite third, college educated and liberal, and one third working class. 61% of the population have some college, and 45% have a college degree of some sort. The Democratic party has increasingly moved from its base in the working class to this educated portion of the electorate, and we should own up to what is in essence a battle between two elites- the business elite and the educated elite. These have very different ethics, at least at this late phase of capitalism, when business leaders (if we can call the president that) seem just as interested in business models of grift and fraud (subprime loans, lying about fossil fuels, crypto) as those that build the country. 

Face the past

This diametric difference in ethics is why the divisiveness is at such a fever pitch now. But Democrats made a lot of mistakes as well, of which I can mention four. First, immigration. The utter loss of control at the southern border was highly unpopular. It was unfair to everyone who participated in the (highly unfair, and punishing) legal immigration system. It showed scenes of chaos and lawlessness. And it was an economic threat to everyone on the lower end of the economic spectrum- exactly those demographics the Republicans were aiming at. Second, extreme woke. With the best of intentions, liberal elites set up increasingly abstruse and extreme theaters of correctness, demanding oaths of DEI adherence from faculty, celebrating every deviance from tradition- in the political sphere, in children's literature, and the ever-extending letters of the LGBT... etc. community. All this strenuous virtue signaling was highly distracting and estranging from the bedrock of political coalition-building: unity and common sense. Third was foreign policy, principally the disastrous Afghan withdrawal. For all of the Biden administration's competence in policy over a vast gamut, this was handled very poorly. Granted, it was a cake that was baked by the Trump administration's bad deal with the Taliban, and the management on the ground was run by the military, not the White House, but whatever the cause, it looked bad to catastrophic for US standing the world. And fourth is regulatory gridlock, sapping our ability to build anything, driving up housing costs specifically.

Democrats can not just wait out the madness in Washington, and expect to be elected as the default governing party. They have to face up to ways they have strayed from a winning coalition, and think deeply about fixing it and offering a narrative and program that is both responsible and welcoming to most Americans. 

Face the future

The basic problem of US politics is that we have some unpleasant truths to face. The frontier is gone, the climate is rapidly heating up, US international power is declining. We have rapidly switched from a rising, expansive, and optimistic power to a conservative and somewhat crabby power. Our last tango with a new frontier, that of space, ended up cruelly fruitless. Robotic scientific missions have been spectacularly productive, but manned spaceflight has gone from the height of optimism to another rote exercise in great power sclerosis. There is no there there, in terms of any economic, let alone demographic, frontier. And on top of that, the planet we are thus stuck on is becoming increasingly uncomfortable, even hostile. The future is looking a bit constricted, and more so because the rich elites have lost any sense of collective dedication, and devote themselves to screwing everyone else.

Republicans have addressed these issues by lying, denial, and fantasy, powered by their rabid media sphere. Obviously, Democrats have done and will do better. The current administration makes it clearer than ever what the regulatory state is about, and it is about helping people and restraining the powerful. Democrats need to keep beating the drum that financial protection, drug regulation, food inspection, securities fraud investigation.. these are all aimed at keeping the system fair for the regular citizen. They are not "the deep state", they are not very expensive, either. They are hated only by their antagonists- fraudulent businesspeople. More broadly, inequality is culturally corrosive and calls for taxing the rich more, not less. It is insulting to working people that income from capital gains (let alone carried interest) is taxed less than labor. Everything should be taxed the same- investments, labor, estates, changes in trust membership ... everything.

Build the future

At the same time, practicality has to take a front seat. Democrats need to alter course to promise that regulators keep their eye on the ball of efficiency- fulfilling their mission without tying the economy in knots. This applies particularly to building and zoning. The most liberal areas of the country are also the most conservative, in terms of real estate. The dread of "sprawl" has excused total stasis and lack of building, even as immigrants are allowed to flood in. The result is predictable enough. 

Next, the environment. Democrats did the responsible and foresighted thing with climate bills of the Biden administration. And that helped to build up the green transition. Too little, too late, but these were broadly popular, and need to be continued. The problem is that we are still facing a difficult transition, particularly from gas cars, and on a wide spectrum of other harms such as plastics, habitat loss, resource scarcity. Democrats need to take the long view here that sustainability is the ultimate goal. Everyone can see that our current practices are not fundamentally sustainable. Catastrophes ramp up in frequency and intensity. Democrats have to be the truth-tellers here, while pointing out that building green is beneficial across the board.

Lastly, education. Our education system lays the golden eggs, powering the future of better living standards and international competitiveness. It should be easy for Democrats to make the case against the war on education being carried out by the current administration. Yet, some rebalancing is needed. We have neglected the working class end of the system, and should strengthen community colleges to provide more low-cost trades education. It is appalling that the poor are preyed upon by fly-by-night trade schools in a rich country that supposedly has high standards of education and workmanship. 

Additionally, Democrats might address the deeper malaise among the young and in the culture at large by calling for national service. Two years for young people spent on serving others would be highly beneficial. Choices could range from military to conservation corps, and medical assistance. The main condition would be that these would be non-specialized, mixed groups so that participants work with people from all walks of life in the US, (or in the Peace Corps), broadening their experience and vision of what it means to be part of this country. Call this a form of civic education, essential to a democratic society.

Democrats have the tools, and need to reach out a bit to form a bigger coalition. The right wingosphere has successfully demonized learning, knowledge, and regulation, and it is all coming home to roost in the current administration. But the regulatory state was built originally not out of a college educated coalition, but a progressive working class movement, revolted by the corruption and inequality of the gilded age. Republicans can't help themselves but serve the rich and powerful. Democrats can re-align with their natural coalition.

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