How do You Get a Hula Hoop onto DNA?

DNA synthesis relies on a hoop-shaped complex, or "clamp" around DNA to keep it on track.

Processivity is a big issue for biological polymerases. An RNA polymerase needs to stay on its template until it reaches the end. Otherwise, the mRNA might be truncated and the resulting protein would be incomplete. Such proteins frequently have an activity opposite to that which the complete protein has, either because of a particular domain structure that puts key domains at the end, or just because it gums up the works instead of being a well-oiled cog in those works.

Likewise for DNA polymerases that replicate genomes. DNA polymerases that do small repairs may jump in for only a few nucleotides, but for earnest replication of entire genomes, you need a polymerase that chugs along reliably, for long distances. Evolution has come up with an elegant, if obvious, solution- mate the polymerase to another protein complex that firmly encircles the DNA like a hula hoop and doesn't let go. But this leads to other questions ... how does such a complex assemble where it is supposed to, and what happens to it later on?

Whatever the answers to those questions, this solution has been around for a very long time. The structures of the replication-associated sliding clamp from bacteria and from humans look virtually identical:

Human (left), and E. coli (right) sliding clamp protein complexes that facilitate DNA replication. Where does the DNA go? Obviously through the middle. These proteins do not bind to the DNA in any sequence specific way, but have positive charges arrayed around the inner ring, to gently stay in contact with negatively charged DNA.

The clamp complexes assemble into extremely stable rings right after they are synthesized off the ribosomal assembly line. That means that they have to be pried apart again to get put on DNA. That is the job of "clamp loaders"- yet another protein complex that orchestrate the proper placement of these clamps. While the clamp proteins are pretty simple affairs- pure structures lacking any enzymatic activity- the clamp loaders are ATP-ases and quite dynamic. 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683903/

An extremely simplistic model of clamp loading, with the loader prying open the clamp to allow it to admit double stranded DNA.

The job of a clamp loader is to recognize the correct location in DNA, to bind a free clamp complex, to pry open that complex, transiently allowing the double stranded DNA to enter, to detect when the DNA has successfully been loaded into the clamp, and lastly to detach again from the resulting complex. It is a big job, obviously. What is the correct location? It is a fork where single stranded DNA meets double stranded DNA- i.e. where DNA replication has been primed by a special replication origin selection, opening, and priming process. Clamp loaders are also rings, with five highly related subunits all around. Their ATPase activity (active on each subunit) allows them to twist, which is how they manage to twist the clamp as well, to open it up.

A recent paper extended structural knowledge about clamp loaders. These authors used the new cryo-electron microscopy methods to obtain high resolution structures of these complexes in a variety of states. These states were made available by virtue of using a slowly hydrolyzing form of ATP that gave them the whole spectrum, then frozen and photographed with electrons. They capture beautifully the sequence of events, where the loader first tentatively binds a clamp, and then opens it up wide enough to accept DNA. The DNA binding groove is not open until the clamp is bound, enforcing this forward sequence of clamp binding, then DNA binding (availability), then clamp opening. The authors do not, however, provide reason to think that this opening is dependent on DNA being already bound, so it is possible that this opening complex can be futile- opening and closing repeatedly before encountering the right replication fork. This is probably unlikely in practice, though, because the clamp loader complexes are quite rare and probably have other interactions that position them at replication forks before this process even begins. Additionally, the loader + clamp complex may remain open as long as necessary, until it encounters the right fork location.

An early form of the clamp loading complex is shown, when it first binds a clamp. The five subunits of the clamp loader are shown in color, while the clamp is shown at bottom in gray. In B, the contacts between the two complexes are shown in color, on the clamp, which is composed of proteins called PCNA. The different structures are of various stably inhibited forms, and the A-gate is where DNA is bound during the loading process.

Later on, after the loader has expended some energy, it has more contacts apparent with the clamp (right) and has pried it open, wide enough to accept a strand of DNA.

With DNA, the whole complex contracts a little again, and you can see the continuous flow of DNA (yellow) through the cleft of the loader complex down through the clamp complex. 

The last step is closure, when the clamp closes fully around the DNA, and the loader complex unbinds. This seems to be when ATP is actually hydrolyzed, implying that the opened complex is the ATP binding state, the closed (free) loader complex is the ADP bound state, and that successful DNA binding to is what stimulates ATP hydrolysis. Finally, another structure- a closeup of the DNA as bound, shows that the end of the primer strand, which sits right at the crux of the DNA fork, is specifically bound with its last base flipped around into the protein. This provides part of the mechanism of how the clamp loader feels its way to the right place at replication forks, after the briefly interacting polymerases that create such primers have fallen off. It is likely that this clamp loader complex engages in interaction with the final processive DNA polymerase to help it find the fork and clamp, but notably, the same face of the clamp interacts with both the loader and with that polymerase, so the handoff can not involve both binding at the same time at the same place.

A blowup of just the DNA within the fully bound complex above. Note how the top base of the yellow primer strand is flipped out from the rest of the helix, due to interactions with the loader protein complex.

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DNA Mambo in the Nucleus

Some organizational principles for nuclear DNA to organize genes for local regulation.

There has been a long and productive line of research on the mechanisms of transcription from DNA to RNA- the process that reads the genome and translates its code into a running stream of instructions going out to the cell through development and all through life. This search has generally gone from the core of the process outwards to its regulatory apparatus. The opening of DNA by simple RNA polymerases was one of the first topics of study, followed by how the polymerase is positioned at the start site by "promoter" DNA sequences, with ever more ornate and distant surrounding machinery coming under scrutiny over time, as researchers climbed the evolutionary trajectory of life, from viruses and bacteria to mammals. 

But how this process fits into the larger structure of the nucleus, and how it is globally organized eukaryotes has long been an intriguing question, and tools are finally available to bring this level of organization into focus. For example, genes are known to be activated by direct contact with "enhancer" elements located thousands, even many tens of thousands, of basepairs away on the DNA- so why can't those enhancers activate other genes elsewhere in the nucleus, rather than the genes they are nearest to on the one-dimensional DNA? The nucleus is a small place with a lot of DNA. Roughly 1/100 of its physical space is taken up by DNA, and it is highly likely that such enhancers could be closer in 3-D space to other genes than the ones they are supposed to regulate, if everything were arranged randomly. Similarly, how do such enhancer elements find their proper targets, amid the welter of other DNA and proteins? A hundred thousand base pairs is long enough to traverse the entire nucleus.

So there has to be some organization, and new techniques have come along to illuminate it. These are crosslinking methods where the cells are treated with a chemical to crosslink / freeze a fraction of protein and DNA interactions in place, then enzymes are introduced to chop everything up, to various degrees of completeness. What is left are little clumps of DNA and protein that hopefully include distant cross-links, between enhancers and promoters, between key organizational sites and the genes they interact with, etc. Then comes the sequencing magic. These clumped stray DNAs are diluted and ligated together (only to local ends), amplified and sequenced, generating a slew of DNA sequences. Those hybrid sequences can be interpreted, (given the known sequence of the reference genome), to say whether some genomic location X got tangled up with some other location Y, reflecting their 3-D interaction in the cell when it was originally treated.

A recent paper pushed this method forward a bit, with finer-grained enzymatic digestion and deeper sequencing, to come up with the most detailed look ever at a drosophila genome, and at some particular genes that have long held interest as key regulators of development. This refined detail, plus some experiments mutating some of the key DNA sites involved, allowed them to come up with a new class of organizing elements and a theory of how the nuclear tangle works.

Long range contacts in the Antennapedia locus of flies. Micro-C refers to the crosslinking and sequencing method that maps long-range DNA contacts mediated by proteins. Pyramids in the top diagram map binary location-to-location contacts. Local contacts generally predominate over distant ones, but a few distant connections are visible, such as between the ends of the ftz gene. TAD stands for topologically associating domain, mapping out the connections seen above between pink sites. This line also lists the genes residing in each zone (Deformed, micro RNA 10, Sex combs reduced, fushi terazu, and Antennapedia promoters P1 and P2). The contacts track shows where the authors map specific sites where organizing factors (including Trl (trithorax-like) and CP190 (centrosomal protein of 190 kDa)) bind. The overall idea is that there are two kinds of contacts, boundaries and tethers. Boundaries insulate one region from the next, preventing regulatory spill-over to the wrong gene. Tethers serve as pro-regulatory staging points, helping enhancers contact their proper promoter targets, even though the tether complex does not itself promote RNA transcription.

Insulator elements have been recognized for some time. These are locations that seem to block regulatory interactions across them, thus defining, between two such sites, a topologically associated domain, (TAD). How they work is not entirely clear, but they may stitch themselves to the nuclear membrane. They are thought to interact with a DNA pump called cohesin to extrude a loop of DNA between two insulator sites, thereby keeping that DNA clear of other interactions, at least temporarily, and locally clumped. The authors claim to find a new element called a distal tethering element (DTE), which works like an enhancer in promoting interaction between distant activating regulatory sites and genes, but doesn't actually activate. They just structure the region so that when a signal comes, the gene is ready to be activated efficiently. 

One theory of how insulator elements work. The insulator sites "CTCF motif" are marked on the DNA with dark blue arrow heads. They control the boundaries of action by the protein complex cohesin, which forms dimeric doughnuts around DNA and can pump DNA. Cohesins are central to the mechanisms of meiosis and mitosis. The net effect is to produce a segregated region of DNA as portrayed at the bottom, which should have a much higher rate of local interactions (as seen in the Micro-C method) than distant interactions.

At the largest scale, these authors claim that there are, in the whole fly genome and at this particular (early) point in development, 2034 insulator locations (TADs) and 620 tethering elements (TEs or DTEs). They show that DTEs in the locus they study closely play an active role in turning the nearby genes on at early times in development, and in directing activation from enhancers near the DTE, rather than ones farther away. What binds to the DTEs? So-called "pioneer" regulatory factors(such as Zelda) that have the power to make way through nucleosomes and other chromatin proteins to bind their target DNA. The authors say that these tether sites, once set up, are then stable on a permanent basis, through all developmental stages, even though the genes they assist may only be active transiently. 

The "poised" nature of some genes had been observed long ago, so it is not entirely surprising to see this mechanism get fleshed out a little, as a structural connection that is made between genes and their regulatory sites in advance of the actual activator proteins arriving at the associated enhancers and turning them on.

 

Final model: the normal case around the Antennapedia locus is shown at top, with insulator sites shown in pink, and tethering sites shown in teal. If one of the tethering elements is removed (middle), then the enhancer EE has less effect on the gene Scr, whose expression is reduced. If an insulator is removed (bottom), the re-organized domain allows the ftz gene's regulators, including the enhancer AE1, to affect Scr expression, altering its timing and location of expression.

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Origins in the Other: Moses the Egyptian

Stray notes on what Judaism owes to Egypt.

It is a bitter historical irony that Jesus was a Jew, (as were all the founding Christians), yet his religion was taken up by non-Jewish communities who turned its stories against its originators, casting Jews as the betrayers, stubborn heretics, and generally the other, with disastrous consequences. Well, something similar may have happened at the origin of Judaism, as there is a fascinating thread of historical scholarship and speculation that suggests that Moses was Egyptian, and that much of the religion of Judaism was generated as a mixture of inheritances from, or inversions of, the religion and cults of Egypt.

While the Bible is full of Moses stories, no other historical, let alone archeological, attestation exists. Thus the many authors who have striven to unearth the truth of what happened have had to be creative. The whole thing may be an out and out myth, or unrecognizably reworked. Freud wrote "Moses and Monotheism" as an exercise in retro-psychoanalysis, cutting his totemic father figure down in an imagined Oedipal paroxysm of murder, followed by remorse. Jan Assmann more recently, in "Moses the Egyptian", wrote an eliptical tale of cultural hints and suppressed memories and trauma continually expressed and re-interpreted over time in the "othering", adoption, and inversion of cultural patterns. Manetho, a third century BCE Egyptian priest, wrote a history that puts Moses, originally an Egyptian priest named Osarseph, at the head of a renegade (and leper, for good measure) army which terrorized Egypt sometime in the 1300-1500 BCE period, ending in their expulsion and exile. 

The Hyksos, a semitic people, had a distinctive look, in Egypt of ~1900 BC.

Ever since the reign of Akhenaten was unearthed in the late 1800's, it has been tempting to tie his monotheistic revolution (1353-1336 BCE) to that of Judaism, which was putatively founded in the same general time period, when Egypt was at its height of power and regional influence. In both cases, monotheism was a tough sell, and created antagonism that characterized both episodes. The Amarna period ended with complete reversal- Akhanaten being erased from his monuments and records, and Egypt returning to its traditional ways. Judaism, according to its own documents, and despite Moses's teaching, endured a lengthy period of conflict and consolidation before the monotheistic faction gained ascendence in the post-Babylonian exile period. It also generated the enduring enmity of neighboring polytheists, ultimately resulting in the military defeat and dispersal of the Jewish nation.

So what is the evidence? Moses is an Egyptian name. Like Tutmosis, Ahmose, Ramses, and many others, it means "is born", or "is child of". While there are both Hebrew and Egyptian etymologies possible, Moses is also described as practiced in all the arts of Egypt, including various forms of magic and the secret symbols, i.e. hieroglyphs. Egyptians practiced circumcision, which Judaism obviously adopted with gusto. Egyptians worshipped the ram (representing the leading god, Amun) and the bull (Osiris), which the Jews turned around and sacrificed in their rituals. Cooking meat in milk was an Egyptian practice, which may have been the source of the contrary interdiction in Kosher law. Judaism was anti-iconic, completely contrary to the abundant icons of, frankly, all the other polytheistic religions, though new icons have been snuck in, in the form of the ark of the covenant, the Torah scrolls, wailing wall, etc. The Thummim is a judicial badge and device for divination, taken from the Egyptians. It is indeed likely that originators of Judaism were assimilated Egyptians who left, whether by choice or not. The historian Tacitus noted the inverted character of Judaism vs the Egyptian religion. And Maimonides argued that the laws were a form of treatment for withdrawal from idol-addiction, in his case against the "Sabians", which in reality were the Egyptians, if they were any actual culture at all. But it served other purposes as well, such as cultural glue, which continues to be functional even when all other reasons have become irrelevant and many of the less convenient laws have been cast aside.

Whichever pharoh was the one described in Exodus, its Egyptological details, though accurate, come from a substantially later time, the 600's BC, when it was written, not from the time of the events. And Assmann argues that Manetho, for one, conflated several historical episodes to come up with his account. One was a Hyksos colony of semitic peoples that occupied northern (lower) Egypt through the second intermediate period (~1800 BC) to their defeat, about 1540 BC, by Kamose and his successor Ahmose, who were based in the south. There may have, however, been other incursions of semitic peoples from time to time, especially as records through the less organized periods of Egyptian history are sparse. A second episode was the Amarna period, which was officially suppressed, but which Assmann argues remained vivid as a traumatic memory of religious and existential revolution, informing an Egyptian official's view of "pollution" of the Egyptian culture by outsiders. 

Similarly, one can imagine that the idea of monotheism, so suddenly sprung upon the Egyptians, is something that was knocking around for longer periods of time, both before and since. Assmann goes through a long argument by Ralph Cudworth (who wrote long before the hieroglyphs were deciphered) about a possible "esoteric" theology of the Egyptians, which was monotheistic, while the cult for public consumption was polytheistic. That makes little sense, as all royal tombs and decorations hew (religiously!) to the standard story, and so clearly embody a full cast of characters, and their belief in the Osiris story and hope of continued life in the land of the un-dead. Nevertheless, even without such an esoteric/demotic split, it is natural to wonder about origins, such as where this family of gods arose from, which in turn would send thoughts in the direction of possible monotheism. Perhaps the incredible conservatism of Egyptian culture caused such thoughts to be ruthlessly stamped out, but also prone to occasional eruption in incovenient forms. We in our own time are experiencing the thrill of normative inversion, when a subculture decides that black is white, that all norms should be trampled, and a new god worshipped. 

Even if the Amarna period did not directly foster Moses and the Jewish form of monotheism, the latter owes a great deal to Egyptian culture, likely including some glimmer of the monotheistic idea. Within Judaism, it took a second (and certainly real) exile, in Babylon, to bring the monotheistic idea to fuller fruition, as the last set of prophets called for purification and repentance, the Torah was written down, and the second temple built.

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Mate Choice and the Origin of Species

The definition of a species is somewhat murky, because speciation is a continuous process. Which says something about race and racism as well.

Santa Claus brought me a delightful book about bird evolution, over the holidays. The writing is workman-like, and the treatment thorough. It covers the diversity, origins, and current status of birds, with an emphasis on how they inform evolutionary theory generally. One issue that is still live in evolutionary biology is the definition of a species. It is clear enough that birds and bees are different species, even different phyla. But are the six species of herring gull separate, or just sub-species? This is not just a headache for birders filling out their lists, but very much for biologists as well, in conservation, taxonomy, and evolutionary theory. How different do species have to be? Does any interbreeding / hybridization disqualify two populations from being different species? If not, how much does? And more importantly, at which point do such populations behave like different species, avoiding mating with each other, and maintaining distinct traits? This remains a difficult question, and is generally relevant, including for our own increasingly ramified family tree of ancestors.

One point that the author (Douglas Futuyma) makes, which I had not appreciated fully before, is that in the process of speciation, genetic barriers to successful mating between two species (or nascent species) come very late. In contrast, behavioral, ecological, or geographical forms of separation come early and are the real drivers. The examples are all around us as hybrid forms that can occur at range overlaps, not only among birds, but mammals as well. Polar bears can interbreed with other bears, though they don't want to! Species are recognized as separate by traits or molecular isolation long before they differ enough that they can't make viable hybrid offspring.

"So far, there doesn't seem to be any detailed evidence about how specific genes interact to cause incompatibility in hybrids, and why the genes diverged. But genetic incompatibility is seldom the cause of speciation in birds simply because premating isolation almost always evolves long before incompatibility does. Hybrids seem to be fully fit in about half of 254 different crosses between closely related species, many of which diverged as long as 5 million years ago. We can learn more about the causes of speciation if we focus on premating isolation, especially behavior. What causes behavioral isolation to occur?" p.169

This has some significant implications. Speciation turns out to arise largely from mate choice decisions, which we know in birds are highly discriminating. Why do they have all the plumage colors, song singing, and other mating behaviors, but to carry out the most careful evaluation of mates? The ideal mate is not very closely related. There are instinctive barriers to sibling mating, for instance. On the other hand, the ideal mate is also not very distantly related- not part of a differently colored sub-population or allied species. The plumage colors of birds are usually the clearest marks of speciation, both to us, and evidently to themselves- a way to keep straight who is who. On top of all that, the ideal mate has other properties that it may display during the courtship period, like vigor, courage to sing from the top-most branches, the ability to bring gifts of food, or to to construct an intricate bower.

The typical differentiation of nascent species happens due to physical separation, such as a founder finch flying to Hawaii, and starting an amazing adaptive radiation of honeycreepers and other birds. But sometimes, (as must have happened within the Hawaiian radiation of these birds), separation can be by habit or specialization within the same location- which is called sympatric speciation, or speciation in place. While difficult to demonstrate and reconstruct, this has been documented to occur, and again must be attributed to some kind of behavioral specialization and mating preference that overrides the heretofore mixed mating system of a local population.

A happy pair of horned puffins- not to be confused with tufted puffins!

It is evident that mate choice is a deeply significant and influential force. It causes all the decorative features typically displayed by males, but more deeply embodies and enforces the very concept of species as experienced by the species themselves. When a tufted puffin declines to mate with a horned puffin, it may not know whether offspring would have been possible or infertile, but it just knows that something isn't quite right about that prospective mate, and finds someone more suitable. And this is naturally relevant in humans as well. We are always making fine distinctions among ourselves in status and innumerable other qualities. Therefore it should come as little surprise that we have racist impulses, despite the fact that humans are, due to our rapid evolution and rampant internecine warfare, one of the least diverse species in existance, with no trace of genetic differentiation to support any kind of genetically based sub-speciation, races, etc.

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Covid Rings a Bell

How Covid causes such intense inflammatory reactions, via Toll receptors.

Those who have gotten a Covid vaccine might have an appreciation of our adaptive immune system. This comprises the B and T cells that learn about detailed features on pathogens that invade us, and then form, through an amazing feat of genetic reprogramming, novel proteins (such as antibodies) that can precisely zero in on those pathogens and call in lethal powers such as killer cells, phagocytes, neutrophils, etc. But there is another part, called the innate immune system, which is not adaptive, is evolutionarily older, and plays key roles in our day-to-day defense as well as in the beginning phases of the adaptive response. For how, after all, are pathogens recognized in the first place so that the adaptive response can even get started?

The innate immune system has many layers and actors, being very ancient. But one of its themes is that it recognizes "patterns", rather than the ultra-specific "epitopes" that the adaptive immune system gears itself up against. These patterns come in a two general forms, called pathogen-associated molecular patterns (PAMPs), which as the name implies come from outside, and damage-associated molecular patterns (DAMPs), which are typically cellular debris of various kinds that give notice that an injury has occurred. (There are also MAMPs and XAMPs for microbial and xenobiotic patterns, respectively.) PAMPs can include bacterial cell wall components like lipopolysaccharides and peptidoglycans, flagellin, fungal chitin, and a variety of nucleic acids such as double-stranded RNA characteristic of viruses, single stranded RNA, and DNA without special host markings.

What detects these patterns? Well, there are cell surface receptors for that, called the Toll-like receptors, or TLRs. Toll is a fly gene that was originally found to play important roles in development, (in German, it means "fantastic"), helping (together with its developmental ligand, Spätzle) embryos figure out their up/down, or dorsal/ventral polarity, and express the appropriate cell types to those locations. It was later found that fly adults use Toll for a totally different purpose- the activation of their innate immune systems, even though what pathogens it recognizes in that setting is still not known. All animals have TLRs, and plants have related systems, so this kind of thing is very ancient. Humans have at least 13 TLRs, so several bases are covered, making it difficult for a pathogen to completely escape this method of relatively crude detection.

The TLRs mediate immune responses against four general types of molecular patterns comprising damage/danger-associated molecular patterns (DAMPs), microbial/microbe-associated molecular patterns (MAMPs), pathogen-associated molecular patterns (PAMPs), and xenobiotic-associated molecular patterns (XAMPs). They do this by directly binding them. For instance, long double-stranded RNA, which is a common viral component, is detected by human TLR3. The TLR proteins form little hooks facing out from the cell. It takes two of them to fully bind one of the pattern molecules they detect, and when such a pair of TLRs forms, then their internal domains dimerize and set off a cascade of signaling inside the cell that serves as a danger alert. In this case, the antiviral program of interferon production, among many other genes, is activated. 

A structural model of a pair of TLR receptors, binding in this case a double stranded RNA. One TLR is blue, the other purple. One RNA strand is green, the other red. Binding the pathogenic pattern molecule drives dimerization of the receptor, which then causes its cell-internal domains to pair up and drive an activating signaling cascade.

Among the genes activated are signaling molecules called cytokines that alert and attract cells of the adaptive immune system ( T-cells and antigen presenting cells). Further genes are activated that process and present pieces of the pathogen pieces to those adaptive immunce cells. So the innate immune system, which has many other components, is a critical stage of our defensive system, both in its own right and as an initiator of the adaptive system.

This gets us back to Covid, where the SARS CoV2 virus causes extraordinary immune system activation in some people, a condition that is far worse than the original infection. A recent paper (review) suggested that the spike protein- the very same protein that is the first to dock to our respiratory epithelial cells, and the one that all the vaccines are made against, also binds in combinations of TLR2, TLR1, and TLR6. They do not show direct binding, but show that the spike protein by itself generates high TLR activity, and that this activity is composed of dimers of either TLR2+TLR6 or TLR2+TLR1. These receptors are known for the following activities:

• TLR2: Binds glycolipids from bacteria and also zymosan from fungi.
• TLR1: Binds lipopeptides, components of bacterial cell walls.
• TLR6: Binds diacyl lipopeptides, components of bacterial cell walls.

This is somewhat odd, since a pathogen would normally try to avoid triggering these sorts of receptors and responses. And a virus would particularly have little business activating a series of bacterial-specific TLR receptors. Covid may yet evolve towards evasion rather than activation, as it evolves to become endemic in humans and trade virulence for transmissibility. But for now, it is a here-and-gone kind of pathogen which seems to value immune activation and the associated respiratory expectoration that goes along with it. The authors also suggest that this activation may be a diversionary tactic, setting off the anti-bacterial alarms, and thus quieting the antiviral alarms, which the virus has more reason to fear. SARS CoV-2 is a wily antagonist, apparently purposefully activating some front-line immune defenses and landing many victims in a form of immune system over-activation called the cytokine storm.

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Some Theological Aspects of Modern Economics

Economics remains in a difficult intersection between science and humanities, with distinctly political and ideological conflicts.

We seem to be in a passion play about inflation right now. It is skyrocketing, or zooming, etc. It is a huge crisis. But, since it is measured year-over-year, maybe it is just a simple bounce from the depths of the pandemic when demand and prices, especially for gasoline, were negligible, and some businesses shut down. Now demand is back, but some sectors of the economy are having a hard time meeting demand, especially for workers, so prices are going up, by modest amounts. Some stories say that "inflation is never temporary". Others say the structural dislocations will pass and things will get back to normal. One can tell the ideology quite clearly from the story line. Conservatives have double motives to paint it a crisis, to disparage the current president (tax cuts had nothing whatsoever to do with this!), to support the preservation of capital and capitalists, and to generally box in policy makers from spending money on truly momentous objectives, like addressing climate change.

Why is this such a drama? Why isn't economics more of a science? In real sciences, you do not see competing schools of thought, such as the Chicago and the Keynesian schools of economics, the New Keynsians and the Modern Monetary Theorists, which last for decades and never seem to resolve their warfare. Maybe that is because real sciences don't study anything important. But more likely, real sciences have methods to efficiently describe and resolve their differences- with reference to reality- that economists do not seem to have. For in the macroeconomics realm, there is not a lot of experimentation that one can do. It is a field more like history, from which scholars and observers tend to draw the lessons they want to draw, not the ones that would serve them best. Or theology, whose subject is wholly illusory, such that its practitioners are not really in the business of studying anything observable at all, (or even discernible!), but in social management- how to build ideologies and propagandize with effective rhetoric, how to build churches, how to sermonize, whom to target in their weaker moments, what and whom to value, which social hierarchy to support, and how to do so.

Economics is far from illusory, and plenty of economists do the truly scientific work of describing the economy as it is, giving us the grist of statistics from which the theorists can spin their opinions. It is at the policy and macro level where things get theological, where moral and ideological commitments outweigh technocratic sense. For economics at the policy level is fundamentally Darwinian- how one wants to split the pie depends on who you think is worthy- morally and operationally. Economics is not intrisically democratic- far from. There are some who are worth more to the system, depending on one's standpoint. The Ricardians (with the Chicago school carrying on its supply-side banner) deemed production and producers the only important parts of the mechanism. Demand would take care of itself as long as producers were given maximum latitude to conduct business and trade as they wished. As the ideological cycle turned, entrepreneurs were once again the vanguard and watchword in the eighties and nineties. 

When it comes to inflation, similarly vast ideological forces are at work. The progressive Kenyesian policy environment of the 1960's was eroded, then eviscerated by Milton Friedman's and the Chicago school's general neo-Ricardian attacks during the 1970's, in our period of stagflation. It was genuinely destructive to experience inflation at relatively high levels, and the solution ended up being deep recessions ultimately authored by Jimmy Carter via his appointment of Paul Volker. The power of workers to bid for higher pay and inflation-protected pay was destroyed by de-unionization, outsourcing and off-shoring. Those forces largely remain today, suggesting that the current inflation blip will be transitory. 

Inflation is measured in consumer prices, so it largely reflects low-end wages that are spent most readily, rather than the stock market or other places where the rich invest. As long as wages are kept down, then inflation will be kept down as well. The big question is how the economy splits the pie- between wages at the low and middle levels, versus returns on capital / wealth and executive pay. This balance has been heavily out of kilter over the last few decades. This may have been great for keeping inflation down, but has obviously had highly corrosive effects on much else, from the opioid epidemic, to our great dependence on China for goods and supply chains, and our political breakdowns. So economics is not just about the economy, but about a great deal more- who we value and what vision we have for the future.

Keynes in his magnum opus had some wry comments on this phenomenon, in 1936:

"The completeness of the Ricardian victory is something of a curiosity and a mystery. It must have been due to a complex of suitabilities in the doctrine to the environment into which it was projected. That it reached conclusions quite different from what the ordinary uninstructed person would expect, added, I suppose, to its intellectual prestige. That its teaching, translated into practice, was austere and often unpalatable, lent it virtue. That it was adapted to carry a vast and consistent logical superstructure, gave it beauty. That it could explain much social injustice and apparent cruelty as an inevitable incident to the scheme of progress, and the attempt to change such things as likely on the whole to do more harm than good, commanded it to authority. That it afforded a measure of justification to the free activities of the individual capitalist attracted to it the support of the dominant social force behind authority."- John Maynard Keynes, The General Theory of Employment, Interest, and Money

• Resisting the lies is harder than you think.
• Sustainability is the big issue, and our politics are too small to address it.
• Democracy is hanging by a thread.
• And each side seems to think it is saving democracy, apparently. Though only one side does so undemocratically.
• Of course.. Republicans dedicated to state destruction will support crypto.

Choices, Choices

Hippocampal maps happen in many modes and dimensions. How do they relate to conscious navigation?

How do our brains work? A question that was once mystical is now tantalizingly concrete. Neurobiology is, thanks to the sacrifices of countless rats, mice, and undergraduate research subjects, slowly bringing to light mechanisms by which thoughts flit about the brain. The parallel processing of vision, progressively through the layers of the visual cortex, was one milestone. Another has been work in the hippocampus, which is essential for memory formation as well as mapping and navigation. Several kinds of cells have been found there (or in associated brain areas) which fire when the animal is in a certain place, or crosses a subjective navigational grid boundary, or points its head in a certain direction. 

A recent paper reviewed recent findings about how such navigation signals are bound together and interact with the prefrontal cortex during decision making. One is that locations are encoded in a peculiar way, within the brain wave known as the theta oscillation. These run at about 4 to 12 cycles per second, and as an animal moves or thinks, place cells corresponding to locations behind play at the trough of the cycle, while locations progressively closer, and then in front of the animal play correspondingly higher on the wave. So the conscious path that the animal is contemplating is replayed on a sort of continuous loop in highly time-compressed fashion. And this happens not only while the animal is on the path, but at other times as well, if it is dreaming about its day, or is resting and thinking about its future options.

"For hippocampal place cells to code for both past and future trajectories while the animal navigates through an environment, the hippocampus needs to integrate multiple sensory inputs and self-generated cues by the animal’s movement for both retrospective and prospective coding."

These researchers describe a new piece of the story, that alternate theta cycles can encode different paths. That is, as the wave repeats, the first cycle may encode one future path out of a T-maze, while the next may encode another path out of the same maze, and then repeating back to A, B, etc. It is evident that the animal is trying to decide what to do, and its hippocampus (with associated regions) is helpfully providing mappings of the options. Not only that, but the connecting brain areas heading towards the prefrontal cortex (the nucleus reuniens, entorhinal cortex, and parahippocampal gyrus) separate these path representations into different cell streams, (still on the theta oscillation), and progressively filter one out. Ultimately, the prefrontal cortex represents only one path ... the one that the rat actually chooses to go down. The regions are connected in both directions, so there is clearly top-down as well as bottom-up processing going on. The conclusion is that in general, the hippocampus and allied areas provide relatively unbiased mapping services, while the cortex does the decision making about where to go, and while it may receive.

    "This alternation between left and right begins as early as 25 cm prior to the choice point and will continue until the animal makes its turn"

A rat considers its options. Theta waves are portrayed, as they appear in different anatomical locations in the brain. Hippocampal place cells, on the bottom right, give a mapping of the relevant path repeatedly encoded across single theta wave cycles. One path is encoded in one cycle, the other in the next. Further anatomical locations (heading left) separate the maps into different channels / cells, from which the prefrontal cortex finally selects only the one it intends to actually use.

The hippocampus is not just for visual navigation, however. It is now known to map many other senses in spatial terms, like sounds, smells. It also maps the flow of time in cognitive space, such as in memories, quite apart from spatial mapping. It seems to be a general facility to create cognitive maps of the world, given whatever the animal has experienced and is interested in, at any scale, and in many modalities. The theta wave embedding gives a structure that is highly compressed, and repeated, so that it is available to higher processing levels for review, re-enactment, dreaming, and modification for future planning. 

Thus using the trusty maze test on rats and mice, neuroscientists are slowly, and very painfully, getting to the point of deciphering how certain kinds of thoughts happen in the brain- where they are assembled, how their components combine, and how they relate to behavior. How they divide between conscious and unconscious processes naturally awaits more insight into what this dividing line really consists of.

• Biochar!
• More about the coup attempt.
• Yes, there was electoral fraud.
• The more you know about fossil fuels, the worse it gets.
• Graph of the week. Our local sanitation district finds over a thousand omicron genomes per milliliter of intake, which seems astonishing.

Desperately Seeking Calcium

How cells regulate internal calcium levels.

Now that we are getting a crash course in molecular biology and evolution courtesy of the pandemic, many will be familiar with the intricate and dynamic activities of some proteins. The SARS spike protein doesn't just dock at a particular receptor on our pulmonary epithelial surfaces, but goes through a gymnastic routine to facilitate membrane fusion as well. Many other proteins have dynamic behaviors as well- something that was not fully appreciated back when structural biology was in its infancy and knowing anything about the structure of a protein or DNA or RNA required it to be locked into crystaline form for X-ray diffraction studies.

Another example came up recently, involving calcium regulation within cells. Calcium is a hugely important ion and regulator, central to core signaling cascades in all eukaryotic cells- to neuronal function, and to muscle activation, among many other roles. Our blood levels of calcium are tightly regulated, (to within a 20% range), mostly by way of an axis of parathyroid hormone between the parathyroid gland and the kidney, with additional effects from factors such as vitamin D, calcitonin, and estrogen. So our cells can rely on having a constant level of calcium on the outside. How do they maintain their levels internally?

One way is to have a large store socked away, as we have in bones for the body generally. Within cells, the endoplasmic reticulum (ER) turns out to have far higher concentrations of calcium than the rest of the cytosol, up to 10,000 fold. In muscle cells, the ER gets a special name- as the sarcoplasmic reticulum. Many calcium regulatory events rely on calcium being released briefly from the ER, having some effect, and then gradually getting pumped back in. But what if the ER is short of calcium? That would be a crisis!  

It turns out that we have a sensor system for that, llinking an ER protein called STIM1, which senses levels of Ca++ in the ER with a plasma membrane channel called ORAI1, which can open to let in Ca++ from the outside. A recent paper, (review), in combination with much other past work, demonstrates how STIM1 works. The two proteins turn out to interact directly, thanks to the fact that the ER, which is a huge organelle that extends all over the cell, always has some spots that interact with the plasma membrane, called membrane contact sites. These are strucured by other proteins, so there is a set distance between the two membranes, which must never fuse together. This means that while STEM1 can get very close to ORAI1 in the plasma membrane, there will still be a gap between them. How to bridge it?

Overall model for how STIM1 works. The luminal side sticks into the ER and binds calcium (red dots). If levels are low, the protein dimerizes at the transmembrane and internal domains, causing extensive refolding of the external domains residing in the cytosol. This causes them to straighten out and span the space of the contact structure between the ER and the plasma membrane, where it activates the ORAI1 calcium channel protein by direct contact.

The STIM1 protein turns out to provide the bridge, in the form of a transformer-style mechanism that shifts it from a compact blob on the ER when calcium levels are high, to an extended rod that pokes into ORAI1, activating it, when calcium levels are low. Since it is the ER-internal level of calcium that needs to be sensed, it is the ER-internal (or luminal) portion of the STIM1 that does this sensing. It has about five calcium binding sites that, if filled, prevent its dimerization, but which if empty, promote it. Internal dimerization induces a dramatic refolding of the cytoplasmic portion of STIM1 into the active, extended rod. 

These authors were faced with a situation where the full STIM1 protein was apparently impossible to crystalize, so no full structure was available. Worse, some of the prior structural studies of fragments of STIM1 conflicted with each other. So they turned to very clever method to probe structural dimensions point by point, called fluorescence (or Förster) resonance energy transfer, (FRET). If by mutation or chemical modification one installs fluorescent molecules on a protein of interest, indeed installs two different ones, one of whose absorbtion spectrum overlaps with the emission spectrum of the other, one can measure quantitatively the distance between them.

How the FRET fluorescence method works. Different fluorophores are placed on the protein of interest, here the EFSAM luminal domain of STIM1. The absorption spectrum of one (acceptor) overlaps the emission spectrum of the other fluorophore (donor). In the first graph, the green graph shows that when the two are combined on the same molecule, emission from the acceptor goes up dramatically, due to its proximity-dependent absorbance of emissions from the donor fluorophore. The second graph shows how this protein responds to calcium, by increasing interaction (absorbance-emission intensity at 620 nm, reflecting the physical distance between the fluorescence probes) as Ca++ concentration goes down.

 

By placing fluorescence probe pairs all over the external regions of STIM1, these authors were able to definitively refute one of the prior structural models, and then outline the probable sequence of events by which STIM1 opens up into its active form. The image above ably summarizes their model, by which the ORAI1-interacting domain (CC2/CC3) is stored upside-down and inside out in the inactive conformation. It is quite a proposal, all carried out by domains which are alpha helixes hinged at strategic locations and obviously highly sensitive to slight changes in the structure, induced by the dimerization outlined above, in low calcium conditions.

Finally, they investigated a mutation which in humans causes Stormorken syndrome, a wide-ranging set of deficiencies including bleeding, dyslexia, muscle weakness, and hypocalcemia. In molecular terms it is a "gain of function" mutation. It weakens the interactions that keep STIM1 closed during high calcium conditions, so promotes its stimulation of ORAI1 and excess uptake by cells all over the body. The mutation changes argenine at position 304 in STIM1 to tryptophan, which has much different characteristics. It is genetically dominant, meaning that a single allele, combined with a wild-type allele on the other chromosome, gives the syndrome. Thus it is a powerful mutation, tweeking the sensitivity of this system just enough to screw up a lot of physiology. Deletions of this gene are not lethal, however, in part because there is also a STIM2 gene that encodes a similar function.

Analysis of the effect of the Stormorken mutation (R304W) on the physical proximities and overall shape of the STIM1 protein. The FRET graphs track different probe pairs that were placed all over the cytosolic (folding) portion of STIM1. In these graphs, degree of FRET relative frequency shift/communication is on the X axis, while photon counts are on the Y axis. They show noticeable shifts in distances, reflected in the structural model. The mutation significantly loosens up the high-calcium folded state, inducing more Ca++  influx when it is not needed.

So, we are just full of little machines, developed and refined over the billions of years in the ongoing race to live a little better, keep things humming, and to defend ourselves against all the other machines, such as parasitic viruses.

• How the new space telescope is unfolding, and where.
• The history of another pandemic- tuberculosis.
• The importance of relationships, and trust.
• Nuttery and religion go together.
• How to plan and build a city.

Eugenics is All the Rage

Animal breeders have no qualms directing intensive systems of artificial selection.

Eugenics is defined with reference to humans, as any consideration or implementation of artificial selection. There is little doubt that it would be effective, but there is some disagreement about what an "improvement" would represent. We are not cattle to be bred to specification, but organisms with dignity and freedom- specifically freedom from meddling by others in our reproduction. Wild animals have this freedom as well, by default. But domestic animals- that is a different story. For all our "humane" societies and pampering of some, our treatment of others is distinctly undignified. And that includes their breeding. 

Across the domestic animals, from racing horses and show dogs to dairy cows and chickens, breeding these days is carried on at unprecedented intensity, with the most advanced scientific and statistical techniques. For farm animals, this has led to inbreeding and alarming malformations, such as chickens that can't walk, and cows with chronic udder infections. For dogs, the creation of fundamentally malformed breeds also leads to chronic suffering, (short snouts, short legs), as does lack of care in breeding for temperamental health.

These animals have serious problems, of a genetic nature.

Animal breeding has progressed through three major stages. First is the traditional approach, using hunches and personal judgements- using the best animals, and perhaps cross-breeding with animals from other farms to retain diversity, if any directed breeding is done at all. With a relaxed approach, this led to generally good results, establishing the great dog breeds and other livestock, where hardiness and health were always prominent values. But in pigeon, cat, dog, and other casual breeding since Victorian times, amateur breeding like this can also go rather astray. 

In modern livestock breeding, this was superseded by the use of Estimated Breeding Value, or EBV, which is a systematized way to account for the genetic, rather than phenotypic trait quality in animals, by accounting for their relatives, as far as they have been measured, and also by accounting for uncertainties around heritability and systematic and environmental effects on the trait of interest. This concept puts breeding on a far more scientific basis, with quantification of traits, and of pedigrees. One result is that the breeding value can be estimated for animals who do not even have the trait, such as male dairy cattle. Another has been that animal breeding has been even more relentlessly driven to meet commercial and consumer objectives, even ones that shift over time as tastes change.

Naturally, the EBV method has now been supplemented by DNA-based evaluations in more recent times. The ability to "see" into the genome by sequencing some or all of it, thereby establishing a landmark map based on variants distributed throughout, allows the traits (if linked to such landmarks) to be tracked in all individuals, regardless of phenotype, and even in individual gametes and fetuses. This dramatically reduces the lottery that otherwise is genetics. However, its value is significantly bounded by the fact that most interesting and desirable traits are usually not genetically simple (like, say, eye color), but are complex, influenced in very small amounts by many different loci / genes. 

This is a frontier for animal rights and humane policy development, that animals not only should be treated well, but bred well. In livestock breeding, European countries have some relatively aspirational standards and laws, the US lacks even that. The "standards" used by such organizations are the American Kennel Club are worse than nothing, as they drive breeding for looks alone, and welcome the most obscure and unhealthy breeds, regardless of grave malformations, temperamental disasters, and inbreeding. While health of the animal needs to be paramount, other issues such as the ability of animals to live without special care and infrastructure, and genetic diversity, also need to be addressed, if we are going to be serious stewards of animals in our care.

• Our inert politics.
• The sleaze never let up.

The Cycle of Kingship

History shows a repeated cycle between democratic forms and autocratic forms of politics.

Before their history, the Greeks had another long history- the Mycenaean age. As reflected in the Homeric epics, it was an age of kings, palaces, and courts. But by the time of classical Greece, their politics had progressed to a spectrum of aristocracies, oligarchies, and democracies. In Athens, democracy was a sort of aristocracy, as it was far from including all the people of the city. But at any rate, the relatively egalitarian system established in Athens spurred an age of innovation and empire, still culturally influential down to today. Rome likewise progressed in its early days from kings to a republic of the most complex and rigorous kind. Again, it was largely a collective aristocracy of the well-to-do, but represented significant political progress, and again spurred several centuries of growth and empire.

How surprised they would have been to know that kingship would make a comeback as the norm of European political organization for almost the next two thousand years. The decline and fall of the Roman empire is a story of rising autocracy, from the Augustan arrangement that saved the appearance of the Senate and Republic, down to the frank autocracy of Constantine and his successors through the Byzantine Empire. 

In recent times, we have experienced a similar burst of innovation and growth from the egalitarian political systems modeled on Enlightenment ideals. It has been two and a half centuries of social progress and movement towards greater democracy. Indeed, with the end of the Cold War, we had thought a New World Order was on the horizon, and history itself had come to a conclusion. That all countries would inevitably adopt the Western model and live happily ever after, tended to by a European Union-style bureaucracy. 

Well, perhaps not total democracy.

What hubris! Our shock is most keen in the cases of Russia and China, which went through existential crises in their conflicts with the West and with basic economics, and were thought would inevitably open up politically as they freed themselves from the shackles of the various communist -isms of the twentieth century. But deeper political patterns were at work. Neither country had ever experienced functional democracy. Unlike the West with its fleeting (if glorious) experiences of Republican and Democratic systems, neither had ever gotten even to that point. Indeed their traumatic experiences of communism had originated as innovative, supposedly democratic political ideas from the West, which immediately curdled into the most cruel sorts of despotism.

And obviously the US itself, at the very height of its political dominance, is now beset by a would-be king. Autocracy turns out to have numerous points in its favor. First is a fundamental psychological archetype, based on the family and richly embodied by religions like Christianity. One must have a god, a father- someone to bow down to and beseech. In our infant democracy, George Washington took the truly radical step of stepping down from the presidency and retiring to private life. But now, the "base" can not fall over themselves fast enough to worship their leader, to withstand any lie or abuse as long as their side beats the other side, and installs itself in power, however corrupt.

Autocracy has, additionally, a sort of Darwinian logic to it. Our own founders in the Federalist papers and elsewhere explicitly feared the rise of a demagogue who could so twist the people from their better judgement, best interests, and hallowed institutions as to leave the constitutional system in tatters. Well, here we are. And we see it all over- in Julius Caesar and the disintegration of the Roman triumverate, in the rise of Napoleon, in the rise of Vladimir Putin and Xi JinpIng- the truly talented leader with the ability and the desire can shake the foundations of current institutions and remake the political system. Our current would-be king may be a man of fewer talents, yet seems to excite many to insurrection and destruction of democratic political institutions. It may look like a farce, but it is only a farce until they get to write the history books. Then it is glorious.

One must also take a hard look at democracy. There seems to be a dynamic where early democracies are limited to an elite, like the early United States. As the franchise is broadened by the natural / internal logic of democracy, the elites have less of a stake in it, and may indeed be attacked by the state. That leads to the danger of a leader like Julius Caesar taking an anti-elite position, using people against the elites, and installing, not a better democracy, but a dictatorship. Where does the defect lie? The fact is that true democracy is unworkable. We see in California the poor decisions frequently made by the ballot box on referendums. Without controlling the media and education environment in a rigorous fashon for the public interest, good public policy has little chance against moneyed interests, propaganda, and apathy. So the search through history has been for representative or other delegating systems that raise the most talented and public-spirited people to decision-making positions. But there can be no permanent proof against shamelessness, greed, and the other inherent vices of humanity.

• How's the next king doing?
• Ukraine is about the coherence of the whole Russian-dominated eastern sphere, like keeping Belarus from cracking up.

The RNAs Shall Protect Us

The humble skin mole has at least one oncogenic mutation. But it is not cancer- why not?

We know that mutations cause cancer. But we also know that it takes multiple mutations, not just one, in virtually all cases. This is one reason why age is such a strong risk factor, providing the time to accumulate multiple "hits". One place where this is particularly apparent is the skin. Most people have moles (nevi) and other imperfections, which are no cause for alarm. We are also on the lookout for the unusual signs and forms that indicate melanoma- which truly is a cause for alarm. Moles typically have one of the key oncogenic mutations for melanoma, however: BRAF V600E (which means the 600th amino acid in its protein chain has been changed from valine to glutamic acid). So what is behind the difference? What systems do cells and organs have to keep this train on the tracks, despite a wheel or two coming off?

A recent paper (review) explored this issue, and tells a complicated technical and scientific story. But the bottom line is that certain miRNAs- a novel form a gene regulator discovered just in the last couple of decades- form a firewall against further proliferation. The BRAF mutation is an activating change, which disrupts the normal "off" state of this protein kinase. BRAF is a protein kinase that attaches phosphate groups to serines and threonines on other proteins. And some those other proteins are specifically other (MAP) protein kinases that form cascades promoting cell proliferation and differentiation. In the case of melanocytes in the skin, the BRAF mutation promotes just that: proliferation, mole formation, and, in some cases, progression to full blown melanoma. 

What is a skin mole? Well, it clearly is composed of lots of cells, so whatever is arresting the mutant BRAF-activated proliferation is taking its sweet time. Proliferation goes for a while, but then stops for an unknown reason. It had been thought in the field (and by these researchers as well) that mole cells had gone into senescence- an irreversible division arrest that is frequently activated in cancer cells and is similar to age-dependent cell cycle arrest. But they show now that senescence is not the explanation. If the BRAF mutation state is reversed, the cells resume dividing. And they also have other hallmarks of a different form of (G2/M) cell division arrest. So something more dynamic is going on.

They do a few technical tours de force of modern DNA sequencing and large-scale molecular biology to find what unusual genes are being expressed in these cells, and find two:  MIR211-5p and MIR328-3p. These are miRNAs, which are short RNA pieces that repress the expression of other genes. We have thousands of them, and each can repress hundreds of other genes, forming a somewhat crazy interdigitated regulatory network. They evolved from an immune function of repressing the expression of viruses and other foreign DNA, but have been repurposed to have broad regulatory effects, often in development and disease.

In BRAF-activated skin mole cells, these miRNAs have one effective target, which is AURKB (Aurora B kinase), another protein kinase that is needed for cell division. No AURKB, no cell division. Indeed, skin mole cells have a high rate of cells stuck in the last phase of cell division, with 4 genome equivalents. They found that AURKB has low expression in skin mole cells, but high expression, as expected, in melanoma cells, while the miRNAs had the reverse pattern. And tellingly, artificial inhibition of these miRNAs released mole cells from their proliferation arrest and allowed the BRAF mutation to have its way with them.

Model of this paper's findings about melanocytes. Starting with stem-like melanocytes, mutated BRAF can cause oncogenenic or pre-oncogenic proliferation. Separately, TPA, or some local tissue factor like TPA, can encourage stem melanocytes to grow and differentiate properly into mature melanocytes. But those same activators (TPA and its natural analog) increase miRNA expression of particularly MIR211-5p, which (by inhibiting AURKB) arrests growth as part of the differentiation program, and also shuts down proliferation caused by mutated BRAF, (at late mitosis / G2 arrest), at least most of the time.

But there was still a problem- what activates the miRNA gene expression in the natural setting? It isn't the mutated BRAF protein, since it routinely drives cells through several replication cycles to form moles, and didn't have any regulatory effect on the miRNAs. The researchers focused on the kinds of local secreted hormones, like endothelin, that might locally inhibit overgrowth of cells, and logically lead to a mole-like pattern. What they hit on was TPA, an artificial analog of diacylglycerol, which is an activator of yet another protein kinase, PKC. TPA is paradoxically a tumor promoter, and is routinely used in cell culture systems to goose the proliferation of melanocytes. But for the mutated BRAF- driven cells from moles, TPA arrests their growth, and it does so because PKC activates the expression of MIR211-5p. They showed that taking TPA out of their cell culture mixes dramatically restarted the growth of mole-derived and other BRAF mutation-driven cells. So this closes the circle in some degree, explaining how it is that skin moles form as sort of arrested mini-cancers.

Unfortunately, TPA is not a natural chemical, and diacylglycerol is not hormone, though many hormones, such as thyroid hormone and oxytocin, do affect PKC activity. So the natural PKC and miRNA activator, and inhibitor of excess proliferation in these BRAF mutation-driven melanocytes remains unknown. I am sure that this research group will be hunting diligently for it, since it is an extremely interesting issue not just in oncology, but in skin and tissue development generally.

• The walls may be closing in.
• Jung on Trump.
• Yeah, there was voter fraud.
• Electrification.
• Those precarious human-soil relations.
• Plastic rain.
• Efficient housing.