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. 

• Total lies come naturally to RFK Jr., as to so many in this administration.
• With the help of crypo, our banks are not-so-unwitting conduits for crime.

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.

• What the hell is it with ivermectin?
• Christian is as Christian does.
• 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.

• Crime and lead.
• Community medicine.
• Who needs psych meds? RFK seems to.
• Rules? Who ever said there were rules?
• Taco Tariffs.
• Vaccines are being cancelled.

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.

• In many cases, going backwards requires lying.
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• Hospice, or tax cuts?
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• And carrying water for Russia.
• The next frontier, after zero carbon.
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• Another reason why crypto is so great.

An Arms Race at the Tiniest Scale

Defense and anti-defense against genetic attack by plasmids.

Bacteria have a pretty active sex life. And like for us, this involves defense and offense, in a complicated tango of genetic exchange. Only, for bacteria, conjugation mechanisms are overwhelmingly used for attack, even though they are also the conduit of great innovations like horizontal gene transfer from different species, and antibiotic resistance. The top priority of most bacteria most of the time is to defend against alien DNA, which most of the time are selfish genetic elements and viruses, and they have several mechanisms to do so.

Plasmids are a very common feature of bacteria, and fundamental to genetic engineering, as the primary form of manipulated DNA. Plasmids can be amplified to high copy number in bacteria, can be cut, altered, and ligated back together- the very essence of engineering. This dates me, but I remember preparing plasmids from bacterial cultures by cesium chloride isopycnic (high speed) centrifugation, after which the plasmid (marked by the poison ethidium) would end up swimming in the middle of the gradient, and have to be sucked out with a syringe, before further steps to clean up the purified DNA. It was a rather messy, expensive, uncertain, slow, and unsafe process. 

Summary of the current paper, outlining plasmid transfer, plasmid genome structure, and some components (genes and promoters) of the leading strand as it is transferred to target cells.

Anyhow, plasmids in the wild are typically aggressive genetic elements that carry (encode) some or all of the components needed to form an injection attack complex (i.e. type IV secretion system) that conjugates with other bacteria. Plasmids can also carry many other things, like antibiotic resistance, or stray genes from prior bacterial hosts, or transposing genetic elements. So most of the time, bacteria want to defend themselves against this kind of invasion, even though some of the time the gifts they bring can end up being highly beneficial.

One of their defenses is the restriction system. Another foundation of genetic engineering was and remains the restriction enzyme. These are enzymes that cut DNA at a particular sequence. One can imagine how useful it can be to have such specific scissors for these infinitesimal molecules, and most labs would have a freezer filled with a large library of such enzymes that could be used for breaking and reconstructing new (plasmid) molecules, and also for analyzing them by their pattern of "restriction" sites. In the wild, these enzymes are paired with DNA methylation enzymes that make the host genome invisible to the restriction enzyme, leaving only newly arrived alien DNA susceptible to cleavage, and thus destruction, by these enzymes. 

Another defense is the now-famous CRISPR system. Bacteria capture small bits of invading genomes, and, assuming they survive, knit them into special genetic modules in their own chromosomes. Then they express these small modules as RNAs that latch onto an enzyme called Cas9, (or related enzyme plus RNA systems), which are nucleases that are guided by these RNAs to cut and inactivate invading DNA that matches the RNA sequence. This system is noted as a sort of adaptive immune system that learns from experience, and passes its knowledge down genetically to future generations.

More detailed maps of three example plasmid genomes, showing the distribution of anti-defense and other genes at the leading edge of the genome (left end). Red and yellow colored genes are anti-defense genes of various kinds. The tiny arrows are promoters, each of the early-start kind that can operate on single-stranded DNA.

A recent paper discussed specialized anti-defenses that plasmids have against these and other bacterial defenses, including toxin/suicide systems and inducible stress responses. For it is naturally an arms race with innovation on both sides. Plasmids have an origin of replication where new DNA strands start, and this new strand is what is injected into the target cell. So there is a linear order of DNA and thus genes going into the target cell, head to tail. These researchers find that the anti-defense genes of plasmids tend to be bunched up at the head of the genome, where they get into the target cell first. These regions also have a special way to fold up their single-stranded DNA (into a cruciform shape) that forms immediate promoters that allow these "early" genes to be transcribed by the host RNA polymerase, before replication in the host cell has regenerated normal double-stranded DNA. 

The authors do a very wide survey of species and plasmids, and find that there is a wide variety of anti-defense genes, many of which have unknown functions. Among the known ones are: anti-restriction inhibitors, which directly bind and inactivate restriction enzymes, methyltransferases (MTase) that methylate restriction sites to make them look like host DNA, single strand binding proteins (SSB), which coat the single stranded DNA and protect it from detection as single stranded DNA, and may assist in repair after Cas9 or restriction cleavage, and antitoxins that inhibit the toxin systems or the SOS system. There are also anti-CRISPR proteins, which degrade the Cas9 enzyme, or inhibit its binding to target DNA.

Experiment to demonstrate the importance of being first... into the target cell. Targeting means that the bactierial cell has a CRISPR system targeting the incoming plasmid. acrIIA4 is an anti-CRISPR protein that effectively blocks cleavage of plasmid DNA by the CRISPR/Cas9 enzyme. The petri plate exhibits bacteria that can grow only if plasmid transfer was successful. See text for further details.

They finish with an elegant experiment that asks how important it is for the anti-defense gene to be at the front of the plasmid. The gene they chose was acrIIA4, which is an anti-CRISPR that very efficiently inhibits Cas9 cleavage of targeted DNA. The petri plate at top shows the growth of infected bacteria after infection by the experimental plasmid, selected for plasmid presence on antibiotic medium. The grey bar, in both diagrams is the control, which are cells whose CRISPR system does not target this plasmid. The plasmid transfers fine, and the cells grow fine. In contrast, if the cell's CRISPR system does target the plasmid, lack of acrIIA is fatal, (top), decreasing plasmid transfer by about three logs, or a thousand fold. Putting the acrIIA gene at the tail end of the plasmid (middle experiments) helps a little, and transfer is knocked down only a hundred fold. Putting the defense gene at the front of the plasmid, (leading), though, corrects plasmid defense almost fully, and transfer is down a few fold only. Lastly, if acrIIIA is placed in opposite orientation, (inverted), such that plasmid replication is needed before this gene can be expressed (it can't use the cruciform single-stranded promoter), it is virtually useless. So indeed, being first off the block when invading a target cell is critically important, since the host cell makes its defenses all the time- they are ready and waiting.

While most of these transfers are unwanted, sometimes plasmids integrate into bacterial genomes, and then when they start transferring themselves into other cells, they can bring along huge amounts of their host genomes. That starts to look like serious genetic exchange that begins to approximate sex in eukaryotes. So, some balance of defense, offense, and beneficial exchange is the lifeblood of ecology and evolution at this most ancient scale of life.

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A Very French Star Wars

Bruno Dumont's "The Empire" touches on the true meaning of Sci Fi. (Spoiler alerts!)

The online reviews are not very good, but to me, this film was both hilarious and profound. A bunch of scuzzy French villagers go about their normal business, fishing, arguing, flirting, driving around. Then, though the magic of acting, they betray another plot entirely. Some are extraterrestrials just taking human form, deeply engaged in some cosmic battle and sponsored by hulking space ships above, in the heavens. The kicker is that the space ship is topped by ... a gothic cathedral. At first, this just seems like a hilarious way to cut special effects expenses. Why not use the local cathedral to shoot the space ship interiors?!? But as you revolve all this in your mind, it starts to appear as though Dumont is making a more interesting point.

By the traditional theological story, aren't we all extra-terrestrials, trapped in human bodies, constantly fighting with the flesh and destined to return to a better realm? Conversely, isn't the standard science fiction story full of magical wonders and grand dramas and theologies? What if religion and science fiction are ultimately, as L. Ron Hubbard appreciated, the same thing? Transporter, resurrection; medical miracle, laying on of hands; Borg, Satan; tomato, tomahto. 

Unfortunately, sometimes the humanity takes charge, sex first and foremost fouling up the neat good vs bad dynamic. In the plot, neither side really does anything bad or good, reinforcing the absurdity of a film that comes off as a sort of French Terry Gilliam masterpiece. The "1"s come from the flying gothic cathedral and think of themselves as good, while the "0"s come from a flying Versailles, (which makes for a particularly ungainly space ship), and know they are demons. But they are all equally distracted by those human bodies.

The ending was, as far as I understood it, a disappointment. The armadas of mini-cathedral and mini-Versailles fighters are lined up for the final battle, à la Star Wars. But suddenly, they all get sucked into a wormhole, and ... end of film. It is almost as though Dumont holds out hope that there is a real deity, or at least higher being out there, to save humanity from this battle between these wonderfully absurdist extra-terrestrials. After the wind dies down, the local policemen and villagers are left to puzzle over the wreckage, and what these signs and wonders could have meant.

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Histones Require a Towtruck With a Winch

Motorized remodelers adjust and open up chromatin for gene expression.

Wouldn't it be nice if, on a stop-and-go congested highway, you could just plow through all the obstructing cars and go where you want? That is what polymerases get to do on our DNA, once they are set in motion. They plow right through chromatin, histones, DNA-binding transcription regulators, and everything else in their way. But getting them to that point is a different matter. Origins of replication need to be carefully cleared and set up. Promoters of genes need to be activated by the convergence of enhancer-binding proteins, promoter-binding proteins, and mediators of various kinds to get that RNA polymerase set on its path. And that is not so easy in a chromatin environment where the DNA is almost all covered by something, principally histones that wind up our DNA in tight little 146 base pair loops.

A basic schematic of nucleosome cores (yellow), composed of histone proteins, and how they wind up DNA and pack with each other.

So a class of "chromatin remodelers" have evolved that move histones around, and exchange histones in a way that facilitates transcription. It became apparent a couple of decades ago that regions of active transcription have altered histone composition, H2A.z/H3.3 instead of the regular H2A and H3 histones. These histones are looser, allowing regulatory proteins better access to the DNA as well as easing the passage of the polymerases. But how do they get there? It has also gradually become apparent that regulatory proteins come in different types, with some "pioneer" regulators able to bind in the midst of packed chromatin. These in turn recruit additional regulators, including enzymes that loosen up histones by chemically altering them with methyl, acetyl, and other modifications, and remodeling enzymes that push histones around, revealing DNA where other regulators can bind, and popping out conventional histones for more weakly-binding ones.

A few recent papers revealed the structure of a few of these remodeling enzymes, and compare them between yeast cells and human cells. There are a variety of these machines, which specialize things like nudging nucleosomes into regular spacing, or evicting/moving nucleosomes from particular regions, such as near transcription regulators, or exchanging nucleosomes in active regions. It is the latter that is being studied here. In yeast, this SWI/SNF family remodeler comes in two parts, NuA4, which is a histone acetylase, and SWR1, which uses ATP to winch out H2A and replace it with H2A.z. These protein complexes cooperate with each other and have related effects in opening local chromatin to be more transcription competent. In humans, these complexes are combined into one super-complex, TIP60-C, which weighs in at 1.8 million daltons, a dalton being the mass of one hydrogen atom. One can appreciate here, as in so many other details of biology, the nature of evolution- the tension between conservation and change.

Chromatin remodelers from yeast (top) and human (bottom). SWI/SNF is on the left, and RSC is on the right. Both of these remodelers have wide capabilities of moving or replacing nucleosomes. DNA is in orange, and the histone is at top, within the DNA coils. See text for further description.

At top are the structures of two yeast remodelers, SWI/SNF and RSC. At bottom are structures of the corresponding human remodelers, BAF and PBAF. One can appreciate how similar they are in overview, while being very different in detail. All of these remodelers function in detailed transcriptional control in collaboration with other regulators. The orange structure is the DNA wrapped around a nucleosome, while the large blobs at the bottom of these structures are relatively loose regions where they interact with other transcription regulators, which recruit them to the proper locations. The ATP-driven motor is shown at the top in green, and grabs tightly, with two RecA domains (that is to say, with two hands) to bits of the DNA circling the nucleosome. Given that the whole apparatus is anchored to the histone and other nearby structures, this enables the motor to pull on the DNA. It is pretty slow, moving only 1 or 2 base pairs per ATP used, but with enough copies (of these motors) and time, great things can be done. The structure in (b) indicates where the DNA enters (top) and where it gets pulled towards (bottom) as the motor works. This action can nudge the histone to some new location, relative to the DNA. Alternately, with other forms of anchoring, it can also pop the histone entirely off the DNA, and, since this large protein complex can bind alternative histone complexes, can bring in a new histone for exchange.  

Downloadable animation of nucleosome movement (source). 

Downloadable animation of overall assembly and nucleosome spacing activity (source).

So in short, what we have here is a DNA winch, which in various configurations can adjust, evict, or exchange nucleosomes, as directed by various signals. One signal is the sequence of the DNA itself, another is the standard spacing between chromosomes that is set by some of these motors that have a suitably sized extension, extending out to touch the next histone, and establishes the default nucleosome pattern, genome-wide. But more significant are the various pioneer regulators and histone modifiers that direct these motors to specific areas to reshape the local chromatin to control gene expression. Here is where the regulatory action is, and these proteins have been found to be determinants for cancer progression, and indeed are targets for some investigational anti-cancer drugs.

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An Uneasy Relationship With the Air

Review of Airborne, by Carl Zimmer. 

The pandemic was tough on everyone. But it had especially damaging effects on the political system, and on its relationship to the scientific community. Now the wingnuts are in charge, blowing up the health and research system, which obviously is not going to end well, whatever its defects and whatever their motivations.

While the scientific community had some astounding wins in this pandemic, in virus testing and vaccine production, there were also appalling misses. The US's first attempt at creating a test failed, at the most critical time. We were asleep at the wheel of public health, again at the earliest time, in controlling travel and quarantining travelers. But worst of all was the groupthink that resisted, tooth and nail, the aerosol nature of viral transmission of Covid. That is, at the core, what Zimmer's book is about, and it is a harrowing story.

He spends most of the book strolling through the long history of "aerobiology", which is to say, the study of microbes in the air. There are the fungal spores, the plant pests, the pollen, the vast amount of oceanic debris. But of most interest to us are the diseases, like tuberculosis, and anthrax. The field took a detour into biowarfare in the mid-20th century, from which it never really recovered, since so much of that science was secret, and in its shadow, the sporadic earlier public studies that looked carefully into disease transmission by aerosols were, sadly, forgotten. 

So it became a commonplace at the CDC and other public health entities, among all the so-called infectious disease specialists, that respiratory viruses like influenza, colds, and coronaviruses spread not by aerosols, but by contact, surfaces, and large droplets. This made infection control easy, (at least in principle), in that keeping a few feet away from sick people would be sufficient for safety, perhaps plus surgical masks in extreme situations. There was a curious disinterest in the older studies that had refuted this concept, and little interest in doing new ones, because "everyone knows" what the virus behavior is.

It is hard to explain all this in purely scientific terms. I think everyone knew at some level that the true nature of respiratory virus transmission was not well-understood, because we clearly had not managed to control it, either in residential or in hospital settings. It is hard to grapple with invisible things, and easy to settle into conventional, even mythical, trains of thought. First there were miasmas, then there were Koch's postulates and contact by fluids. It was hard to come full circle and realize that, yes, miasmas were sort of a thing after all, in the form of aerosols of infectious particles. It was also all too easy to say that little evidence supported aerosol spread, since the work that had been done had been forgotten, and the area was unfashionable for new work, given the conventional wisdom.

Even more significantly, the implications of aerosol spread of viruses are highly unpleasant, even frightening. The air we need every minute of our lives is suspect. It is a bit like the relationship we have with food- deeply conflicted and fraught, with fears, excesses, and rituals. One has to eat, but our food is full of psychological valences, possible poisons, cultural baggage, judgement, libraries full of advice. No one really wanted to go there for air as well. So I think scientists, even those calling themselves infectious disease specialists, (of all things), settled into a comfortable conventional wisdom, that droplets were the only game in town.

But what did this say about the larger research enterprise? What did it mean that, even while medical/bio research community was sequencing genomes and penetrating into obscure and complex regions of molecular biology, we had not done, or at least not appreciated and implemented, the most basic research of public health- how infectious diseases really spread, and how to protect people from them? It constituted gross negligence by the medical research community- no two ways about it. And that appears to have caused the public at large to question what on earth they were funding. A glorious enterprise of discovery, perhaps, but one that was not very focused on actual human health.

A timeline of research/policy

• Sprawling work on airborne infection in 1955. 

• Earliest glimmer in latest pandemic, May 2020. 

• Deeper followup, October 2020.

• Even deeper followup, August 2021. Later, 2023.

• Critics still can not believe aerosol spread, November, 2022.

• Hospital HVAC requirements currently go to MERV 8, not enough.

• Current CDC guidance mentions aerosols only from "procedures", not from people, though masks are recommended.

Aerosol spread of disease requires two things- that aerosols are produced, and that the infectious microbes remain infectious while in those aerosols. The former is clear enough. We sneeze, after all. Even normal breathing creates fine aerosols. The latter is where scientific doubt has been more common, since many viruses are not armored, but have loose coats and membranes derived from our own, delicate cells. Viruses like HIV don't survive in aerosols, and don't spread that way. But it turns out that Covid viruses have a half life of about two hours in aerosols. 

The implications of that are quite stunning. It means that viruses can hang around in the air for many hours. Indoor spaces with poor ventilation- which means practically all indoor spaces- can fill up with infectious particles from one or a few infected people, and be an invisible epidemic cloud. No wonder everyone eventually got Covid. 

What to do about it? Well, the earliest aerobiology experiments on infectious disease went directly to UV light disinfection, which is highly effective, and remains so today. But UV light is dangerous to us as well as microbes, so needs to be well-shielded. As part of an air handling system, though, UV light is an excellent solution. Additional research has found that far-UV, at 222 nm, is both effective against airborne microbes and safe for human eyes and skin, creating an outstanding way to clear the air. Another approach is HEPA filtration of air, either as part of an air handling / exchange system, or as stand-alone appliances. Another is better ventilation overall, bringing in more outside air, though that has high energy costs. Lastly, there are masks, which are only partially effective, and the place no one really wants to go. But given a lack of responsibility by those in charge of our built environment, masks are the lowest common denominator- the one thing we can all do to protect ourselves and others. And not just any mask, but the N95 high-quality filtration mask or respirator.

The pandemic threw some sharp light into our public institutions. We sequenced these viruses in a hurry, but couldn't figure out how they spread. We created vaccines in record time, but wasted untold effort and expense on cleaning surfaces, erecting plexiglass shields, and demanding masking, rather than taking responsibility for guarding and cleaning public air spaces in a more holistic way. It is a disconcerting record, and there remains quite a bit yet to do.

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Donald Trump is no Andrew Jackson

A few notes about the Jacksonian era.

One common historical touchpoint for our current epoch is the Jacksonian era, when a populist president presided over a significant increase in presidential power, carried into the White House by a ragtag rabble. Andrew Jackson stood against the elite power centers of the time, having been denied the presidency earlier by a shady deal that gave John Quincy Adams the office. Nor did he have much more love for the aristocrats of Virginia. He came from the backwoods of Tennessee, and a long career of fighting Indians as well as the English. Once in office, Jackson cleaned house and installed a patronage system that led to decades of increasing corruption, till the civil service was instituted. He also used the veto power, and made his cabinet secretaries subservient, to an unprecedented degree.

Jackson strengthened the party system and cultivated friendly media in a way that people at the time decried as divisive and dangerous. And, perhaps most strikingly, he oversaw the mass expulsion of Native Americans from the South. Jackson was a slaveowner and had no issue with the white supremacy of his day, whether against African Americans or Native Americans. Ironically, when France decided to not honor a treaty with the US, Jackson spared no effort to defend the nation's honor and rights. But when it came to the many treaties the US had signed with indigenous nations, many expressly meant for perpetuity, they were waved away like so much smoke.

On the other hand, Jackson was a successful general and businessman and won all the major battles of his presidency. And he was successful enough to anoint a successor, Martin van Buren. He was surprisingly eloquent and well-written and had a core set of principles that guided him and the nation. One principle was the importance of the constitution and the union. While previous presidents had thought the veto power should be confined to extreme legislative acts they regarded as unconstitutional, Jackson saw nothing in the constitution against using the veto on a policy basis, to weigh in on substantive issues as a popularly elected co-equal branch of government.

More importantly, he guided the nation through a nullification crisis with South Carolina with a sure hand. Always a hotbed of resistance and secession, South Carolina took particular issue with federal tariffs, which were set quite high to favor domestic industry. Industry generally located in the North. Jackson laid the groundwork for federal military intervention, promoted a tariff reduction, and issued a forceful and closely argued denunciation of "nullification" and secession that, in combination, squelched the movement of southern states against federal supremacy. This put off for a generation the crisis that Lincoln was fated to deal with.

One of Jackson's most interesting fights was against the Second Bank of the United States. Congress had chartered, from the Washington administration onwards, a national bank that was the sole interstate financial institution of the US. It was charged with facilitating the finances of the federal government, and with providing credit for internal improvements crossing state lines. But it was in essence a private bank that had only a fraction of its board appointed by the government and otherwise ran its business on a private basis as a commercial bank. In its opening years, it was generally undersized and not well run, and by the time of the second bank, had caused a couple of recessions due to its mismanagement. 

Finally, by the Jackson administration, it had come under competent management and was both expanding in all directions and doing a reasonable job of controlling the money supply and credit in the US, by limiting expansion of the state banks, (a significant source of opposition). It had, indeed, become the largest single financial institution in the world. But to Jackson, these were hardly points in its favor. He viewed it as a dangerous center of power, as though in our day JP Morgan were the only commercial bank allowed to do nation-wide business, with no competitors. The whole idea of a publicly-run central bank had not yet arisen at this time, and the national bank was more or less modeled on the Bank of England, which was a similar hybrid private entity. Unfortunately, instead of seeking reform of the national bank into a more modern and public-interest institution, Jackson pulled the only levers he had, which were to veto the rechartering of the Second National Bank, and then to follow that up with removing all federal deposits and putting them into state banks, effectively killing it. This had the unfortunate effect of dooming the US to almost a century of financial instability and poorly regulated banking. But on the whole, I am quite sympathetic to Jackson's position in killing the bank. It was a nascent form of anti-monopoly policy, which should have been taken up more systematically later in the century.

So, Jackson was very much of his time, not a visionary who could prepare the government for the vast growth in population, social institutions, and technology that were coming. But at the same time, he was not trying to drag the US backwards in time either. He did not cruelly run rampant through federal agencies, or foster international trade wars in search of a happier dream time of mediocre jobs and pay. The economic crisis that happened during his administration was not a tantrum he threw, but rather was caused by the national bank, as it consciously fostered a recession by withdrawing credit in an attempt to turn the people against Jackson. An attempt that failed because everyone knew what was going on, and which indeed showed the kind of power that Jackson was fighting against. Andrew Jackson did not view the federal government as an extortion racket or a throne from which bootlickers could be alternately fawned over and kicked in the teeth. He was thus, despite a few parallels, quite unlike the current occupant.

I am taking most of this material from an enjoyable biography by Jon Meacham. It is based mostly on correspondence, thus is quite chatty and focused on Jackson's domestic affairs. It is, conversely, frustratingly weak on the larger historical and policy issues of his day, particularly when it comes to the bank fight, which was so important for the country's future. 

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Covid Builds a Fortress Within

 Viral proteins build peculiar vesicles to hide the viral replication apparatus.

SARS-CoV is still with us, a brutal addition to the already extensive army of respiratory viruses infecting humanity. While most people clear it, we have a hard time doing so, a testament to a tough evolutionary arms race. A fair portion of our extremely complicated immune system is devoted to viruses, including basics like recognizing double-stranded RNA and viral replication structures. A trick that coronaviruses and allied species possess has gradually come to light, which is the formation of vesicular structures that appear to host their replication apparatus. 

Coronavirus-infected cells display a variety of vesicular structures, including "zippered" endoplasmic reticulum, convoluted membranes (CV), dense membrane spherules (DMS) and double-membrane vesicles (DMV). The endoplasmic reticulum (ER) is the cellular organelle where membrane proteins and secreted proteins are first made, before they are sorted out to various other membrane systems and the outside (and where the bulk of membrane lipid production happens, among much else). Coronaviruses appear to commandeer the ER and divert its membranes to the new structures. It is the DMV that turns out to have an important function- hosting viral replication. How do we know this? Researchers recently turned to a classic technique- radioactive labeling of new RNA production in infected cells, followed by electron microscopy combined with auto-radiography. The image below shows in stunning detail various organelles within an infected cell, and the black dots are film grains turned by the radioactive RNA to mark synthesis sites. They are quite closely aligned with the DMV structures.

Exquisite auto-radiograph and electron micrograph of a SARS-CoV-infected cell. The mitochondria (m) are most apparent, followed by the viral replication organelles (RO, aka DMV), followed by the endoplasmic reticulum (ER), lipid droplets (LD), nucleus (N), and virion-containing region (VCR). The black dots from photo-sensitive film exposed by radioactive RNA is clustered around the DMV structures.

This finding leads to several questions. How do these structures form? And, given the need for replication to both get inputs such as nucleotides and to export outputs like the virus's genomic RNAs, why use membranes that are impermeable to such molecules? Why use two membranes, when one suffices for most cellular organelles like the ER, lysosomes, peroxisomes, etc? This had puzzled the field for some time. Now, it turns out (in another recent paper) a couple of powerful viral proteins solve both questions at once. Coronavirus products nsp3 and nsp4 have long been known as important for viral success, but recent work puts them at the heart of DMV formation, into what is now called a replication organelle (RO), as well as a DMV. They are expressed in the ER and seem to play the leading role (along with several host proteins and lipids) in curving its membranes into the DMV shape. They also form dimeric pairs (nsp3 on one membrane, and nsp4 on a facing membrane) that seal two membranes together, as seen in the DMV structure. And thirdly, they, once fully assembled and mated, form a pore which keeps out pretty much everything big, but lets through single stranded RNA and small molecules.

Structure determination of the multimeric nsp3/4 pore structure from purified DMV vesicles, several views. Note the tight pore going through the center, and differential sizes of the inner and outer membrane rings. It is a protein complex that both bends the membrane and keeps only the most essential traffic going through it.

This structure is beautiful in a way. The central pore, at about 1.5 nm, is lined with positive charges like lysine and asparagine, the better to conduct negatively charged RNA. The inner membrane structure is tighter than that of the outer membrane, the better to curve those membranes into the observed spherical size. While it is a little hard to believe that such DMV vesicles, even studded with such a bespoke pore, can conduct the kind of traffic, both in and out, needed to sustain high rates of viral replication, that is quite evidently how it works. These researchers make a few mutations in the newly revealed key positively charged central pore amino acids to show that, if those charges are lost, replication of the virus was "abolished". This creates an obvious drug target as well- some chemical that plugs this pore or otherwise blocks the assembly of this ornate structure.

Additionally, the assembly of all this out of flat ER was also studied. The nsp3/4 proteins are originally connected end-to-end and do a delicate dance of pulling on each other (after cleavage) to dramatically curve the membrane between them, forming a tight loop from the (future) outside DMV membrane to the (future) inside one. On the other hand, another way they can assemble (right side in diagram below) is from separated (ER) membranes, leading to the "zippered" ER conformation that is also seen in infected cells. Whether the latter can be transformed into the former remains a question. 

Models for assembly of the linked nsp3-nsp4 proteins into the curved membranes of the DMV pore, with super-curvature at the pore junction between outside and inside membranes. TM stands for transmembrane domain, NTD for N-terminal domain (front), CTD for C-terminal domain (rear), and Ecto for the ecto-domains of each protein that are not within the membrane.

It is naturally implicit in this work that, if the pores of nsp3/4 allow through the absolute essentials of viral replication, they also block the various cellular sensors of viral presence, such as the RIG and TLR proteins, thus delaying the host response. Perhaps the RNAs allowed out are modified prior to exit to make them look more host-like. All those assumptions have yet to be nailed down explicitly. At any rate, viral assembly takes place elsewhere, so it is not entirely clear yet what exactly is being hidden here.

There were some technical innovations along the way to these results. These researchers tagged the nsp proteins in a way that allowed them to easily purify DMV vesicles out of whole cells, speeding their cry-electron microscopy work of getting these structures. Did they just use the Alpha fold program and do all this the easy way? Not at all. They did use Alpha fold to refine some of the structures, to extract more atomic detail. But they notably did not trust the AI to cook this kind of finding up from scratch. Some things still need to be done empirically, if you really want the truth.

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