A Brief History of DNA Sequencing

Technical revolutions that got us to modern DNA sequencing.

DNA is an incredibly elegant molecule- that much was apparent as soon as its structure came out. It is structurally tough, and its principles of information storage and replication are easy to understand. It is one instance where evolution came with, not a messy hack, but brilliant simplicity, which remains universal over all the life that we know. While its modeled structure was immediately informative, it didn't help to figure out its most important property- its sequence. Methods to sequence DNA have gone through an interesting evolution of their own. First were rather brutal chemical methods which preferentially cut DNA at certain nucleotides. Combined with the hot new methods of labeling the DNA with radioactive P32, and of separating DNA fragments by size by electically pushing them (electrophoresing) through a jello-like gel, this could give a few base pairs of information.

A set of Maxam-Gilbert reactions, with the DNA labeled with 32P and exposed to X-ray film after being separated by size by electrophoresis through a gel. Smallest are on the bottom, biggest fragments on on the top. Each of the four reactions cleaves at certain bases, as noted at the top. The intepretation of the sequence is on the right. PvuII is a bacterial enzyme that cleaves DNA, and this (palindromic) sequence noted at the bottom is the site where it does so.

Next came the revolution led by Fred Sanger, who harnessed a natural enzyme that polymerizes DNA in order to sequence it. By providing it with a mixture of natural nucleotides and defective ones that terminate the extension process, he could easily develop far bigger assortments of DNAs of various lengths (that is, reads) as well as much higher accuracy of base calling. The chemistry of the Maxam-Gilbert chemical process was quite poor in base discrimination. This polymerase method also eventually used a different isotope to trace the synthesized DNAs, S35, which is less powerful than P32 and gave sharper signals on film, which was how the DNA fragments were visualized after laid out and ordered by size, by electrophoresis.

The Sanger sequencing method. Note the much longer read length, and cleaner reactions, with fully distinct base specificity. dITP was used in place of dGTP to help clarify G/C-rich regions of sequence, which are hard to read due to polymerase pausing and odd behavior in gel electrophoresis. 

There have been many technological improvements and other revolutions since then, though none have won Nobel prizes. One was the use of fluorescent terminating nucleotides in place of radioactive ones. In addition to improving safety in the lab, this obviated the need to generate four different reactions and run them in separate lanes on the electrophoretic gel. Now, everything could be mixed into one reaction, with four different terminating fluorescent nucleotides in different colors. Plus, the mix of synthesized DNA products could now be run through a short bit of gel held in a machine, and a light meter could see them come off the end, in marcing order, all in an automated process. This was a very significant advance in capacity, automatability, and cost savings.

Fluorescent terminating nucleotides facilitate combined reactions and automation.

After that came the silicon chip revolution- the marriage between Silicon Valley and Biotech. Someone discovered that silicon chips made a good substrate to attach DNA, making possible large-scale matrix experiments. For instance, DNA corresponding to each gene from an organism could be placed at individual positions across such a chip, and then experiments run to hybridize those to bulk mRNA expressed from some organ or cell type. The readout would then be fluorescent signals indicating the level of expression of each gene- a huge technical advance in the field. For sequencing, something similar was attempted, laying down all possible 8 or 9-mers across such a chip, hybridizing the sample, thereby trying to figure out all the component sequences of the sample. The sequences were so short, however, that this never worked well. Assembling a complete sequence from such short snippets is nearly impossible.

What worked better was a variation of this method, where the magic of DNA synthesis was once again harnessed, together with the matrix layout. Millions of positions on a chip or other substrate have short DNA primers attached. The target DNA of interest, such as someone's genome, is chopped up and attached to matching primers, then hybridized to this substrate. Now a few amplification steps are done to copy this DNA a bunch of times, all still attached in place to the substrate. Finally, complementary strands are all melted off and the single DNA strands are put through a laborious step-by-step chemical synthesis process, similar to how artifical DNA is made to order, across the whole apparatus, with chemicals successively washed through. No polymerase is used. Each step ends with a fluorescent signal that says what the base that just got added was at that position, and a giant camera or scanner reads the plate after each pass, adding +1 to the sequence of each position. The best chemical systems of this kind can go to 150 or even 300 rounds (i.e. base pairs), which, over millions of different DNA fragments from the same source, is enough to then later re-assemble most DNA sequences, using a lot of computer power. This is currently the leading method of bulk DNA sequencing.

A single DNA molecule being sequenced by detecting its progressive transit through a tiny (i.e. nano) pore, with corresponding electrical readout of which base is being wedged through.

Unfortunately, our DNA has lots of repetitive and junky areas which read sizes of even 300 bases can not do justice to. We have thousands of derelict transposons and retroviruses, for instance, presenting impossible conundrums to programs trying to assemble a complete genome, say, out of ~200 bp pieces. This limitation of mass-sequencing technologies has led to a niche market for long-read DNA sequencing methods, the most interesting of which is nanopore sequencing. It is almost incredible that this works, but it is capable of reading the sequence of a single molecule of single stranded DNA at a rate of 500 bases per second, for reads going to millions of bases. This is done by threading the single strand through a biological (or artifical) pore just big enough to accommodate it, situated in an artifical membrane. With an electrical field set across the membrane, there are subtle fluctuations detectable as each base slips through, which are different for each of the four bases. Such is the sensitivity of modern electronics that this can be picked up reliably enough to read the single thread of DNA going through the pore, making possible hand-held devices that can perform such sequencing at reasonable cost.

All this is predicated on DNA being an extremely tough molecule, able to carry our inheritance over the decades, withstand rough chemical handling, and get stuffed through narrow passages, while keeping its composure. We thought we were done when we sequenced the human genome, but the uses of DNA sequencing keep ramifying, from forensics to diagnostics of every tumor and tissue biopsy, to wastewater surveillance of the pandemic, and on to liquid biopsies that promise to read our health and our future from a drop of blood.

• Is originalism progressive?
• Crypto is insane- a climate disaster, ponzi-ridden, crime-facilitating, sanctions-busting.
• Who lies, and who believes them? It tends to be a consistent demographic.
• The insurrection is not over.
• "The Capitalist system was never designed to satisfy societal needs."
• How do you pronounce "mayonnaise"?
• Big box and big parking lot solar? Obviously a good thing.
• Jane Fonda is onboard.

Alzheimer's: Wnt or Lose

A molecular exploration of the causes of Alzheimer's disease.

What causes Alzheimer's disease remains a bit of a mystery, as there is no simple and single molecular explanation, as there is with, say, Huntington's disease, which is caused by a single gene defect. There is one leading candidate, however, which is the amyloid protein, one of the accumulated molecular signatures of the disease in post-mortem brains. Some genetic forms of Alzheimer's start with defects in the gene that encodes this protein, APP (amyloid precursor protein). And a protease processing system that cleaves out the toxic amyloid beta protein from the much larger original APP protein is also closely involved with Alzheimer risk. So while there are many other genetic risk factors and possible causes relating to the APP and other systems, this seems to be the dominant causal element in Alzheimer's disease.

The naming of this protein is rather backwards, focusing on the pathological roles of defective forms, rather than on what the normal protein does. But we don't really know what that normal function is yet, so have had little choice. A recent paper described one new function for the normal APP protein, which is as a receptor for a family of proteins called WNT (for wingless integration site, an obscure derivation combining findings from fly and mouse genetics). APP had long been known to interact with WNT functions, and a reduction of WNT signaling is one of the pathologic (and possibly pathogenic) hallmarks of Alzheimer's, but this seems to be the first time it has been tabbed as a direct receptor for WNT.

What is WNT? These proteins track back to the dawn of multicelled animals, where they first appear in order to orchestrate the migration and communication of cells of the blastopore. This is the invagination that performs the transition (gastrulation) from an egg-derived ball of cells to the sheets of what will become the endoderm and mesoderm on the inside, and the ectoderm on the outside. The endoderm becomes the gut and respiratory organs, the mesoderm becomes the skeleton, muscles, blood, heart, and connective tissue, and the ectoderm becomes the skin and nervous system. WNT proteins are the ligands expressed in one set of cells, and their receptors (Frizzled and a few other proteins) are expressed on other cells which are nearby and need to relate for some developmental / migration / identification, or other purpose. One other family, the NOTCH proteins and their respective cell surface receptors, have a similar evolutionary history and likewise function as core developmental cell-cell signaling and identification systems. 

Rough structure of the APP protein. The membrane  spanning portion is in teal at the bottom, showing also some key secretase protease cleavage sites, which liberate alpha and beta portions of the protein. The internal segment is at bottom, and functions, when cleaved from the rest of the protein, as a nuclear transcription activator. Above are various extracellular domains, including one for "ligand binding", which is thought by at least one research group to bind WNT. The dimerization domain can bind other APP proteins on other cells, and heparin, another binding partner is a common component of the extracellular environment.

Fast forward a billion years, and WNT family members are deeply involved in many decisions during animal development and afterwards, particularly in the brain, controlling nerve cell branching and synapse formation in adults. WNT, NOTCH, and APP are each ligand+receptor systems, where a ligand from one cell or in soluble form binds to a receptor on the surface of another cell, which "receives" the signal and can do a multitude of things in response. The usual receptors for WNT are a family of Frizzled proteins plus a bunch of other helper proteins, the receptors for NOTCH are Jagged proteins, and the APP protein is itself a receptor whose ligand has till now been unclear, though it can homodimerize, detecting APP on other cells. APP is a large protein, and one of its responses to signals is to be cleaved in several ways. Its short cell-interior tail can be cleaved, (by gamma secretase), upon which that piece travels to the nucleus and with other proteins acts as a transciption regulator, activating, among other genes, its own gene, APP. Another possible cleavage is done by alpha secretase, causing the release of soluble APP alpha (sAPPα), which has pro-survival activities for neurons and protects them against excessive activity (excito-toxicity). Lastly, beta-secretase can cleaves APP into the toxic beta (Aβ), which in tiny amounts is also neuro-protective, but in larger amounts is highly toxic to neurons, starting the spiral of death which characterizes the hollowing out of the brain in Alzheimer's disease.

The cleavages by alpha secretase and beta secretase are mutually exclusive- the cleavage sites and products overlap, so cleavage by one prevents cleavage by the other, or destroys its product. And WNT signaling plays an important role in which route is chosen. WNT signals by two methods, called canonical or non-canonical, depending on which receptor and which ligand meet. Canonical signaling is neuro-protective, opposed to Alzheimer development, and leads to alpha secretase cleavage. Non-canonical signaling tends to the opposite, leading to internalization of APP from the surface, and beta secretase cleavage which needs acidic conditions that are found in the internal endsomes where APP ends up. So the balance of WNT "tone" is critical, and is part of the miscellaneous other risk factors that make up the background for Alzheimer's disease. Additionally, cleavage by gamma secretase is needed following cleavage by beta secretase in order to make the final forms of APP beta. The gene for gamma secretase is PSEN1 (presenilin-1), mutations in which are the leading genetic cause of Alzheimer's disease. Yet these mutations have no clear relation with the activity of the resulting gamma secretase or the accumulation of particular APP cleaved forms, so this area of causality research remains open and active.

But getting back the WNT story, if APP is itself a WNT receptor, then that reinforces the centrality of WNT signaling in this syndrome. Indeed, attempts to treat Alzheimer's by reducing the toxic amyloid (APP beta) build up in various ways have not been successful, so researchers have been looking for causal factors antecedent to that stage. One clue is that a key WNT inhibitor, DKK (for dick-kopf, derived from fly genetics, which have had some prominent German practitioners), has been experimentally an effective therapy for mice with a model form of Alzheimers. DKK is an inhibitor of the canonical WNT pathway, (via the LRP6 co-receptor of Frizzled), shunting it towards more non-canonical signaling. This balance, or "tone" of WNT signaling seems to have broad effects in promoting neurite outgrowth and synapse formation, or the reverse. Once this balance is lost, APP beta induces the production of more DKK, which starts a non-virtuous feedback cycle that may form the core of Alzheimer's pathology. This cycle could be started by numerous genetic defects and influenced by other environmental risk factors, leading to the confusing nature of the syndrome (no pun intended!). And of course the cycle starts long before symptoms are apparent and even longer before autopsy can verify what happened, so getting to the bottom of this story has been hugely frustrating.

• Even Forbes is covering these molecular details these days.
• A new low for the US- as a sleazy tax haven.
• No hypocrisy at the Bible museum!
• Senator from coal is now in control.
• Facebook has merely learned from the colleagues at FOX- the Sith network.
• But does add its own wrinkles.
• Bill Mitchell on the Australian central bank accounts.

Covid Will Never End

But it will be a very small problem, once everyone is vaccinated.

It should be obvious by now that Covid-19 is endemic and will be with us forever. At a fatality rate of roughly 2% for the unvaccinated, it is better than the bubonic plague (50%) and smallpox (30%), but far worse than influenza (0.1%), not to mention colds and other minor respiratory infections. With vaccination, the fatality rate is reduced to, in very rough terms, 0.05%. Thus, with vaccination, Covid-19 is a much less significant public health problem, superceded by influenza, whose vaccine is much less effective.

(This calculation, of the death rate, in vaccinated people, is rather fraught, because the infection rate is hard to gauge. But assuming that over the four months when roughly one third to one half the population has become vaccinated, and exposure rates of this population similar to that of the unvaccinated and productively infected population, the overall death toll was roughly 50,000 people, of which 1,263 were vaccinated, for a ratio of 40:1)

While breakthrough infections and consequences like hospitalization and death (and possibly long covid, though that is unclear) are not impossible for vaccinated people, they are rare enough that we can resume normal activities. Current policies to limit the spread of the virus, even by vaccinated people who can carry and transmit it via light infections, is mostly aimed at the remaining unvaccinated population, who will be ending up in the hospital at much higher rates, and creating the public health burden. So no wonder patience is wearing thin with the unvaccinated, who will eventually just be cut loose to take their chances while the rest of society moves on in a new world where covid is as or even more manageable than influenza.

Why is Covid less severe in children? ACE2, the key receptor for the virus seems to have lower expression naturally, and is driven even lower by incidental conditions like asthma and allergies. Other cold viruses, to which children are widely exposed, may have "pre-vaccinated" them to the new coronavirus. And children seem to produce fewer inflammatory cytokines, producing a less exaggerated immune response, which is the main factor in later Covid pathology.

Why all the breakthrough infections? One issue is that vaccination primes the immune system, which does not prevent infection, actually. What it does is to shorten the time that the body needs to fight an infection that has already occurred, by pre-educating the immune system about the target it is facing. So vaccinated people are going to be infected at normal rates, but they just won't show symptoms nearly as frequently. And second, as widely discussed, the vaccines have great, but not perfect effectiveness. It stands to reason, as has been widely reported, that more vaccines are better than fewer, and as the virus mutates to meet our weapons of social distancing and vaccination, new editions of covid vaccines will be needed. There can never be enough education of our immune systems against these evolving threats. With the advent of successful mRNA vaccines that can be rapidly programmed with new immunogens, we have the opportunity to increase our protection against both new threats, in form of yearly (or more) covid boosters, and against old threats, like influenza, whose vaccines are stuck in a time warp of antiquated technology and poor effectiveness.

This all implies that we (the vaccinated population) will be spreading around covid on an ongoing basis. It will be endemic, and our protection will be by vaccination rather than isolation. The virus has little interest in killing us, so it will likely evolve to be more benign, as our countless cold viruses have done, thereby spreading more effectively in a well-mixed population.

The extremely urgent need for universal vaccination raises the question of why the FDA has not been faster in its authorizations. All children should have already been cleared for vaccination, and full authorization should already have been granted for adults. The safety and efficacy data is present in overwhelming amounts, and if not, (in the case of children), the studies should have been started much sooner, and run on compressed schedules. One gets the impression that this is a bureaucracy that is overly wedded to process, rather than data- particularly the critical interpretation of data that comes from actual use in the field, rather from corporate reports. And this slowness has implications for future vaccines, such as ones against influenza, as well. We deserve better from our public institutions.

• R0, vaccination rates, etc.
• Can vaccinated people get long covid? Maybe.
• What are those breakthrough cases like?
• Two is better than one.
• Variants and vaccines.
• Ever wonder why religious people are gullible?
• Crypto is not a currency, it is a gamble and tax dodge.
• Gene editing is now a thing.

Sneakey Eating

An evolutionary perspective on overeating syndromes.

Most animals have a simple problem in life- find enough food to live and survive. But social animals, if they are even slightly advanced, share food, and thus alter this basic equation. They have to find ways to store and share food in a way that sustains the group, whether that is starving the old, or feeding the helpless larvae that can not feed themselves. Humans have always faced this dilemma, but don't have the rigid programming that insects do.

Humans can lie, and steal, and then lie some more. It isn't pretty, but sometimes it gets the job done. Humans can regard rules as optional, a flexibility that is a perpetual threat to institutions, norms, cultural patterns, and ultimately to group success. We recently went through an administration that regarded norms as suggestions, laws as annoyances, and then wondered why their behavior attracted so much hatred, and such low historical esteem.

This dynamic comes to mind more concretely in the case of overeating syndromes, which exemplify the conflict between the individual and the group. In a prehistoric setting, food was almost always scarce and precious. In all native cultures there are elaborate practices of public food sharing and eating, which contribute to surveillance by the community of what everyone is eating. Anyone who violates such social structures must have been severely penalized.

Public, communal eating is a fundamental human practice.

Imagine then that someone feels a compulsion to eat more than their share. Such a compulsion would be highly advantageous- if successful- to enable survival when the others in the group might be starving or malnourished. Some extra weight might well mean the difference of making it through the next winter or not. But being caught could dramatically alter the calculus. Primitive societies had harsh punishments for violating critical norms, including ostracism or execution. What then? 

I would suggest that this background sets the stage for overeating syndromes that commonly combine secret eating, often at night, stealth, and stealing. In a world of plenty like today, it is stigmatized and medicalized, and due to the abundance of food, relatively easy to navigate and thus easy to gain weight from. But pre-historically, it would have been far more fraught, and challenging, probably less likely to result in easily observable weight gains. Like other issues in social life, this conflict would take the form of an arms race between cheaters and rule-enforcers. It would be a cognitive battle between effective surveillance and punishment, vs stealth and the intelligence required to not get caught. So one can view it as one impetus among many other evolutionary forces that shaped human intelligence, and in light of its considerable incidence in modern populations, an arms race that was never resolved. Indeed, it is the type of trait that comes under balancing selection, where a high incidence in a population would be self-defeating, while a low incidence yields a much more successful outcome.

• Satire- not so funny when you are the target.
• Making every home a part of the energy solution.
• Constitution? Who ever heard of enforcing it?

Way Too Much Dopamine

Schizophrenia and associated delusions/hallucinations as a Bayesian logic defect of putting priors over observations, partly due to excess dopamine or sensitivity to dopamine.

It goes without saying that our brains are highly tuned systems, both through evolution and through development. They are constantly active, with dynamic coalitions of oscillatory synchrony and active anatomical connection that appear to create our mental phenomena, conscious and unconscious. Neurotransmitters have long been talismanic keys to this kingdom, there being relatively few of them, with somewhat distinct functions. GABA, dopamine, serotonin, glutamate, acetylcholine are perhaps the best known, but there are dozens of others. Each transmitter tends to have a theme associated with it, like GABA being characteristic of inhibitory neurons, and glutamate the most common excitatory neurotransmitter. Each tends to have drugs associated with it as well, often from natural sources. Psilocybin stimulates serotonin receptors, for instance. Dopamine is central to reward pathways, making us feel good. Cocaine raises dopamine levels, making us feel great without having done anything particularly noteworthy.

As is typical, scientists thought they had found the secret to the brain when they found neurotransmitters and the variety of drugs that affect them. New classes of drugs like serotonin uptake inhibitors (imipramine, prozac) and dopamine receptor antagonists (haloperidol) took the world by storm. But they didn't turn out to have the surgical effects that were touted. Neurotransmitters function all over the brain, and while some have major themes in one area or another, they might be doing very different things elsewhere, and not overlap very productively with a particular syndrome such as depression or schizophrenia. Which is to say that such major syndromes are not simply tuning problems of one neurotransmitter or other. Messing with transmitters turned out to be a rather blunt medical instrument, if a helpful one.

All this comes to mind with a recent report of the connection between dopamine and hallucinations. As noted above, dopamine antagonists are widely used as antipsychotics (following the dopamine hypothesis of schizophrenia), but the premier hallucinogens are serotonin activators, such as Psilocybin and LSD, though their mode of action remains not fully worked out. (Indeed, ketamine, another hallucinogen, inhibits glutamine receptors.) There is nothing neat here, except that nature, and the occasional chemical accident, have uncovered amazing ways to affect our minds. Insofar as schizophrenia is characterized by over-active dopamine activity in some areas, (though with a curious lack of joy, so the reward circuitry seems to have been left out), and involves hallucinations which are reduced by dopamine antagonists, a connection between dopamine and hallucinations makes sense. 

"... there are multiple genes and neuronal pathways that can lead to psychosis and that all these multiple psychosis pathways converge via the high-affinity state of the D2 receptor, the common target for all antipsychotics, typical or atypical." - Wiki

So what do they propose? These researchers came up with a complex system to fool mice into pressing a lever based on uncertain (auditory) stimuli. If the mouse really thought the sound had happened, it would wait around longer for the reward, giving researchers a measure of its internal confidence in a signal which may have never been actually presented. The researchers thus presented a joint image and sound, but sometimes left out the sound, causing what they claim to be an hallucinated perception of the sound. Thus the mice, amid all this confusion, generated some hallucinations in the form of positive thinking that something good was coming their way. Ketamine increased this presumed hallucination rate, suggestively. The experiment was then to squirt some extra dopamine into their brains (via new-fangled optogenetic methods, which can be highly controllable in time and space) at a key area known to be involved in schizophrenia, the striatum, which is a key interface between the cortex and lower/inner areas of the brain involved in motion, emotion, reward, and cognition.

Normal perception is composed of a balance of bottom up observation and top-down organization. Too much of either one is problematic, sometimes hallucinatory.

This exercise did indeed mimick the action of a general dose of ketamine, increasing false assumptions, aka hallucinations, and confirming that dopamine is involved there. The work relates to a very abstract body of work on Bayesian logic in cognition, recognizing that perception rests on modeling. We need to have some model of the world before we can fit new observations into it, and we continually update this model by "noticing" salient "news" which differs from our current model. In the parlance, we use observation to update our priors to more accurate posterior probability distributions. The idea is that, in the case of hallucination, the top-down model is out-weighing (or making up for a lack of) bottom-up observation, running amok, and thus exposing errors in this otherwise carefully tuned Bayesian system. One aspect of the logic is that some evaluation needs to be made of the salience of a new bit of news. How much does it differ from what is current in the model? How reliable is the observation? How reliable is the model? The systems gone awry in schizophrenia appear to mess with all these key functions, awarding salience to unimportant things and great reliability to shaky models of reality. 

Putting neurotransmitters together with much finer anatomical specification is surely a positive step towards figuring out what is going on, even if this mouse model of hallucination is rather sketchy. So this new work constitutes a tiny step in the direction of boring, anatomically and chemically, into one tiny aspect of this vast syndrome, and into the interesting area of mental construction of perceptions.

• Another crisis of overpopulation.
• And another one.
• Getting China to decarbonize will take a stiff carbon price, about $500 to $1000/ton.

Various policy scenarios of decarbonization in China, put into a common cost framework of carbon pricing (y-axis). Some policies are a lot more efficient than others. 

Transmission of SARS-CoV2

Reflections on viral spread.

This is a brief update based on studies of SARS-CoV2 transmission over the course of the pandemic. They mostly nail down features that we already know, and offer a comparison with influenza, which has interesting differences in its transmission. One observation is that influenza has been eradicated to an astonishing degree by our efforts to prevent SARS-CoV2 transmission, a testament not only to the lower transmissibility of influenza, but also to the regular round of death and illness that we have been putting up with for millennia without much complaint.

After all the hand-wringing about hand-washing, we gradually learned that this new virus is almost exclusively passed by aerosols through the air, with limited range in space and time. Also that, despite the infinitesimal size of the virus, that face masks of many kinds are effective in knocking down both emission and reception of viral innocula by several-fold. This is doubtless because both the viruses with their lipid coats, and the moist aerosols they reside in, are quite sticky, prone to capture by even rough cloth filters with channels many times the size of a viral particle. The notorious superspreader events are characterized by 

• indoors, close physical proximity to others
• limited air circulation
• an infected person, typically asymptomatic, engaging in
• vocal activity, like singing or loud talking
• with no mask

Scale drawing of surgical mask fibers, against viral and aerosol particle sizes.

Meditation is not conducive to transmission, nor do most infected people transmit their infection. Superspreaders seem to have a very high viral load in key areas of their vocal or respiratory tracts that leads to abundant aerosol emissions with high viral counts. For recipients, it takes numerous viruses to establish an infection- something like 300 for influenza, and something similar for SARS-CoV2. This is a live virus count, not counting inactive viruses, which are always part of the produced and transmitted population of particles. The reason is probably due to our various innate clearance mechanisms, both physical and molecular, meaning that only one virus may get through to successfully infect someone, out of a population of thousands that that person breathed in. 

SARS-CoV2 transmission vs influenza. SARS-CoV2 seems to survive longer in air, leading to more infections in enclosed spaces. Being outdoors subjects the aerosols to getting blown away, and to purifying UV light. This graph does not show it, but SARS-CoV2 also differs in having high viral loads prior to symptom onset, or sometimes without any symptoms, making isolation and contact tracing very difficult.

Additionally, infection by one or few viruses may present a speed problem, where they can not grow an infection fast enough relative to the ability of the immune system to respond and put out small fires. Only if the inoculum immediately generates a large conflagration (think Molotov cocktail) is the fire department overwhelmed, at least for a few days. This leads in turn to the fascinating prospect of mass inoculation with small doses of the virus. Understandably, this is not a popular idea, with its similarity to playing roulette. It resembles the old-fashioned method of small pox inoculation, which used to be done with small doses of actual small pox, not cow pox as was later introduced by Jenner. 

But it may be a significant explanation behind the enormous conundrum of the low impact of Covid-19 on tropical and low-income countries. These countries (India, Central America, Nigeria) show quite high seropositive rates, indicating wide-spread infection. But their death rates and hospitalization rates are very low, and they have escaped this pandemic with relative ease. While reporting issues and pre-existing immune exposure are possible explanations, so is a possible warmer outdoor culture with lower innocula and lower-severity infections. An interesting aspect of inoculum size is that it can have far-reaching consequences, with lower-level infections leading to smaller viral counts in the aerosols emitted, thereby causing smaller, less-severe infections in the next recipients.

The study of viral transmission and infectivity could have profound effects on how we deal with this and similar diseases, and one has to say that it has been frustrating that our knowledge of it remains haphazard, and has been so slow in coming, with such mediocre experiments, false starts and poor messaging.

• Restaurants? Don't do it.
• Reclining on an airplane? Don't bet on it.
• Future nuclear power.

Tale of an Oncogene

Research on a key oncogene of melanoma, MITF, moves from seeing it as a rheostat to seeing it as a supercomputer.

The war on cancer was declared fifty years ago, yet effective therapies are only now trickling in. And very few of them can be characterized as cures. What has been going on, and why is the fight so slow? Here I discuss one example, of melanoma and one of its drivers and central players, the gene MITF.

Melanocytes are not really skin cells, but neural crest cells, i.e. originating in the the embryonic neural tube and giving rise to various peripheral neural structures in the spine, gut, and head. One sub-population migrates off into the epidermis to become melanocytes, which generate skin pigment in melanosome packets, which they distribute around to local keratinocytes. Evolutionarily, these cells are apparently afterthoughts, after originally having developed as part of photoreceptor systems. This history, of unusual evolution and extensive developmental migration and eventual invasion into foreign tissues, has obvious implications for their capacity to form cancers later in life, if mutations re-activate their youthful propensities.

 

Above is shown a sketch of some genes known to play roles in melanoma, and key pathways in which they act. In red are oncogenes known to suffer activating mutations that promote cancer progression. In grey are shown additional oncogenes, ones whose oncogenic mutations are simpler loss-of function, not gain of function, events. And green marks ancillary proteins in these pathways that have not (yet) been found as oncogenes of any sort. MITF is a transcription regulator that drives many genes needed for  melanocyte development and melanosome formation. It also influences cell cycle control and cytoskeletal and cell surface features relevant to migration and invasion of other tissues. This post is based mostly on reviews of the molecules active in melanoma, and the more focused story of MITF.

MITF binds to DNA near target genes, often in concert with other proteins, and activates transcription of the local gene (in most cases, though it represses some targets as well). The evidence linking MITF with melanoma and melanocytes is mostly genetic. It is an essential gene, so complete deletions are lethal. But a wide variety of "mi" mutations in mice and in humans lead to unusual phenotypes like white hair color, loss of hearing, large head formation, small blue eyes, osteopetrosis, and much else. Originally researchers thought there were several different genes involved, but they all resolved down to one complex locus, now called MITF, for mi transcription factor. Certain hereditary mutations also predispose to melanoma, as do some spontaneous mutations. That the dose of MITF also correlates with how active and aggressive a melanoma is also contributes to the recognition that MITF is central to the melanocyte fate and behavior, and also one of the most central players in the disease of melanoma.

The MITF gene spreads over 229,000 base pairs, though it codes for a protein of only 419 amino acids. The gene contains nine alternate transcription start sites, 18 exons (coding regions), and five alternate translation start sites, as sketched above. This structure allows dozens of different forms of the protein to be produced in different tissues and settings, via alternative splicing. The 1M form (above, bottom) is the main one made in melanocytes. Since the gene is essential, mutations that have the phenotypes mentioned above tend to be very small, affecting one amino acid or one splice site, or perhaps truncating translation near the end of the protein. Upstream of the MITF gene and in some of its introns, there are dozens of DNA sites that bind other regulators, which either activate or repress MITF transcription in response to developmental or environmental cues. For example, a LEF1/TCF site binds the protein LEF1, which receives signals from WNT1, which is a central developmental regulator, driving proliferation and differentiation of melanocytes from the stem neural crest cells.

That is just the beginning of MITF's complexity, however. The protein contains in its sequence codes for a wide array of modifications, by regulatory protein kinases (that attach phosphate groups), and other modifiers like SUMO-ylation and ubiquitination. Key cellular regulators like GSK3, AKT, RSK, ERK2, and TAK kinases each attach phosphates that affect MITF's activity. Additionally, MITF interacts with at least a dozen proteins, some of which also bind DNA and alter its target gene specificity, and others that cooperate to activate or repress transcription. One of the better-known signaling inputs is indirectly from the kinase BRAF1, which is a target of the first precision melanoma-fighting drugs. BRAF1 is mutated in half of melanoma cases, to a hyper-active form. It is a kinase responsive to growth factors, generally, and activates a core growth-inducing (MAP) kinase cascade (as shown above), among other pathways. BRAF1 has several effects on MITF by these pathways, but the dominant one seems to be its phosphorylation and activation of PAX3, which is a DNA-binding regulator that activates the MITF gene (and is, notably, absent from the summary figure above, showing how dynamic this field remains). Thus inhibition of BRAF1, which these precision drugs do, effectively reduces MITF expression, most of the time.

Then there are the gene targets of MITF, of which there are thousands, including dozens known to have significant developmental, cell cycle, pigment synthesis, cytoskeletal, and metabolic effects. All this is to say that this one gene participates in a bewilderingly complex network of activities only some of which are recognized to date, and none of which are understood at the kind of quantitative level that would allow for critical modeling and computation of the system. What has been found to date has led to a "switch", or rheostat hypothesis. One of the maddening aspects of melanoma is its resistance to therapy. This is thought in part to be due to this dynamic rheostat, which allows levels of MITF to vary widely and send individual cancer cells reversibly into several different states. At high levels of MITF, cancer cells are pigmented and proliferative (and sensitive to BRAF1 inhibition). But at medium levels of MITF, they revert more to their early migratory behavior, and become metastatic and invasive. So melanoma benefits from a diversity of cell types and states, dynamically switching between states that are both variable in their susceptibility to therapies like anti-BRAF1, and also maximally damaging in their proliferation and ranging activities (diagrammed below).

The theme that comes out of all this is enormous complexity, a complexity that only deepens the more one studies this field. It is a typical example in biology, however, and can be explained by the fact that we are a product of 4 billion years of evolution. The resulting design is far from intelligent- rather, it is a compendium of messy contraptions, historical compromises, and accreted mechanisms. We are very far from having the data to construct proper models that would critically analyze these systems and provide accurate predictions of their behavior. It is not really a computational issue, but a data issue, given the vast complexity we are faced with. Scientists in these fields are still thinking in cartoons, not in equations. 

But there are shortcuts of various kinds. One promising method is to analyze those patients who respond unusually well to one of the new precision treatments. They typically carry some hereditary alteration in some other pathway that in most people generates resistance or backup activity to the one that was drug-treated. If their genomes are fully sequenced and analyzed in depth, they can provide insight into what other pathway(s) may need to be targeted to achieve effective combination treatment. This is a lesson from the HIV and tuberculosis treatment experiences- that the redundancy and responsiveness of biological systems calls for multiple targets and multiple treatments to meet complex disease challenges.

• Bill Mitchell on the evasions of main stream economists.
• Senate budget committee chair: Bernie.
• Stuttering in high office.
• Amanda Gorman ... complete performance art.
• Cultists feeling lost.

Coronavirus M protein

Why is SARS-CoV2 such a protean virion?

Some viruses have a crystaline structure- a capsid that is absolutely uniform, dictated by the proteins that make it up in orderly arrays. The classic T4 bacteriophage is like that, and one of its features is that it stuffs its DNA contents into the capsid under extremely high pressure (20 atmospheres). That allows those contents to shoot out later with great force, into its victim- a bacterium with a strong cell wall. Coronaviruses and related influenza viruses are not like that at all. Their RNA contents are loosely collected, and the capsid assembled in cellular membranes, ending up with a variable, almost floppy, shape. The way into the next target cell is not via a physical power-injection system, but much more gently, by using one of the eukaryotic cell's several mechanisms for endocytosis to slip inside, and then veer off from the fate of being digested like food, and instead set up shop inside its own custom-made system of vesicles. 

The coronavirus surface (envelope) is composed of only a few proteins- the spike (S) protein that sticks out and attaches to, and initiates fusion with, target cells; the envelope (E) protein, which is a minor helper towards the virus's final shape; and the membrane (M) protein, which makes up most of the envelope. E is not essential, and S is not essential at all for virus formation, but M is quite essential. It gathers in huge amounts at internal membranes (called ERGIC) in rafts in preparation for virus assembly. Meanwhile the N protein has bound to special sequences on the genomic RNA, forming a sort of chromosome. The genomic RNA also has a particular tail that can bind directly to M protein. It is M that really orchestrates the whole assembly process, binding to the genomic RNA, to N, to E, and to S proteins, not to mention membrane lipids.

Schematic of the coronavirus envelope structure. The spike (S) protein in light gray sticks outwards. The nucleocapsid (N) protein in dark gray organizes the strands of genomic RNA. The envelope (E) protein in yellow occurs occasionally to impose curviture on the envelope, encouraging it to adopt a spherical shape. And the membrane (M) protein in black forms the bulk of the envelope, binding to and organizing all the other components. These authors find that occasional M proteins (red) lack inward projections. 

Incidentally, a recent paper came out about influenza M protein, which plays a very similar role, except that influenza virions are even more variable, forming into both spheres and filaments. These authors were able to generate remarkable images of the influenza M protein forming into very regular filament arrays, seen in cross-section, below. 

Filaments of M1 protein from influenza virus, see in cross-section, with a littering of half-helices lying about. The core of these filaments would be filled with infectious genomic RNA. Scale bar is 1000 nm.

So- why so unstructured? First, the viral envelope is developed from cellular membranes, and the final virus envelope still has some amount of lipids from those membranes. These tend to be quite fluid, limiting the structural regularity that can be achieved. Also, since there is no need for pressurized injection to the target, there is no need to obsess about the virus container as a totally defined, rigid body. It is apparent, from the micrographs below, that virion volumes can vary substantially, at least two-fold, suggesting that sometimes two genomes can get packed into one virion, or even more. Where is the harm in that? Given some spread by aerosols, twice the virus dose is perhaps a reasonable tradeoff for a slightly bigger and less spread-able virion container.

Is M a target for the immune system or for vaccines? No, no one seems to care about it, since it is the S protein that is most exposed on the surface and the key for attaching to and infecting new cells. Yet all parts of the virus life cycle are interesting, and it is certainly possible that small molecule drugs directed against M could be highly disruptive, to virus assembly if not to already- formed virions. Drug targets or not, these proteins play a humble structural role, knitting together virions so that they can go out into the hostile world on a puff of air and survive a few hours, enough to enter new hosts incautious enough to be gabbing in enclosed spaces.

A comparison of virions grown without S protein (top) or with S protein (bottom). Nor is N protein required for virion formation, really. It is M that organizes everything. These experiments use another coronavirus, mouse hepatitis virus (MHV).

• Green hydrogen may be the storage medium & fuel we need.
• Weakness is strength.
• We are not alone.
• Trump's Reichstag moment, in gestation.
• Snowflakes on the Supreme Court can not deal with freedom.

SARS E!

How does this virus assemble and get out of the cell? The key proteins are named S, M, N, and E.

True to their tiny size, viruses typically have short genomes and short names for their genes, which are relatively few. Coronaviruses generally have two halves to their genomes- a big polyprotein that gets translated right away from the genome RNA, and encodes key proteins, some of which interfere with host functions, and others of which include its own replicase, and proteases that cleave itself into those pieces. The other half of the genome is expressed later, into the proteins that make up the baby virions- the envelope and nucleocapsid, along with a slew of smaller proteins that have other, and sometimes still unknown, functions.

Once all this has gotten going, the virions have to assemble and escape from the cell- a complicated and interesting process, not completely understood, though blowing up the cell through inflammation, apoptosis, and general tissue destruction certainly helps. Genomic viral RNAs, as they are made in the cell cytoplasm by the viral replicase, get bound by the N protein, which is the viral protein that binds and packages the genome, and also has binding sites for the M protein, which organizes the outside envelope. N has other roles in controlling host processes, but this is its major function. These N+genome RNA complexes (which are regarded as the nucleocapsid) find their M partners sticking out of the endoplasmic reticulum (ER, or actually a post-ER compartment called ERGIC) that is the major site in cells of protein synthesis of membrane proteins and secreted proteins. Binding promotes budding of the genome complex into the ER, forming a nascent virion, now inside the ERGIC.

Final portion of the SARS life cycle. Virion proteins are made by ribosomes (green) from gene-sized portions of the viral genome, at lower left. The nucleocapsid protein (yellow) gathers up the replicated RNA genomes into packages, and then docks onto the membranes of the ERGIC, or endoplasmic reticulum-golgi intermediate membrane compartment, specifically on the M proteins (red) exposed there, that have been translated into the endoplasmic reticulum and formed homogenous rafts. Then all that is left is to follow the endocytic pathway out of the cell, or wait for the cell to blow up by other means. In actual virions, there are many more M proteins than S proteins, and extremely few E proteins.

The M protein has in the mean-time been synthesized in vast amounts and has several important properties, being the main protein that constitutes and drives formation of the viral envelope. It is an integral membrane protein, and associates with itself, in a particular array that prefigures the complete virion and excludes non-viral membrane proteins. M protein also, in these rafts of itself, makes space for the S protein- the surface spike which gives the virus its name (corona) and which binds to the next target cell- in someone else's lung tissue. And it binds to the N protein, so that the virus envelope engulfs the packaged genome as it docks from the cytoplasm.

E protein from original SARS. That is it! Red denotes hydrophobic amino acids, blue hydrophilic, and stars the charged amino acids.

That leaves the E protein.. what is it doing? It is a tiny (76 amino acid) membrane protein, important, though not essential, for viable viruses. Indeed it is so important that viruses with this gene experimentally removed, while able to limp along at low levels, quickly evolve a new one from scratch. But it is present in virions only in very small amounts. Its structure indicates one transmembrane domain, but predictions have been ambiguous- some methods predict two, some only one. This may suggest that this protein truly has somewhat ambiguous membrane localization, which might suggest a key function in the budding process, encouraging the last, critical transition from membrane invagination to true, fully enclosed virion.

You might not need many E proteins to do this, just a small ring around the final lip of the M-protein led vesicle. E binds to M protein, and the two of them alone are sufficient to make virion-like particles in experimental cells. N protein is not needed at all, nor a genome! Yet E is thought to also be able to bind to S, helping anchor it in the viral envelope. E can also bind to itself in complexes form membrane pores, one of whose effects is to promote inflammation and apoptosis, i.e. cell death. As if that weren't enough, E protein also contains a regulatory domain (PBM) that can bind hundreds of cellular proteins to regulate cell function, particularly dysregulating cell-cell junctions to form multi-cellular synctia that allow viral spread to neighboring cells, while impeding immune responses. A lot to do for such a small protein!

Virions lacking E, made with only M, are abnormally shaped, and ones made with mutant E proteins have novel, still abnormal, shapes. This leads to the idea that M forms flat sheets, and E helps the viral envelope curve, as it must to form the spherical virion. As mentioned above, it is also quite possible that E helps with the ultimate encirclement of the virion, the final membrane-fusing stage of budding that is actually rather tricky to accomplish and requires specialized machinery in the cell and in most membrane-envelope viruses. So there remains quite a bit to learn about the machinery of this virus, for all we know so far. And we are naturally even more curious about more practical matters, like whether all this can help create a vaccine, how exactly it spreads, whether it provides immunity after infection and for how long, and how much each of our protective measures, like masks, gloves, washing, disinfecting, etc., really help.

• Social networks, evolution, the friendship paradox, and epidemic modeling.
• Coronaviruses remain viable for over an hour in aerosol, and for many hours on hard surfaces. So they spread everywhere, mask or no mask.

How do we Get Out of Here?

It is hard to tell just yet how the coronavirus lockdown will end. Some scenarios.

With the US having frittered away its early opportunity to contain incoming travel and the spread of SARS-CoV-2, we lost containment and now have an endemic pandemic. Nor are our health authorities pursuing definitive contact tracing and quarantine of all cases/contacts- some regions of the country are even well beyond this possibility. Time lines for the lockdown are being progressively extended, without a clear end-game in sight. Where will it end?

The China Solution
China has done draconian quarantines and close tracking, contact tracing, and isolation. And they have stamped out the epidemic, other than a tickle of cases, supposedly mostly coming from abroad. How ironic, but also impressive. They have used institutions and norms of close social control, sometimes rather blunt and indiscriminate, to get the upper hand over this contagion. The prospects for us doing the same are dim. Neither our public officials nor population have the stomach for it. Thus this is not a realistic scenario as an endgame for the US pandemic.

Slow burn
No, we take a more relaxed approach, hoping that the pandemic will magically recede. But that is unlikely to happen, given the vast reservoir of uninfected people, and the virus's high infectivity. So far, the US has ~300,000 cases, and ~8,000 deaths. Assuming that the reported case rate is one-tenth of actual cases, there might be three million people who have been exposed and recovered, out of a population of over 300 million. Exposing everyone would thus result in roughly a million deaths. This will happen no matter how good our social isolation is, or how long it lasts, because the minute anyone pokes their head out, they will be exposed. Without comprehensive tracking and isolation of cases/contacts, our laissez-faire approach leads to a slow burn (also termed flattening the curve) where our hospitals might be able to keep up with the extended crisis, but we still take an enormous hit in illness and death.

Exposure testing
One supplement to the slow burn scenario is the addition of exposure testing, for antigens to SARS-CoV-2. If these tests were broadly offered, like at grocery stores and by home delivery, we could at least recognize a large population that is immune and thus can move freely, (perhaps wearing a scarlet letter!), helping to re-establish economic and other essential activities. This is like having some amount of herd immunity, without waiting for the entire population to have been exposed. But it would not significantly curtail the slow burn, since we are still unwilling to keep everyone else out of circulation in a comprehensive fashion.

County quarantine
Some areas of the country are doing much better than others, and could set up local clean zones and boundaries. Once cases were reduced to a small trickle, the health departments could do what they failed to do at the outset, which is to block and test at all borders, and comprehensively trace contacts and enforce isolation internally. Given the large and necessary traffic of deliveries of goods, especially food, this is quite unlikely to happen, and would represent a sort of breakdown of our political society. But the behavior of the Federal administration, giving a "you're on your own" message to states and localities, does make this scenario more likely. It also ends up being a sort of slow burn, since any locality can not forever keep up such isolation. It would have to continue until the advent of a final solution- a vaccine or treatment.

Vaccine or treatment
This is the magic solution everyone is waiting for. The antivax movement isn't looking so good at this moment,when everyone's attention is focused on virology, epidemiology, and public health. Candidate vaccines are easy to dream up- any protein from the virus could be expressed in some heterologous system (like in E. coli cells or yeast cells) in massive amounts, and injected into people to generate immune responses. But effective vaccines are another story. Coronaviruses and other respiratory viruses tend not to generate strong and durable immune responses. That means that their ingredients just are not that immunogenic- they have devious ways to hide from immune surveillance, for one thing. Indeed, we still do not have good vaccines (or treatments) against the common cold. So a good vaccine will need to use all the tricks of the trade, such as multiple protein pieces, both invariant and variable, and immune-stimulating adjuvants/additives, to make an effective vaccine. It may take a year, but it may also take several years.

It looks like we will be in this lockdown for a very long time, with reduced economic and social activity. And the more effective our social distancing, the longer we will have to stay isolated, as the flatter curve extends out in time. If we go down the China route with more draconian methods to stamp it out before it burns through the whole population, we will be in a very precarious situation until a treatment emerges, given the wide-spread, now endemic, presence of this virus world-wide if not in continuing hot spots in the US.

• For those locked in ..
• How China is controlling spread while getting back to work.

Coronavirus Testing Update

A review of how testing is done, and where we are at.

We in the US are flying blind through the current epidemic, with cases popping up all over, testing done on very few people, and the rest ranging between nervousness and panic. What is the death rate? We still do not know. Did China contain its outbreak by draconian measures, or by wide-spread infection and natural burnout? How about South Korea, or Taiwan? Everyone claims the former, but it far from certain what actually happened. We need more testing, and particularly scientifically sampled population testing, and post-infection exposure testing. The basics of epidemiology, in other words.

SARS-CoV-2 is the virus, and COVID-19 is the disease. Most people do not seem to have mortality risk from infection, other than the elderly and infirm. In these respects, and in its great infectiousness, this disease resembles influenza. Testing from patient samples is done by RT-PCR, which stands for reverse-transcription polymerase chain reaction. The reverse transcription part employs specialized enzymes to copy the viral genomes, which are RNA, from the patient sample, into DNA, the more stable molecule that can be used in PCR. And PCR is the revolutionary method that won a Nobel prize in 1993, which uses a DNA polymerizing enzyme, and short segments of DNA (primers), to repetitively (and exponentially) replicate a chosen stretch of DNA. In this way, a minuscule amount of a pathogen can be processed to an easily detectable amount of DNA. The FDA mandates using three target regions of the new Coronavirus N protein encoding gene for its tests, but will accept one target, if the test is otherwise properly validated. They point test makers to the NAID resource that provides positive control material- RNA genomes from SARS-CoV-2.

 Just the primers, Ma'am. These tubes contained dried DNA- the short primers with specific sequences needed to amplify specific portions of the SARS-CoV-2 viral genome. Using these requires quite of bit of other laboratory equipment and expertise.

Schematic of PCR, the exponential amplification of small amounts of DNA to huge amounts. Primers are in green, nucleotides are light blue, and the target template is dark blue.

So far, so good. But there are a range of test technologies and ways to do this testing, from the bare-bones set of primers, to a roboticized, fully automated system, each appropriate to different institutions and settings. To use the basic primer set, the lab would have to have RNA extraction kits or methods to purify the viral genomes from patient samples, then a reverse transcription kit or method, then a PCR machine and the other materials (nucleotides, high-temperature DNA polymerase, purified water and other proper solution ingredients). The PCR machine is basically a heater that cycles rapidly between the low temperature required for polymerizing and primer annealing, and the higher temperature required to melt all the DNA strands apart so that another round of primer annealing can take place. And all this needs to happen in very clean conditions, since PCR is exceedingly sensitive (of course) to small amounts of contamination. Lastly, the DNA product is typically detected by trace fluorescent markers that light up only double-stranded DNA, and can generally be detected right in the tube, with an advanced PCR machine.

Automated sample handling machines are used in clinical labs.

Virtually all of this can be mustered by any competent molecular biology lab. Results would take a few days, due to the work involved in all the setup steps. The PCR itself and analysis of its results would take a few hours. But such labs do not operate at the requisite scale, or for this purpose. That is the province of clinical testing labs, which come in various sizes, from a small hospital in-house operation to a multinational behemoth. The latter run these tests on a vast, mechanized scale. They might manufacture the DNA primers themselves, or buy them in bulk, and have the proper logistical structures to do these tests from scratch in a reproducible way, to a high standard. Providers at these scales need different kinds of materials for their testing. A small provider may need a turn-key solution that comes with pre-packaged cassettes that just need the sample added before plugging into the machine, while a larger provider would save costs by using bulk reagents and massively robotized sample handling and PCR machines.

A one-hour test in a turn-key package. But at relatively high cost.

So who are the players and what is the status? The CDC did not, for some reason, use the WHO test, or tests already developed in China, whose capacity for such manufacturing and testing is prodigious. The CDC at first didn't allow anyone else to run the tests, and when they did, they did not work correctly. It has been a bad scene and much valuable time has been lost- time that resulted in the US losing any chance of containment. Now, the FDA is authorizing others to run these tests, with detailed instructions about sampling, extraction, and machinery to be used, and is slowly granting authorization to selected manufacturers and kit makers for more kinds of tests.

Large suppliers like Roche and ThermoFisher have just been approved to supply clinical labs with testing systems. Most significant is Roche, whose tests are pre-positioned and ready to go already at clinical labs around the country. The biggest clinical lab, ominously named LabCorp, offers a home-made test, but only "several thousand tests per day", which is not yet the capacity needed. So capacity for testing will rise very rapidly, and soon enable the diagnostic and surveillance testing that is so important, and has been missing to date.

• Notes on previous pandemics.

Post script:

An aspect I forgot to include is how to select the portions of the viral genome sequence to include in testing kits. Different institutions have clearly come up with primers to different genes, few as they are, and regions within those genes. For example, "The primers currently target the N1, N2, and RP genes of the virus, but these are subject to change."; "In particular, the test detects the presence of SARS-CoV-2’s E gene, which codes for the envelope that surrounds the viral shell, and the gene for the enzyme RNA-dependent RNA polymerase." There is a balance between finding regions and primer sites that are unique to the particular virus you are interested in, so cross-reaction to other viruses is 100% eliminated, and the problem of viral drift and mutation. Some regions of viral genomes mutate much more rapidly than others, but these viruses tend to mutate at pretty high rates overall, so keeping a test current from one year to the next can be challenging. That is also what our immune systems have to deal with, as cold and flu viruses change continually to evade our defenses. So the specific DNA primer targets of a test need to be relatively highly conserved, but not too highly conserved, to put it in evolutionary terms, and the regulating agencies have to keep a close eye on this issue as they approve various test versions, to find a proper balance of high specificity and long-term usability.

Post-Post script:
Yet more significant testing solutions have emerged by late March, including a rapid (~10 minute) system from Abbot, and rapid antigen testing kits that also render results in the ~10 minute range. This speed is enormously helpful, obviously, from the patient, provider, and health system perspectives. The Abbot system is based on something called isothermal PCR, which gets rid of the temperature cycling described above. It is run at an intermediate temperature (~60 degrees C) where the DNA is somewhat loose, and primers can invade duplex strands, and also used a DNA polymerase that can displace duplex DNA as it plows ahead. This plus some other clever tricks allows the DNA amplification process to happen continuously in the reaction tube, going to completion in the rapid time quoted for these tests. These tests also tend to be tough- relatively robust to junk in the samples, and variations in temperature and other conditions.

The antigen tests that are coming on line are particularly significant, since they can be used for wide-spread population surveillance, to figure out what proportion of the population has been exposed, even if no active infection is present. Due to what seems like a complete or virtually complete lack of contact tracing + quarantine, the current pandemic will only stop once most of the population has been exposed, providing herd immunity. Before that point, anytime we give up self-isolation, it will start over again, due to the relatively high rate of low- or asymptomatic cases, and their lengthy course. Health care workers that have been exposed and recovered will have a special role before then by being able to freely staff hospitals that otherwise may be in dire straights.