Ground Truth for Genetic Mutations

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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• We are killers.

Dopamine: Get up and Go, or Lie Down and Die

The chemistry of motivation.

A recent paper got me interested in the dopamine neurotransmitter system. There are a limited number of neurotransmitters, (roughly a hundred), which are used for all communication at synapses between neurons. The more common transmitters are used by many cells and anatomical regions, making it hazardous in the extreme to say that a particular transmitter is "for" something or other. But there are themes, and some transmitters are more "niche" than others. Serotonin and dopamine are specially known for their motivational valence and involvement in depression, schizophrenia, addiction, and bipolar disorder, among many other maladies.

This paper described the reason why cancer patients waste away- a syndrome called cachexia. This can happen in other settings, like extreme old age, and in other illnesses. The authors ascribe cachexia (using mice implanted with tumors) to the immune system's production of IL6, one of scores of cytokines, or signaling proteins that manage the vast distributed organ that is our immune system. IL6 is pro-inflammatory, promoting inflammation, fever, and production of antibody-producing B cells, among many other things. These authors find that it binds to the area postrema in the brain stem, where many other blood-borne signals are sensed by the brain- signals that are generally blocked by the blood-brain barrier system.

The binding of IL6 at this location then activates a series of neuronal connections that these authors document, ending up inhibiting dopamine signaling out of the ventral tegmental area (VTA) in the lower midbrain, ultimately reducing dopamine action in the nucleus accumbens, where it is traditionally associated with reward, addiction, and schizophrenia. These authors use optically driven engineered neurons at an intermediate location, the parabrachial nucleus, (PBN), to reproduce how neuron activation there drives inhibition downstream, as the natural IL6 signal also does.  

Schematic of the experimental setup and anatomical locations. The graph shows how dopamine is strongly reduced under cachexia, consequent to the IL6 circuitry the authors reveal.

What is the rationale of all this? When we are sick, our body enters a quite different state- lethargic, barely motivated, apathetic, and resting. All this is fine if our immune system has things under control, uses our energy for its own needs, and returns us to health forthwith, but it is highly problematic if the illness goes on longer. This work shows in a striking and extreme way what had already been known- that prominent dopamine-driven circuits are core micro-motivational regulators in our brains. For an effective review of this area, one can watch a video by Robert Lustig, outlining at a very high level the relationship of the dopamine and serotonin systems.

Treatment of tumor-laden mice with an antibody to IL6 that reduces its activity relieves them of cachexia symptoms and significantly extends their lifespans.

It is something that the Buddhists understood thousands of years ago, and which the Rolling Stones and the advertising industry have taken up more recently. While meditation may not grant access to the molecular and neurological details, it seems to have convinced the Buddha that we are on a treadmill of desire, always unsatisfied, always reaching out for the next thing that might bring us pleasure, but which ultimately just feeds the cycle. Controlling that desire is the surest way to avoid suffering. Nowhere is that clearer than in addiction- real, clinical addictions that are all driven by the dopamine system. No matter what your drug of choice- gambling, sugar, alcohol, cocaine, heroin- the pleasure that they give is fleeting and alerts the dopamine system to motivate the user to seek more of the same. There are a variety of dopamine pathways, including those affecting Parkinson's and reproductive functions, but the ones at issue here are the mesolimbic and mesocortical circuits, that originate in the midbrain VTA and extend respectively to the nucleus accumbens in the lower forebrain, and to the cerebral cortex. These are integrated with the rest of our cognition, enabling motivation to find the root causes of a pleasurable experience, and raise the priority of actions that repeat those root causes. 

So, if you gain pleasure from playing a musical instrument, then the dopamine system will motivate you to practice more. But if you gain pleasure from cocaine, the dopamine system will motivate you to seek out a dealer, and spend your last dollar for the next fix. And then steal some more dollars. This system shows specifically the dampening behavior that is so tragic in addictions. Excess activation of dopamine-driven neurons can be lethal to those cells. So they adjust to keep activation in an acceptable range. That is, they keep you unsatisfied, in order to allow new stimuli to motivate you to adjust to new realities. No matter how much pleasure you give yourself, and especially the more intense that pleasure, it is never enough because this system always adjusts the baseline to match. One might think of dopamine as the micro-manager, always pushing for the next increment of action, no matter how much you have accomplished before, no matter how rosy or bleak the outlook. It gets us out of bed and moving through our day, from one task to the next.

In contrast, the serotonin system is the macro-manager, conveying feelings of general contentment, after a life well-lived and a series of true accomplishments. Short-circuiting this system with SSRIs like prozac carries its own set of hazards, like lack of general motivation and emotional blunting, but it does not have the risk of addiction, because serotonin, as Lustig portrays it, is an inhibitory neurotransmitter, with no risk of over-excitement. The brain does not re-set the baseline of serotonin the same way that it continually resets the baseline of dopamine.

How does all this play out in other syndromes? Depression is, like cachexia, at least in part syndrome of insufficient dopamine. Conversely, bipolar disorder in its manic phase appears to involve excess dopamine, causing hyperactivity and wildly excessive motivation, flitting from one task to the next. But what have dopamine antagonists like haloperidol and clozapine been used for most traditionally? As anti-psychotics in the treatment of schizophrenia. And that is a somewhat weird story. 

Everyone knows that the medication of schizophrenia is a haphazard affair, with serious side effects and limited efficacy. A tradeoff between therapeutic effects and others that make the recipient worse off. A paper from a decade ago outlined why this may be the case- the causal issues of schizophrenia do not lie in the dopamine system at all, but in circuits far upstream. These authors suggest that ultimately schizophrenia may derive from chronic stress in early life, as do so many other mental health maladies. It is a trail of events that raise the stress hormone cortisol, which diminishes cortical inhibition of hippocampal stress responses, and specifically diminishes the GABA (another neurotransmitter) inhibitory interneurons in the hippocampus. 

It is the ventral hippocampus that has a controlling influence over the VTA that in turn originates the relevant dopamine circuitry. The theory is that the ventral hippocampus sets the contextual (emotional) tone for the dopamine system, on top of which episodic stimulation takes place from other, more cognitive and perception-based sources. Over-activity of this hippocampal regulation raises the gain of the other signals, raising dopamine far more than appropriate, and also lowering it at other times. Thus treating schizophrenia with dopamine antagonists counteracts the extreme highs of the dopamine system, which in the nucleus accumbens can lead to hallucinations, delusions, paranoia, and manic activity, but it is a blunt instrument, also impairing general motivation, and further reducing cognitive, affect, parkinsonism, and other problems caused by low dopamine that occurs during schizophrenia in other systems such as the meso-cortical and the nigrostriatal dopamine pathways.

Manipulation of neurotransmitters is always going to be a rough job, since they serve diverse cells and pathways in our brains. Wikipedia routinely shows tables of binding constants for drugs (clozapine, for instance) to dozens of different neurotransmitter receptors. Each drug has its own profile, hitting some receptors more and others less, sometimes in curious, idiosyncratic patterns, and (surprisingly) across different neurotransmitter types. While some of these may occasionally hit a sweet spot, the biology and its evolutionary background has little relation to our current needs for clinical therapies, particularly when we have not yet truly plumbed the root causes of the syndromes we are trying to treat. Nor is precision medicine in the form of gene therapies or single-molecule tailored drugs necessarily the answer, since the transmitter receptors noted above are not conveniently confined to single clinical syndromes either. We may in the end need specific, implantable and computer-driven solutions or surgeries that respect the anatomical complexity of the brain.

• Thoughts about corruption.
• The Fed is extraordinarily powerful.
• But our Stable Genius is going downhill.
• The real deal with autism.

My Religion is Star Trek

Denial of death and the origin of evil- Ernest Becker on religion.

I have always wondered about the purpose of clothes. Nudists obviously do as well. Sometimes you need to keep warm. But most of the time, clothes are a cultural convention full of signifiers of taste, status ... and something else. That something else is the illusion that we are not animals. Positively, absolutely, something wholly different and on another plane of existence. Not animals. 

Even a century and a half after Darwin explained that we are animals, there are plenty of people who cling to various stories of denial. But these stories have purposes that go well beyond this ontological illusion. Because not only are we animals, but we are animals without meaning. Animals that will die. That is, no meaning is given objectively. So just as we clothe our bodies with fabric, we clothe our spirits with illusions of meaning, for otherwise we could not live. 

I have been following a provocative podcast series, which spent a couple of episodes on Ernest Becker, a mid-20th century philosopher in the US. He posited that we all follow a religion, in the anthropological sense that we live in cultural structures that give us meaning. Structures that are fundamentally illusory, because there is no there there. Meaning has always been generated by us, for us, subjectively by our psychological proclivities for social connection and drama. We are psychologically adapted to make and seek meaning, though in the final analysis, however powerful they feel, these are all conjured, not given. Take Disney as an example. Many people get highly involved with, and take solace from, the narratives Disney puts out, in its parks, cruise ships, movies, merchandise, and other channels. Relentless provision of mechanically assembled archetypes and other psychological triggers that activate / soothe, inspire, and motivate apparently has a substantial market. 

While atheists take no end of potshots at the absurdities and hypocrisies of formal religions, they also live (and must live) in some sort of illusion themselves. The idea that learning and science makes for a more "objective" value system and life of meaning may be less absurd, but is no more objective. These values come with a rationale and a story, one of service to ultimately human ends of knowledge and betterment. But that doesn't make them true- just another set of values that must be gauged subjectively. And when measured by the ironic criterion of Darwinian success in promoting reproduction, they often turn out to be lacking. At the most basic level, getting through the day requires some kind of motivation, and that motivation, when it goes beyond the most animal requirements, requires meaning, which requires us to have some story that narrates a purpose to a life whose end is otherwise irredeemably meaningless. 

There is a problem, however, to Becker. The more enveloping and functional the narrative of meaning, the more any competing narrative becomes alien and threatening. Indeed, threatening narratives become evil. Thus Judaism became the nemesis of Christianity, and Catholicism that of Protestantism. If the meaning of our lives, in a spiritual and eternal sense, is devalued by another story that has competing status, there is no limit to our horror at its doctrines or our dehumanization of its adherents. Thence to crusades, religious wars, pogroms, and the delicately named "communal violence". The management of narratives of meaning thus is perhaps the most critical aspect of human affairs, as all religious leaders have known forever.

One can see the US civil war through this lens. The people of the South, wedded to slavery, justified it through their theology and culture. They were mortally offended by the busybodies of the North who dared cast aspersions on their moral narratives and justifications, not to mention their economic basis. Where "Uncle Tom's Cabin" may have broken through the indifference of Northern culture, it was met with outrage in the South- a stout defense of their powerful cultural and religious narratives. The conflict was spiritual and existential.

Becker did not have terribly novel solutions to the problems of meaning and counter-meaning. Just the meta prescription that arose in the enlightenment, secularism and in all the branches of modern psychology. Which is that understanding this dynamic and taking one's stories less seriously is the path to social peace. It may not be the path to optimal personal meaning, however. How do you compare the smorgasbord of Disney, mainline religion, Western Buddhism, science, and a thousand other sects and value systems to a traditional society with one church, one story, and one universe? The power of social and spiritual unity must have been tremendously validating and comfortable. So there has been a big tradeoff to get to our current state of social and spiritual innovation, plurality, and anomie. It is evident that our political moment is one of deep spiritual revanchism- of revulsion (by the more traditional-minded) against all this plurality, back towards a more benighted unity.

• Only Catholics go to heaven.
• Religious zealots have no clue whatsoever.
• Homelessness as a problem of affluence, gentrification, and too-good policing.
• But crime in DC? We know where that is.
• Cutting off our health to spite our libtards.
• The state of cars.

What Happens When You Go on Calorie Restriction?

New research pinpoints some very odd metabolites that communicate between the gut and other tissues.

The only surefire way to extend lifespan, at least so far, is to go on a calorie restricted diet. Not just a little restricted- really restricted. The standard protocol is seventy percent of a normal, at-will diet. In most organisms tested, this maintains health and extends lifespan by significant amounts, ten to forty percent. There has naturally been a great deal of interest in the mechanism. Is it due to clearance of senescent cells? Increased autophagy and disposal of sub-optimal cell components? Prevention of cancer? Enhancement or moderation of the immune system? The awesome austerities of the ancient yogis and other religious hermits no longer look so far out.

A recent pair of papers took a meticulous approach to finding one of the secret ingredients that conveys calorie restriction to the body. They find, to begin with, that blood serum from calorie-restricted mice can confer longevity properties to cultured cells. Blood from calorie-restricted mice can also confer relevant properties to other mice. This serum could be heated to 133 degrees without detriment, indicating that the factor involved is not a large protein. On the other hand, dialysis to remove small molecules renders it inactive. Lastly, they put the serum though a lipid-binding purification system, which diminished its activity only slightly. Thus they were looking for a mostly water-soluble metabolite in the blood.

Traditionally, the next step would have been to conduct further fractionation and purification of the various serum components. But times have changed, and their next step was to conduct a vast chemical (mass-spectrometry) analysis of blood from calorie restricted vs control mice. Out of that came over a thousand differential metabolites. Taking the most differential and water-soluble metabolites, they assayed them over their cultured cells for a major hallmark of calorie restriction, activation of the AMPK kinase, a master metabolic regulator. And out of this came only one metabolite that had this effect at concentrations that are found in the calorie restricted serum: lithocholic acid.

Now this is a very odd finding. Lithocholic acid is what is known as a secondary bile acid. It is not natively made by us at all, but is a byproduct of bacterial metabolism in our guts, from a primary bile salt secreted by the liver, chenodeoxycholic acid. These bile salts are detergent-like molecules made from cholesterol that have charged groups stuck on them, and can thus emulsify fats in our digestive tract. Bile salts are extensively recycled through the liver, so that we make a little each day, but use a lot of them in digestion. How such a chemical became the internal signal of starvation is truly curious.

The researchers then demonstrate that lithocholic acid can be administered directly to mice to extend lifespan and have all the expected intermediary effects, including decreased blood glucose levels. Indeed, it did so in flies and worms as well, even though these species do not even see lithocholic acid naturally- their digestion is different from ours. They evidently still have key shared receptors that can recognize this metabolite, perhaps because they use a very similar metabolite internally. 

And what are those receptors and the molecular pathway of this effect? The second paper in this pair shows that lithocholic acid binds to TULP3, a member of the tubby protein family of transcription regulators, which binds to lipids in the plasma membrane and shuttles back and forth to the nucleus to provide feedback on the membrane condition or other events at the cell surface. TULP3 also binds to sirtuins, which are notoriously involved in metabolic regulation, aging, and cell suicide. These latter were all the rage when red wine was thought to extend life span, as sirtuins are activated by resveratrol. At any rate, sirtuins inhibit (by de-acetylation) the lysosomal acidification pump that has been known (curiously enough) to go on to inhibit AMPK, and play basic roles on cell energy balance regulation.

So we are finally at AMPK, a protein kinase that responds to AMP, which is the low-energy converse of ATP, the energy currency of the cell. High levels of AMP indicate the cell is low on energy, and the kinase thus phosphorylates and thus regulates a huge variety of targets, which generally increase energy uptake from the blood, (glucose and fats), reduce internal energy storage, and increase internal recycling of materials and energy, among much else. At any rate, the researchers above show that AMPK activation is absolutely required for the effects of lithocholic acid, and also for life extension more generally from calorie restriction. On the other hand, lithocholic acid does not touch the AMP level in the cell, but, as mentioned above, activates through a much more circuitous route- one that goes through lysosomes, which are sort of the stomach of the cell, and are increasingly recognized as a central player in its energy regulation.

Some beautiful antibody staining to different types of myosin- the key motor protein in muscles- in muscle cells from various places in the mouse body (left to right). The antibodies are to slow-twitch myosin (red), which marks the more efficient slow muscle fibers, and blue and green, which mark fast twitch myosins- generally less efficient and markers of aging. The bottom two rows are from a AMPK knockout mouse, where there is little difference between the two rows. The pairs of rows are from normal (top) and (bottom) a specific mutant of the lysosomal proton pump that has its acetylation sites all mutated to inactivity, thus mimicking in strongest form the action of the sirtuin/lithocholic acid pathway. Here, (second row, especially last frame), there is a higher level of red vs blue, and of blue vs green staining/fibers, suggesting that these muscles have had a durable uptick in efficiency and, implicitly, reduced atrophy and higher longevity.

These are some long and winding roads to get to positive effects from calorie restriction. What is going on? Firstly, biology does not owe it to us to be concise or easy to understand. Lithocholic acid is going to always be around, given that we eat food, but has mostly, heretofore, been regarded as toxic and even carcinogenic. It, along with the other bile acids, bind to dedicated transcription regulators that provide feedback control over their synthesis in the liver. So they are no strangers to our physiology. The authors do not delve into why calorie restriction durably raises the level of lithocholic acid in the gut or the blood, but that is an important place to start. It might well be that our digestive system squeezes every last drop of nutrition out of meagre meals by ramping up bile acid production. It is a recyclable resource, after all. This could have then become a general starvation signal to tissues, in advance of actual energy deficits, which one would rather not face if at all possible. So it is basically a matter of forewarned is forearmed. One is yet again amazed by the depth of biological systems, which have complexity and robustness far beyond our current knowledge, let alone our ability to replicate or model in artificial terms.

• A critique of public health politics.
• Morality and community.
• What are we going to do about corruption?
• Department of injustice, lying, and corruption.
• China does solar.

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.

• Poor (and personalized) policy means poor science and poor vaccines.
• Department of injustice and thought crime.
• Who needs new music?
• Putin and money.
• Another argument for public schools.
• Another biological thing that didn't exist, until it did- lymph drainage in the brain.
• No wonder no one wants to become a primary care physician.
• The Japanese ice age.

The Genome Remains Murky

A brilliant case study identifying the molecular cause of certain neuro-developmental disorders shows how difficult genome-based diagnoses remain.

Molecular medicine is increasingly effective in assessing both hereditary syndromes and cancers. The sequencing approach generally comes in two flavors- whole genome sequencing, or exome sequencing, where only the most important (protein-coding) parts are sampled. In each case, the hunt is for mutations (more blandly called variants) that cause the syndrome being investigated, from among the large number of variants we all carry. This approach is becoming standard-of-care in oncology, due to tremendous influence and clinical significance of cancer-driving mutations, many of which now match directly to tailored treatments that address them (thus the "precision" in precision medicine).

But another arm of precision medicine is the hunt for causes of congenital problems. There are innumerable genetic disorders whose causal analysis can lead not only to an informative diagnosis, and sometimes to useful treatments, but also to fundamental understanding of human biology. Sufferers of these syndromes may spend a lifetime searching for a diagnosis, being shuffled from one doctor or center to another and subject to various forms of hypothetical medicine, before some deep sequencing pinpoints the cause of their disease and founds a new diagnostic category that provides, if not relief, at least understanding and a medical home. 

A recent paper from Britain provided a classic of this form, investigating the causes of neurodevelopmental (NDD) disorders, which encompass a huge range of problems from mild to severe. They comment that even after the most modern analysis and intensive sequencing, 60% of NDD cases still can not be assigned causes. A large part of the problem is that, despite knowing the full sequence of the human genome, its function is less well-understood. The protein-coding genes (20,000 of those, roughly) are delineated and studied pretty closely. But that only accounts for 1 to 2% of the genome. The rest ranges from genes for a blizzard of non-coding RNAs, some of which are critical, to large regulatory regions with smatterings of important sites, to junk of various kinds- pseudogenes, relic retroviruses, repetitive elements, etc. The importance of any of these elements (and individual DNA base positions within them) varies tremendously. This means specifically that exome sequencing is not going to cut it. Exome sequencing focuses on a very small part of the genome, which is fine if your syndrome (such as a common cancer) is well characterized and known to arise from the usual suspects. But for orphan syndromes, it does not cast a wide enough net. Secondly, even with full genome sequencing, so little is known about the remoter regions of the genome that assigning a function to variations found there is difficult to impossible. It takes statistical analysis of incidence of the variation vs the incidence of the syndrome.

These authors used a trove of data- the Genomics England 100,000 genomes project, focusing on the ~9,000 genomes in this collection from people with NDD syndromes. (Plus additional genomes collected elsewhere.) (We can note in passing that Britain's nationalized health system remains at the forefront of innovative research and care.) What they found was an unusually high incidence of a particular mutation in a non-protein-coding gene called RNU4-2. The product of this gene is an RNA called U4, which is an important part of the spliceosome, where it pairs RNA-to-RNA with another RNA, U6, in a key step of selecting the first (5-prime) side of an intron that is to be spliced out of mRNA messages. This gene would never have come up in exome analysis, being non-protein-coding. Yet it is critically important, as splicing happens to the vast majority of human genes. Additionally, differential splicing- the selection of alternative exons and splice sites in a regulated way- happens frequently in developmental programs and neurological cell types. There is a class of syndromes called spliceosomopathies that are caused by defects in mRNA splicing, and tend to appear as syndromes in these processes.

As shown in the images (all based on a large corpus of other work on spliceosomes), RNU4-2/U4 pairs intimately with the U6 spliceosomal RNA, and the mutation found by the current group (which is a single nucleotide insertion) causes a bulge in this pairing, as marked. Meanwhile, the U6 RNA pairs at the same time with the exon-intron junction of the target mRNA (bottom image), at a site that is very close to the U4 pairing region (top image). The upshot is that this single base insertion into U4 causes some portion of the target mRNAs to be mis-spliced, using non-natural 5 prime splice sites and thus altering their encoded proteins. This may cause minor problems in the protein, but more often will cause a shift in translation frame, a premature stop codon, and total loss of the functional protein. So this tiny mutation can have severe effects and is indeed genetically dominant- that is, one copy overrides a second wild-type copy to generate the NDD diseases that were studied.

The U4 RNA (teal) paired with the U6 RNA (gray), within an early spliceosome complex. The mutation studied here is pointed out in black (n.64_65insT - i.e. insertion of a T). Note how it would cause a bulge in the pairing. Importantly, the location in the U6 RNA that pairs with the mRNA (see below) is right next door, at the ACAGAGA (light gray). The authors use this structural work from others to suggest how the mutation they found can alter selected splicing sites and thus lead to disease. Other single nucleotide insertions that cause similar syndromes are marked with black arrows, while single nucleotide substitutions that cause less severe syndromes are marked with orange RNA segments.

The U6 RNA (pink) paired with its mRNA target to be spliced. It binds right at the intron (gray) exon (black) boundary, where the cut will eventually be made the remove the intron. The bump from the mis-paired mutant U4 RNA (see above) distorts this binding, sending U6 to select wrong locations for spicing.

The researchers went on to survey this and other spliceosomal RNA genes for similar mutations, and found few to none outside the region marked in the diagram above. For example, there is a highly similar gene called RNU4-1. But this gene is expressed about 100-fold less in brain and other tissues, making RNU4-2 the principal source of U4 RNA, and much more significant as a causal factor for NDD. It appears that other locations in RNU4-2 (and other spliceosomal RNA genes) are even more important than the one mutated location found here, thus are never found, being lethal and heavily selected against, in this highly conserved gene. 

They also noted that, while this RNU4-2 mutation is severe, and thus must happen spontaneously (i.e. not inherited from parents), it only occurrs on the maternal alleles, not paternal alleles in the affected children. They speculate that this may be due to effects this gene may have in male gametogenesis, killing affected sperm preferentially, but not affected oocytes. Lastly, this set of mutations (in the small region shown in the first figure above) appears to account for, in their estimation, about 0.4 % of all NDD seen in Britain. This is a remarkably high rate for such a particular mutation that is not heritable. They speculate that some mutation hotspot kind of process may be causing these events, above the general mutation rate. What this all says about so-called "intelligent design", one may be reluctant to explore too deeply. On the other hand, this still leaves plenty of room to hunt for additional variations that cause these syndromes.

In this research, we see that clinically critical variations can pop up in many places, not just among the "usual suspects", genetically and genomically speaking. While much of the human genome is junk, most of it is also expressed (as RNA) and all of it is fair game for clinically important (if tragic) effects. The NDD syndromes caused by the mutation studied here are very severe- for more so than the ADD or mild autism diagnoses that make up most of the NDD spectrum. Understanding the causal nexus between the genome and human biology and its pathologies, remains an ongoing and complicated scientific adventure.

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What Causes Cancer? What is Cancer?

There is some frustration in the literature.

Fifty years into the war on cancer, what have we learned and gained? We do not have a general cure, though we have a few cures and a lot of treatments. We have a lot of understanding, but no comprehensive theory or guide to practice. While some treatments are pin-point specific to certain proteins and even certain mutated forms of those proteins, most treatments remain empirical, even crude, and few provide more than a temporary respite. Cancer remains an enormous challenge, clinically and intellectually.

Recently, a prominent journal ran a provocative commentary about the origins of cancer, trashing the reigning model of "Somatic Mutation Theory", or SMT. Which is the proposition that cancer is caused by mutations that "drive" cell proliferation, and thus tumor growth. I was surprised at the cavalier insinuations being thrown around by these authors, their level of trash talk, and the lack of either compelling evidence or coherent alternative model. Some of their critiques have a fair basis, as discussed below, but to say, as the title does, that this is "The End of the Genetic Paradigm of Cancer" is simply wrong.

"It is said that the wise only believe in what they can see, and the fools only see what they can believe in. The latter attitude cements paradigms, and paradigms are amplified by any new-looking glass that puts one’s way of seeing the world on steroids. In cancer research, such a self-fulfilling prophecy has been fueled by next-generation DNA sequencing."

"However, in the quest for predictive biomarkers and molecular targets, the cancer research community has abandoned deep thinking for deep sequencing, interpreting data through the lens of clinical translation detached from fundamental biology."

Whew!

The main critique, once the gratuitous insults and obligatory references to Kuhn and Feynman are cleared away, is that cancer does not resemble other truly clonal disease / population processes, like viral or bacterial infections. In such processes, (which have become widely familiar after the COVID and HIV pandemics), a founder genotype can be identified, and its descendants clearly derive from that founder, while accumulating additional mutations that may respond to the Darwinian pressures, such as the immune system and other host defenses. While many cancers are clearly driven by some founding mutation, when treatments against that particular "driver" protein are given, resistance emerges, indicating that the cancer is a more diverse population with a very active mutation and adaptation process. 

Additionally, tumors are not just clones fo the driving cell, but have complex structure and genetic variety. Part of this is due to the mutator phenotypes that arise during carcinogenesis, that blow up the genome and cause large numbers of additional mutations- many deleterious, but some carrying advantages. More significantly, tumors arise from and continue to exist in the context of organs and tissues. They can not just grow wildly as though they were on a petri plate, but must generate, for example, vascular structures and a "microenvironment" including other cells that facilitate their life. Similarly, metastasis is highly context-dependent and selective- only very few of the cells released by a tumor land in a place they find conducive to new growth. This indicates, again, that the organ setting of cancer cells is critically important, and accounts in large part for this overall difference between cancers and more straightforward clonal processes. 

Schematic of cancer development, from a much more conventional and thorough review of the field.

Cancer cells need to work with the developmental paradigms of the organism. For instance, the notorious "EMT", or epithelial-mesenchymal transition is a hallmark of de-differentiation of many cancer cells. They frequently regress in developmental terms to recover some of the proliferative and self-repair potential of stem cells. What developmental program is available or allowed in a particular tissue will vary tremendously. Thus cancer is not caused by each and every oncogenic mutation, and each organ has particular and distinct mutations that tend to cause cancers within it. Indeed, some organs hardly foster any cancers at all, while other organs with more active (and perhaps evolutionarily recent) patterns of proliferation (such as breast tissue, or prostate tissue) show high rates of cancer. Given the organ setting, cancer "driver" mutations need not only unleash the cell's own proliferation, but re-engineer its relations with other cells to remove their inhibition of its over-growth, and pursuade them to provide the environment it needs- nutritionally, by direct contact, by growth factors, vascular formation, immune interactions, etc., in a sort of para-organ formation process. It is a complicated job, and one mutation is, empirically, rarely enough.

"Instead, cancer can be broadly understood as “development gone awry”. Within this perspective, the tissue organization field theory is based on two principles that unite phylogenesis and ontogenesis."

"The organicist perspective is based on the interdependency of the organism and its organs. It recognizes a circular causal regimen by closure of constraints that makes parts interdependent, wherein these constraints are not only molecules, but also biophysical force."

As an argument or alternative theory, this leaves quite a bit to be desired, and does not obviate the role of  initiating mutations in the process.

It remains, however, that oncogenic mutations cause cancer, and treatments that address those root causes have time and again shown themselves to be effective cancer treatments, if tragically incomplete. The rise of shockingly effective immunotherapies for cancer have shown, however, that the immune system takes a more holistic approach to attacking disease than such "precision" single-target therapies, and can make up for the vagaries of the tissue environment and the inflammatory, developmental, and mutational derangements that happen later in cancer development. 

In one egregious citation, the authors hail an observation that certain cancers need both a mutation and a chemical treatment to get started, and that the order of these events is not set in stone. Traditionally, the mutation is induced first, and then the chemical treatment, which causes inflammation, comes second. They state: 

"The qualitative dichotomy between a mutagenic initiator that creates ’cancer cells’ and the non-genetic, tissue-perturbing promoter that expands them may not be as clear-cut. Indeed, the reverse experiment (first treatment with the promoter followed by the initiator) equally produces tumors. This result refutes the classical model that requires that the mutagenic (alleged) initiator must act first."

The citation is to a paper entitled "The reverse experiment in two-stage skin carcinogenesis. It cannot be genuinely performed, but when approximated, it is not innocuous". This paper dates from 1993, long before sequencing was capable of evaluating the mutation profiles of cancer cells. Additionally, the authors of this paper themselves point out (in the quote below) a significant assymetry in the treatments. Their results are not "equal":

"The two substances showed a reciprocal enhancing effect, which was sometimes weak, sometimes additive, and sometimes even synergistic, and was statistically most significant when the results were assessed from the time of DMBA application. Although the reverse experiment was not in any way innocuous it always resulted in a lower tumor crop than the classical sequence of DMBA followed by a course of TPA treatment. 

However, the lower tumor crop in the reverse experiment cannot be used to prove a qualitative difference between initiators and promoters."

(DMBA is the mutagen, while TPA is the inflammatory accelerant.)

So chemical treatment can prepare the ground for subsequent mutant generation in forming cancers in this system, while being much less efficient than the traditional order of events. This is not a surprise, given that this chemical (TPA) treatment causes relatively long-term inflammation and cell proliferation on its own.

"An epistemic shift towards a biological theory of cancer may still be an uphill battle in the current climate of thought created by the ease of data collection and a culture of research that discourages ’disruptive science’. Here, we have made an argument for dropping the SMT and its epicycles. We presented new and old but sidelined theoretical alternatives to the SMT that embrace theory and organismal biology and can guide experiments and data interpretation. We expect that the diminishing returns from the ceaselessly growing databases of somatic mutations, the equivalent to Darwin’s gravel pit, may soon reach a pivot point."

One rarely reads such grandiloquent summaries (or mixed metaphors) in scientific papers! But here they are truly beating up on straw men. In the end, it is true that cancer is quite unlike clonal infectious diseases, and for this, as for many other reasons, has had scientists scratching their heads for decades, if not centuries. But rest assured that this chest-thumping condescension is quite unnecessary, since those in the field are quite aware of these difficulties. The various nebulous alternatives these authors offer, whether the "epigenetic landscape", the "tissue organization field theory", or the "biological theory of cancer" all have kernels of logic, but the SMT remains the foundation-stone of cancer study and treatment, while being, for all the reasons enumerated above and by these authors, only part of the edifice, not the whole truth.

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Sugar is the Enemy

Diabetes, cardiovascular health, and blood glucose monitoring.

Christmas brought a book titled "Outlive: The Science and Art of Longevity". Great, I thought- something light and quick, in the mode Gweneth Paltrow or Deepak Chopra. I have never been into self-help or health fad and diet books. Much to my surprise, however, it turned out to be a rather rigorous program of preventative medicine, with a side of critical commentary on our current medical system. A system that puts various thresholds, such as blood sugar and blood pressure, at levels that represent serious disease, and cares little about what led up to them. Among the many recommendations and areas of focus, blood glucose levels stand out, both for their pervasive impact on health and aging, and also because there are new technologies and science that can bring its dangers out of the shadows.

Reading: 

• Outlive
• The many facets and forms of damage by high blood sugar, at the level of molecular biology.
• Recent analyses of blood sugar variability, from researchers at Stanford
• ... with tool for anyone with a CGM to upload their own data

Where do cardiovascular problems, the biggest source of mortality, come from? Largely from metabolic problems in the control of blood sugar. Diabetics know that uncontrolled blood sugar is lethal, on both the acute and long-terms. But the rest of us need to realize that the damage done by swings in blood sugar are more insidious and pervasive than commonly appreciated. Both microvascular (what is commonly associated with diabetes, in the form of problems with the small vessels of the kidney, legs, and eyes) and macrovascular (atherosclerosis) are due to high and variable blood sugar. The molecular biology of this was impressively unified in 2005 in the paper above, which argues that excess glucose clogs the mitochondrial respiration mechanisms. Their membrane voltage maxes out, reactive forms of oxygen accumulate, and glucose intermediates pile up in the cell. This leads to at least four different and very damaging consequences for the cell, including glucose modification (glycation) of miscellaneous proteins, a reduction of redox damage repair capacity, inflammation, and increased fatty acid export from adipocytes to endothelial (blood vessel) cells. Not good!

Continuous glucose monitored concentrations from three representative subjects, over one day. These exemplify the low, moderate, and severe variability classes, as defined by the Stanford group. Line segments are individually classed as to whether they fall into those same categories. There were 57 subject in the study, of all ages, none with an existing diagnosis of diabetes. Yet five of them had diabetes by traditional criteria, and fourteen had pre-diabetes by those criteria. By this scheme, 25 had severe variability as their "glucotype", 25 had moderate variability, and only 7 had low variability. As these were otherwise random subjects selected to not have diabetes, this is not great news about our general public health, or the health system.

Additionally, a revolution has occurred in blood glucose monitoring, where anyone can now buy a relatively simple device (called a CGM) that gives continuous blood glucose monitoring to a cell phone, and associated analytical software. This means that the fasting blood glucose level that is the traditional test is obsolete. The recent paper from Stanford (and the literature it cites) suggests, indeed, that it is variability in blood glucose that is damaging to our tissues, more so than sustained high levels.

One might ask why, if blood glucose is such a damaging and important mechanism of aging, hasn't evolution developed tighter control over it. Other ions and metabolites are kept under much tighter ranges. Sodium ranges between 135 to 145 mM, and calcium from 8.8 to 10.7 mM. Well, glucose is our food, and our need for glucose internally is highly variable. Our livers are tiny brains that try very hard to predict what we need, based on our circadian rhythms, our stress levels, our activity both current and expected. It is a difficult job, especially now that stress rarely means physical activity, and nor does travel, in our automobiles. But mainly, this is a problem of old age, so evolution cares little about it. Getting a bigger spurt of energy for a stressful event when we, in our youth, are in crisis may, in the larger scheme of things, outweigh the slow decay of the cardiovascular system in old age. Not to mention that traditional diets were not very generous at all, certainly not in sugar and refined carbohydrates.

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• "Not strictly constitutional"
• Let's go there.