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.
Showing posts with label medicine. Show all posts
Showing posts with label medicine. Show all posts
Saturday, April 4, 2020
Saturday, March 14, 2020
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.
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.
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.
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.
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.
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.
Saturday, January 11, 2020
Shoulder Rehab for Desk Jockeys
Repair your shoulder and keep it healthy.
This is an unusual post, on self-help. It has been revelatory for me to go through this program, and it might be useful for others who experience shoulder pain, weakness, and lack of mobility. What presents as bursitis, impingement, bicep tendonitis, or even frozen shoulder is often a deeper and more common issue of mis-alignment and weakness in the whole shoulder, with chronic cramping of various muscles, brought on by years of hunching in our modern posture of always-forward attention to computers, phones - even books! In my case it was a lab bench that started the process.
It is hard to get a straight answer or analysis about shoulder problems, since it is a complicated and unusual joint. Small issues in the anatomy can cause big issues with soft-tissue irritation and pain, which may take years to develop, but present as sudden pain and debility. But one key concept is scapular rhythym- the fluid rising motion that the scapula should be following when you reach overhead. That can't happen if the scapula is not properly aligned. Which is to say, it should be flat against the back. When sitting in a chair with a solid back, do your shoulder blades lie flat against it? Or do they stick out against it, or even align to the side, not touching the chair back at all? When standing straight with your hands falling loosely to the side, do your hands face backwards? They shouldn't. They should be facing inwards, to your hips. Bad shoulder alignment affects your whole posture, and correcting it takes time, but yields wide-ranging benefits.
The syndrome is well-described here. Knowing shoulder anatomy is somewhat helpful, but not essential, really. The basic idea of the rehab program is to strengthen the back muscles that pull the shoulder blades back into proper position, after they have been stretched and weakened for so long by the hunched posture that over-weights the front-pulling muscles. The first step is to restore mobility and range of motion to all the muscles around the shoulder. So start with a series of stretches. Older people especially need lots of stretching to keep muscles working properly. Both the stretching and the strengthening would then be a life-long program, given that activities with forward posture tend to also be a life-long love affair.
The next step is strengthening, to counteract the typically forward- directed actions we take all day, and make the posture changes permanent. There are many helpful videos and other instructions on the internet.
When walking, attend to posture, leading with the feet, not the shoulders. When sitting, attend to posture, laying scapula flat against the seat.
That is the full program, though many other exercises and stretches can be added. Much of the damage and pain from this syndrome can be assigned to the anterior of the rotator cuff, (supraspinatus, subscapularis, and bicep tendon), and this program will not reverse the damage, but it will prevent further damage and allow effective operation of the shoulder without relying on, and irritating, the front of the rotator cuff so much. I think this issue is endemic and under-recognized. Much of the enthusiasm for muscle "trigger points" and deep massage comes from cramped muscles in the shoulder, neck and back regions. But typically, regular stretching is a better and longer-term solution, even if trigger point release provides rapid relief from pain. Every muscle can be stretched, so when you notice one giving pain or limiting range of motion, do some research on how to loosen it up, and add that to your program.
This is an unusual post, on self-help. It has been revelatory for me to go through this program, and it might be useful for others who experience shoulder pain, weakness, and lack of mobility. What presents as bursitis, impingement, bicep tendonitis, or even frozen shoulder is often a deeper and more common issue of mis-alignment and weakness in the whole shoulder, with chronic cramping of various muscles, brought on by years of hunching in our modern posture of always-forward attention to computers, phones - even books! In my case it was a lab bench that started the process.
It is hard to get a straight answer or analysis about shoulder problems, since it is a complicated and unusual joint. Small issues in the anatomy can cause big issues with soft-tissue irritation and pain, which may take years to develop, but present as sudden pain and debility. But one key concept is scapular rhythym- the fluid rising motion that the scapula should be following when you reach overhead. That can't happen if the scapula is not properly aligned. Which is to say, it should be flat against the back. When sitting in a chair with a solid back, do your shoulder blades lie flat against it? Or do they stick out against it, or even align to the side, not touching the chair back at all? When standing straight with your hands falling loosely to the side, do your hands face backwards? They shouldn't. They should be facing inwards, to your hips. Bad shoulder alignment affects your whole posture, and correcting it takes time, but yields wide-ranging benefits.
The syndrome is well-described here. Knowing shoulder anatomy is somewhat helpful, but not essential, really. The basic idea of the rehab program is to strengthen the back muscles that pull the shoulder blades back into proper position, after they have been stretched and weakened for so long by the hunched posture that over-weights the front-pulling muscles. The first step is to restore mobility and range of motion to all the muscles around the shoulder. So start with a series of stretches. Older people especially need lots of stretching to keep muscles working properly. Both the stretching and the strengthening would then be a life-long program, given that activities with forward posture tend to also be a life-long love affair.
- Door stretch: with arms up and elbows half-way up, like a stick-up, lay them against a door frame and push through forward with your body/chest to open up the shoulder and chest.
- Do the same thing with each arm singly, stretching each arm to 45 degrees back from the plane of the body.
- Facing against a wall, with one arm, reach straight up, then work the arm back through a full circle, turning sideways and stretching against the wall as you go around. Finish with a cross-stretch with the arm going in the front across your chest.
- With a broom handle, place it straight up behind one shoulder with the opposite hand, and reach back to it over the top with the same hand. Then pull forward and up with the opposite hand till you feel a stretch in the subscapularis.
- Brachiation: from a pullup bar, just hang for a few seconds with as much weight as possible.
- During the day, remember to stand and open up your shoulders periodically. Sometimes you can even get a crack out of your sternum, if you have been hunched for a while. A phone app reminder every 10 minutes may be helpful.
- Against a shelf or seat about waist high, lay the front of your arms on it, and lower your trunk till you feel a thorough stretch, then lift about half your weight with your arms- repeat 6 times.
- Hitch arms together behind your back, grasping each opposite elbow. Bend trunk to the sides, stretching the obliques, bend forward and back. Turn neck to each side as far as possible, holding stretch.
- With your back towards a shelf or bar about shoulder-high, grab with your hands, and lower your body to stretch the front of the shoulder. The aim should be to get about horizontal with the arms going straight back, or slightly lower. Next, using the same shelf and position, bend each elbow in turn and lay it/forearm on the shelf behind you, lowering the body again. This is a more intense stretch with the same goal.
- On the floor, on a mat or carpet, make sure your scapula is flat against the ground. Then make angels, swinging arms through full range from sides to overhead, 10X; alternate arms, 10X more.
The next step is strengthening, to counteract the typically forward- directed actions we take all day, and make the posture changes permanent. There are many helpful videos and other instructions on the internet.
- With face down, on a support like a weight-lifting bench or table, lift the arms straight out and up to the sides, as far as possible. Start with no weights, then add weights as possible. 3X 12 repetitions.
- Same posture, but with elbows out and arms pointed forward. 3X 12 repetitions
- Rowing against resistance- using a rowing machine, or resistance band, or rope, pull about 1/2 your weight, 10 times at least. Start slowly with this exercise, as it can cause pain at first.
- With a relatively heavy resistance band, stretch between your hands in front, about shoulder-wide. While stretching apart as much as you can, work your hands up and down a wall, from arms fully up to fully down, 12X. Start slowly with this one as well.
- With a relatively light resistance band, extend arms straight forward and pull wide to the sides, out as far as possible, 12X. While you are at it, while extended, swing your arms back over your head and down to your lower back, for a good stretch.
- With a resistance band anchored to a pole or wall to the side, hold your elbows down at your side with hands straight forward. Pull the resistance band 90 degrees sideways, 20X each direction, strengthening both arms in the rotatory cuff.
- When all that is working OK, raise weights from the side, standing position, to fully overhead, about 10 pounds each side, 10X, strengthening deltoids.
- When all that is working OK, add push-ups and pull-ups.
When walking, attend to posture, leading with the feet, not the shoulders. When sitting, attend to posture, laying scapula flat against the seat.
That is the full program, though many other exercises and stretches can be added. Much of the damage and pain from this syndrome can be assigned to the anterior of the rotator cuff, (supraspinatus, subscapularis, and bicep tendon), and this program will not reverse the damage, but it will prevent further damage and allow effective operation of the shoulder without relying on, and irritating, the front of the rotator cuff so much. I think this issue is endemic and under-recognized. Much of the enthusiasm for muscle "trigger points" and deep massage comes from cramped muscles in the shoulder, neck and back regions. But typically, regular stretching is a better and longer-term solution, even if trigger point release provides rapid relief from pain. Every muscle can be stretched, so when you notice one giving pain or limiting range of motion, do some research on how to loosen it up, and add that to your program.
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Saturday, December 21, 2019
We Are All Special
A study in yeast finds that rare mutations have outsize influence on traits.
The word "mutation" is frowned upon in these politically correct days. While we may have a human reference genome sequence derived from some guy from Buffalo, New York, all genomes are equal, and thus differences between them and the reference are now termed "variations" rather than mutations.
After the first human genome was cranked out, the natural question was- How do we differ, and what do those differences say about our medical futures and our evolutionary past? This led to the 1000 genomes project, and much more, to the point that whole genome sequencing is creeping into normal medical practice, along with even more common panels of a smattering of genes analyzed for specific issues, principally cancers. Well, we differ a lot, and this data has been richly rewarding, especially in forensic and evolutionary terms, but only somewhat in medical terms. The ambition for medical studies has been extremely high- to interpret from our code why exactly we are the way we are, and what our medical fate will be. And this ambition remains unfulfilled. It will take decades to get there, and our code is far from controlling everything- even complete knowledge of our sequences and their impact on our development and medical issues will leave a great deal to accidents of fate.
The first approach to mining the new genomic information, especially variations among humans, for medically useful information was the GWAS study. These put the 1000 genomes (or some other laboriously accumulated set of sequences, which came tagged with medical information) into a blender and asked which variations between people's sequences correlated with variations in their diseases. Did diabetes correlate with particular genes being defective or altered? Despite huge resources and high hopes, these studies yielded very little.
The reason was that the notion of variation (or mutation) and especially the intricate field of evolutionary population genetics, was, among these researchers, in a somewhat primitive state. They only accepted variations that occurred a few times, so that they could be sure they were not just random sequencing mistakes. In a population of, say, 1000, any variation that occurs a few times has a particular nature, which is to say that it must be somewhat stable in the population and have a long history, to allow it to rise to such a (modest, but significant) level of prevalence. This in turn means that it can not have a severe effect, in evolutionary terms, which would otherwise have cut its history in the population rather short. So it turned out that these researchers were studying the variations least likely to have any effect, and for all the powerful statistics they brought to bear, little fruit turned up. It was a very frustrating experience for all concerned.
A recent paper recapitulated some of these arguments in the setting of yeast genetics. The topic remains difficult to approach in humans, because rare variations are, by definition, rare, and hard to link to diseases or traits. Doing so in a clinical study requires statistical power, which arises from the number of times the linkage is seen- a catch-22 unless one can find an obscure family pedigree or a Turkish village where such a trait is rampant. In yeast, one can generate lineages of infinite size at will, and the sequencing is a breeze, with a genome 1/250 the size of ours. The only problem is that the phenotypic range of yeast is slightly impoverished compared to ours(!) Yet what variety they can display is often quantifiable, via growth assays. The researchers used 16 yeast strains from diverse backgrounds as parents, (presumably containing a wide variety of distinctive variations), generated and sequenced 28,000 progeny, and subjected them to 38 growth conditions to elicit various phenotypes.
These researchers claim that they can account for 73% of phenotypic variation from their genetic studies- far higher the rate seen for any complex human trait. They see on average 120 loci affecting each trait across the study, and 12 loci affecting each trait in any one mating. Based on past work with libraries of yeast strains, they could also classify the mutations, er, variations they saw coming from these diverse parents as either common (similar to what was analyzed in the classic GWAS experiments in humans, occurring at 1% or more) or rare. Sure enough, the rarer the allele, the bigger its effect on phenotype, as shown below. In rough terms, the rare variants accounted for half the phenotypic variation, despite comprising only a quarter of the genetic variation.
In an additional analysis, they compared all these variants to their relatives in a close relative of this yeast species, in order to judge which allele (variant / mutant or the reference / normal version) was ancestral, i.e. older. As expected, the rare variations that led to phenotypic effects were mostly of recent origin, and more so the stronger their effect.
The upshot is that to learn about the connection between genotype and phenotype, one needs significant (and typically deleterious) mutations, as geneticists have known since the time of Mendel and Morgan. Thus the use of common variants (with small effects) to analyze human syndromes and diseases has yielded very little, either medically or scientifically, while the study of rare variants has been a gold mine. And we all have numerous rare variants- they come up all the time, and are likewise selected out of existence all the time, due to their significant effects.
The word "mutation" is frowned upon in these politically correct days. While we may have a human reference genome sequence derived from some guy from Buffalo, New York, all genomes are equal, and thus differences between them and the reference are now termed "variations" rather than mutations.
After the first human genome was cranked out, the natural question was- How do we differ, and what do those differences say about our medical futures and our evolutionary past? This led to the 1000 genomes project, and much more, to the point that whole genome sequencing is creeping into normal medical practice, along with even more common panels of a smattering of genes analyzed for specific issues, principally cancers. Well, we differ a lot, and this data has been richly rewarding, especially in forensic and evolutionary terms, but only somewhat in medical terms. The ambition for medical studies has been extremely high- to interpret from our code why exactly we are the way we are, and what our medical fate will be. And this ambition remains unfulfilled. It will take decades to get there, and our code is far from controlling everything- even complete knowledge of our sequences and their impact on our development and medical issues will leave a great deal to accidents of fate.
The first approach to mining the new genomic information, especially variations among humans, for medically useful information was the GWAS study. These put the 1000 genomes (or some other laboriously accumulated set of sequences, which came tagged with medical information) into a blender and asked which variations between people's sequences correlated with variations in their diseases. Did diabetes correlate with particular genes being defective or altered? Despite huge resources and high hopes, these studies yielded very little.
The reason was that the notion of variation (or mutation) and especially the intricate field of evolutionary population genetics, was, among these researchers, in a somewhat primitive state. They only accepted variations that occurred a few times, so that they could be sure they were not just random sequencing mistakes. In a population of, say, 1000, any variation that occurs a few times has a particular nature, which is to say that it must be somewhat stable in the population and have a long history, to allow it to rise to such a (modest, but significant) level of prevalence. This in turn means that it can not have a severe effect, in evolutionary terms, which would otherwise have cut its history in the population rather short. So it turned out that these researchers were studying the variations least likely to have any effect, and for all the powerful statistics they brought to bear, little fruit turned up. It was a very frustrating experience for all concerned.
A recent paper recapitulated some of these arguments in the setting of yeast genetics. The topic remains difficult to approach in humans, because rare variations are, by definition, rare, and hard to link to diseases or traits. Doing so in a clinical study requires statistical power, which arises from the number of times the linkage is seen- a catch-22 unless one can find an obscure family pedigree or a Turkish village where such a trait is rampant. In yeast, one can generate lineages of infinite size at will, and the sequencing is a breeze, with a genome 1/250 the size of ours. The only problem is that the phenotypic range of yeast is slightly impoverished compared to ours(!) Yet what variety they can display is often quantifiable, via growth assays. The researchers used 16 yeast strains from diverse backgrounds as parents, (presumably containing a wide variety of distinctive variations), generated and sequenced 28,000 progeny, and subjected them to 38 growth conditions to elicit various phenotypes.
 |
| The major result, graphing the frequency of variations against their phenotypic effect. The effect goes up quite strongly as the frequency goes down. |
These researchers claim that they can account for 73% of phenotypic variation from their genetic studies- far higher the rate seen for any complex human trait. They see on average 120 loci affecting each trait across the study, and 12 loci affecting each trait in any one mating. Based on past work with libraries of yeast strains, they could also classify the mutations, er, variations they saw coming from these diverse parents as either common (similar to what was analyzed in the classic GWAS experiments in humans, occurring at 1% or more) or rare. Sure enough, the rarer the allele, the bigger its effect on phenotype, as shown below. In rough terms, the rare variants accounted for half the phenotypic variation, despite comprising only a quarter of the genetic variation.
In an additional analysis, they compared all these variants to their relatives in a close relative of this yeast species, in order to judge which allele (variant / mutant or the reference / normal version) was ancestral, i.e. older. As expected, the rare variations that led to phenotypic effects were mostly of recent origin, and more so the stronger their effect.
"Strikingly, no ancient variant decreased fitness by more than 0.5 SD units, whereas 41 recent variants did."
The upshot is that to learn about the connection between genotype and phenotype, one needs significant (and typically deleterious) mutations, as geneticists have known since the time of Mendel and Morgan. Thus the use of common variants (with small effects) to analyze human syndromes and diseases has yielded very little, either medically or scientifically, while the study of rare variants has been a gold mine. And we all have numerous rare variants- they come up all the time, and are likewise selected out of existence all the time, due to their significant effects.
 |
| The scale of the experiments done here in yeast allow high precision genetic mapping. Here, one trait (growth in caffeine) is mapped against correlating genomic variations. The correlations home in on variations in the TOR1 gene, a known target of caffeine and a master regulator of cell growth and metabolism. |
- Stiglitz on neoliberalism.
- Thoughts about Britain's (and our) first past the post voting system.
- Economists have no idea what they are talking about- Phillips curve edition.
- Hayek and the veneration of prices.
- Real trial or show trial?
- The case for Justice Roberts.
- Winning vs success.
- Psychotic.
- Lead from gasoline is being remobilized by wildfires.
- Winter has been averted.
Saturday, March 30, 2019
Addiction, Exorcism, and the American Dream
Review of Beautiful Boy.
Why is drug addiction such a widespread and dangerous scourge? A lot has been made of the despair of the working class especially in declining rural areas- a crisis of meaning and survival at the short end of the capitalist system. But there is higher anxiety everywhere in our unequal, precarious, and atomized system. Even in wealthy Marin, where the story of this movie originates, parents are in what seems like fight to the death to get their offspring into colleges to fulfil an overwhelming set of competitive expectations. No wonder young adults, even when well-to-do, already feel themselves in a rat race which it would be pleasant to check out of, momentarily. Then add in the viciousness of modern drugs like crystal meth and fentanyl, and you have a lethal witches' brew.
We used to regard Russia as a demographic basket case, with declining population riven with alcoholism in the wake of the Soviet collapse. Now we are facing a similar downward dynamic- a social rot punctuated by self-destruction through drugs and suicide. The ultimate source seems to be broad insecurity, which was precisely the point of the economic and cultural reforms of the recent Republican epoch, starting with Ronald Reagan. The benefits of competition and division were trumpeted, and the rich were feted as job creators and entrepreneurs, and given absurd benefits like a tax rate on investment profits half as high as the rate on labor income. Companies developed an ideology of serving profits to the exclusion of all other goals, which meant the destruction of stable life schedules, stable jobs, and stable communities. The Reagan era gave rise to wide-spread homelessness, the ultimate warning to labor to keep its head down. And a broad reduction of safety nets of all sorts, from corporate pensions to onerous rules for welfare, which was divided into a puzzle of ungenerous programs.
How ironic, then, that Donald Trump offered to fix all this for workers, restoring the greatness and jobs of America. Who suspected that he came from a Republican tradition whose first order of business, when given power, has been to hand money to the rich? Who suspected that his policy ideas came more from the tabloid headlines of the 80's and 90's (not to say his fascist forebears in the 30's) than from the issues the working class face today? Who suspected that the greatest epoch in American history, after World War 2, was actually our period of highest taxation, culminating in, not coincidentally, the Apollo space program, which was hardly a capitalist venture?
Reagan, George W. Bush, and Trump each cloaked themselves as shamans for an anxious society, ready to exorcise the demons of economic malaise and insecurity, as well as those of Vietnam. While Democrats offered laundry lists of melioration, Republicans could do no such thing, their object being to strengthen hierarchy and help the better-off. They have instead lighted on a more tribally / religiously tinged approach, offering a broad ideology of conservatism (however radical the implementation, and departure from the existing system) and order, which would by some mystery of compassionate conservatism redound to the benefit of all after generous payouts to the few.
On the military front, they authored a series of military misadventures that climaxed with the criminal debacle in Iraq. On the economic front, they pushed hard-line capitalism as the cure-all to bring economic growth, starving the state with deregulation, outsourcing, and bitter budget / deficit battles as a purgatorial nostrum that would rejuvenate an ailing system. Curiously, however, the treatment never worked for the middle class and poor, keeping them economically static and ever more insecure, while the rich and super-rich pocketed all the proceeds.
Economic vitality needs some dynamism and destruction. But people and communities need stability and a basic level of egalitarianism to feel human and have basic freedoms. The founders foresaw that rising wealth and inequality might make of America the same class-ridden culture they had fled in Europe. Their hopes were tied not just to the new republican structure they were building, but also, in economic terms, to the frontier- the jobs-for-all program of its day- which would continue to offer all Americans (and immigrants from all over the world) the option of a decent and hard-working living, preventing excessive inequality.
Now the frontier is gone, the population continues to rise, and the only solution from the "conservative" right is to squeeze the middle class and poor relentlessly in a spiral of anxiety that drives everyone to work and live under ever less humane conditions. We need a better balance that builds more unifying social structures and public goods, reels back the excesses of extreme capitalism, and gives people breathing space and freedom to dream of being more than cogs in a machine.
Why is drug addiction such a widespread and dangerous scourge? A lot has been made of the despair of the working class especially in declining rural areas- a crisis of meaning and survival at the short end of the capitalist system. But there is higher anxiety everywhere in our unequal, precarious, and atomized system. Even in wealthy Marin, where the story of this movie originates, parents are in what seems like fight to the death to get their offspring into colleges to fulfil an overwhelming set of competitive expectations. No wonder young adults, even when well-to-do, already feel themselves in a rat race which it would be pleasant to check out of, momentarily. Then add in the viciousness of modern drugs like crystal meth and fentanyl, and you have a lethal witches' brew.
 |
| Still from the movie. Timothee Chalamet playing Nic Sheff, and Steve Carrel playing David Sheff. |
We used to regard Russia as a demographic basket case, with declining population riven with alcoholism in the wake of the Soviet collapse. Now we are facing a similar downward dynamic- a social rot punctuated by self-destruction through drugs and suicide. The ultimate source seems to be broad insecurity, which was precisely the point of the economic and cultural reforms of the recent Republican epoch, starting with Ronald Reagan. The benefits of competition and division were trumpeted, and the rich were feted as job creators and entrepreneurs, and given absurd benefits like a tax rate on investment profits half as high as the rate on labor income. Companies developed an ideology of serving profits to the exclusion of all other goals, which meant the destruction of stable life schedules, stable jobs, and stable communities. The Reagan era gave rise to wide-spread homelessness, the ultimate warning to labor to keep its head down. And a broad reduction of safety nets of all sorts, from corporate pensions to onerous rules for welfare, which was divided into a puzzle of ungenerous programs.
How ironic, then, that Donald Trump offered to fix all this for workers, restoring the greatness and jobs of America. Who suspected that he came from a Republican tradition whose first order of business, when given power, has been to hand money to the rich? Who suspected that his policy ideas came more from the tabloid headlines of the 80's and 90's (not to say his fascist forebears in the 30's) than from the issues the working class face today? Who suspected that the greatest epoch in American history, after World War 2, was actually our period of highest taxation, culminating in, not coincidentally, the Apollo space program, which was hardly a capitalist venture?
Reagan, George W. Bush, and Trump each cloaked themselves as shamans for an anxious society, ready to exorcise the demons of economic malaise and insecurity, as well as those of Vietnam. While Democrats offered laundry lists of melioration, Republicans could do no such thing, their object being to strengthen hierarchy and help the better-off. They have instead lighted on a more tribally / religiously tinged approach, offering a broad ideology of conservatism (however radical the implementation, and departure from the existing system) and order, which would by some mystery of compassionate conservatism redound to the benefit of all after generous payouts to the few.
On the military front, they authored a series of military misadventures that climaxed with the criminal debacle in Iraq. On the economic front, they pushed hard-line capitalism as the cure-all to bring economic growth, starving the state with deregulation, outsourcing, and bitter budget / deficit battles as a purgatorial nostrum that would rejuvenate an ailing system. Curiously, however, the treatment never worked for the middle class and poor, keeping them economically static and ever more insecure, while the rich and super-rich pocketed all the proceeds.
Economic vitality needs some dynamism and destruction. But people and communities need stability and a basic level of egalitarianism to feel human and have basic freedoms. The founders foresaw that rising wealth and inequality might make of America the same class-ridden culture they had fled in Europe. Their hopes were tied not just to the new republican structure they were building, but also, in economic terms, to the frontier- the jobs-for-all program of its day- which would continue to offer all Americans (and immigrants from all over the world) the option of a decent and hard-working living, preventing excessive inequality.
Now the frontier is gone, the population continues to rise, and the only solution from the "conservative" right is to squeeze the middle class and poor relentlessly in a spiral of anxiety that drives everyone to work and live under ever less humane conditions. We need a better balance that builds more unifying social structures and public goods, reels back the excesses of extreme capitalism, and gives people breathing space and freedom to dream of being more than cogs in a machine.
Saturday, February 16, 2019
Chromosomes Blown to Smithereens
Where do cancers and cancer relapses come from?
DNA is a treasure trove that keeps on giving. The human genome sequence was a milestone that may not have been self-interpreting, but has provided grist for leaps of technical advancement and knowledge. Ancestry studies are one example, but disease studies are of more immediate interest. Cancer is now understood to be a molecular disease where the DNA suffers mutations that release various brakes on cell proliferation. One of the most influential types of mutations are gene fusions, where one gene that has roles in proliferation is broken from its normal regulatory controls, either within its coding sequence (such as a repressing protein domain) or its upstream expression controls, and hooked up with some other gene that drives its expression in new places and high levels. A recent paper studied several cancers in detail, sequencing samples from various time points and locations, coming up with very interesting findings about the origins of these mutations and the nature of metastasis.
Chromoplexy is one form of a genome blowup, one that is restricted in scope (at least compared with the even more destructive chromothripsis). The best theory about its origin posits that the affected portion of the genome (typically an early-replicating and transcriptionally active region) gets caught outside the normal nucleus, forming a temporary mini-nucleus which is cut off from normal controls, causing the trapped DNA to break up. The cell has strong controls against free DNA ends, and uses end-joining DNA repair to patch things up, pasting ends together essentially at random. This is obviously quite dangerous, and leads to unexpected gene fusions, of which hundreds of different examples are now known that drive various cancers. One such fusion, diagrammed above, is between genes BCLAF1 (upstream) and GRM1 (downstream). GRM1 is a receptor for glutamate, the most prevalent excitatory neurotransmitter. While most highly expressed in the brain, glutamate receptors act throughout the body, and malfunctions are connected with a variety of diseases. Increased expression and activation can drive cell proliferation. The other fusion partner, BCLAF1, is a promoter of cell suicide, or apoptosis. That function will be lost in the fusion, which might have some importance to the disease (though a second copy presumably remains intact elsewhere). The important part is that it is very widely expressed, especially in bone marrow. An earlier paper describing this fusion states:
This pattern of mutation, and the specific fusions that resulted, became apparent due to the deep sequencing the researchers did, taking samples from the patient's tumors and from normal tissues. An important concept here is of mutational signatures. Each mechanism of mutation has its characteristic pattern of mutations left in the genome. Exposure to UV light, which causes C->T mutations, will leave a much different pattern in the genome than the localized chromoplexy blowup mentioned above. So a forensic analysis of the patient's DNA can tell what happened, in some mechanistic detail. For example, the various fusions seen in these samples were not part of extensive copy number variations- reduplications that are common in cancerous cells, which indicated that this blowup took place once as a discrete event, not repeatedly or slowly over a long period of time.
It can also tell when it happened, and here we get to a particularly interesting message from this paper. When they sequenced primary and relapsed tumors, (with comparisons to normal tissue), such tumors shared some key mutations, those which drove the overall cancer. But they failed to share many others. Indeed, the metastatic tumors carried none of several mutations that were uniformly present in the primary tumor. This says that metastases or relapse cancers, (this part of the study was specific only to Ewing's sarcoma, a bone cancer typically arising around ages 1-20), typically do not develop from the primary tumor, but from cells that carry the same driver mutation, but diverged before primary tumor formation. They are independent events, and metastatic prognosis has little to do with the fate of the primary tumor.
Whether this observation about metastisis applies to other tumors is naturally important to follow up. It would alter significantly how we deal with primary tumors, and informs the kind of conservative treatments (lump-ectomies, for instance) that are becoming more common. As sequencing becomes cheaper and more common for all kinds of tumors, the particular drivers, from whatever mutational source, can be identified and used to direct specific, (buzzword: "precision") treatments. GRM1 can be targeted by direct or indirect means. But if one has Ewing's sarcoma, typically associated with a fusion of EWSR1-FLI1, where FLI1 is a transcription factor that drives growth factor production and hence cell proliferation, a different set of therapies would be indicated.
DNA is a treasure trove that keeps on giving. The human genome sequence was a milestone that may not have been self-interpreting, but has provided grist for leaps of technical advancement and knowledge. Ancestry studies are one example, but disease studies are of more immediate interest. Cancer is now understood to be a molecular disease where the DNA suffers mutations that release various brakes on cell proliferation. One of the most influential types of mutations are gene fusions, where one gene that has roles in proliferation is broken from its normal regulatory controls, either within its coding sequence (such as a repressing protein domain) or its upstream expression controls, and hooked up with some other gene that drives its expression in new places and high levels. A recent paper studied several cancers in detail, sequencing samples from various time points and locations, coming up with very interesting findings about the origins of these mutations and the nature of metastasis.
 |
| One example of a genome blow-up, called "chromoplexy". A few regions of the genome got caught in some kind of spindle, and came out with several breaks which then were repaired to form re-joined fusions. In this diagram (right) of one resulting fusion, of genes BCLAF1 and GRM1, the chromosome 6 parts on the outside have rejoined, while the broken parts between the rejoined ends have fused to each other and then to chromosome 16, with one small bit unassigned and perhaps ending up somewhere else. The diagram seems to indicate that GRM1 ends up upstream of BCLAF1, (these are divergently transcribed in the native chromosome), which I think is an error. |
Chromoplexy is one form of a genome blowup, one that is restricted in scope (at least compared with the even more destructive chromothripsis). The best theory about its origin posits that the affected portion of the genome (typically an early-replicating and transcriptionally active region) gets caught outside the normal nucleus, forming a temporary mini-nucleus which is cut off from normal controls, causing the trapped DNA to break up. The cell has strong controls against free DNA ends, and uses end-joining DNA repair to patch things up, pasting ends together essentially at random. This is obviously quite dangerous, and leads to unexpected gene fusions, of which hundreds of different examples are now known that drive various cancers. One such fusion, diagrammed above, is between genes BCLAF1 (upstream) and GRM1 (downstream). GRM1 is a receptor for glutamate, the most prevalent excitatory neurotransmitter. While most highly expressed in the brain, glutamate receptors act throughout the body, and malfunctions are connected with a variety of diseases. Increased expression and activation can drive cell proliferation. The other fusion partner, BCLAF1, is a promoter of cell suicide, or apoptosis. That function will be lost in the fusion, which might have some importance to the disease (though a second copy presumably remains intact elsewhere). The important part is that it is very widely expressed, especially in bone marrow. An earlier paper describing this fusion states:
"The GRM1 coding region remains intact, and 18 of 20 CMFs (90%) showed a more than 100-fold and up to 1,400-fold increase in GRM1 expression levels compared to control tissues. Our findings unequivocally demonstrate that direct targeting of GRM1 is a necessary and highly specific driver event for CMF [bone tumor chondromyxoid fibroma] development."
This pattern of mutation, and the specific fusions that resulted, became apparent due to the deep sequencing the researchers did, taking samples from the patient's tumors and from normal tissues. An important concept here is of mutational signatures. Each mechanism of mutation has its characteristic pattern of mutations left in the genome. Exposure to UV light, which causes C->T mutations, will leave a much different pattern in the genome than the localized chromoplexy blowup mentioned above. So a forensic analysis of the patient's DNA can tell what happened, in some mechanistic detail. For example, the various fusions seen in these samples were not part of extensive copy number variations- reduplications that are common in cancerous cells, which indicated that this blowup took place once as a discrete event, not repeatedly or slowly over a long period of time.
It can also tell when it happened, and here we get to a particularly interesting message from this paper. When they sequenced primary and relapsed tumors, (with comparisons to normal tissue), such tumors shared some key mutations, those which drove the overall cancer. But they failed to share many others. Indeed, the metastatic tumors carried none of several mutations that were uniformly present in the primary tumor. This says that metastases or relapse cancers, (this part of the study was specific only to Ewing's sarcoma, a bone cancer typically arising around ages 1-20), typically do not develop from the primary tumor, but from cells that carry the same driver mutation, but diverged before primary tumor formation. They are independent events, and metastatic prognosis has little to do with the fate of the primary tumor.
 |
| The author's proposed time course of Ewing's sarcoma evolution, placing the origin of metastatic and relapsing tumors well before and outside of the primary tumor at the time of diagnosis. |
Whether this observation about metastisis applies to other tumors is naturally important to follow up. It would alter significantly how we deal with primary tumors, and informs the kind of conservative treatments (lump-ectomies, for instance) that are becoming more common. As sequencing becomes cheaper and more common for all kinds of tumors, the particular drivers, from whatever mutational source, can be identified and used to direct specific, (buzzword: "precision") treatments. GRM1 can be targeted by direct or indirect means. But if one has Ewing's sarcoma, typically associated with a fusion of EWSR1-FLI1, where FLI1 is a transcription factor that drives growth factor production and hence cell proliferation, a different set of therapies would be indicated.
- The Green dream is on.
- Our cold war with China.
- AIPAC is the NRA of foreign policy.
- Bill Mitchell on economics education, especially macro.
- Election crisis.. no problem.
- Give to the rich, take from the poor.
- Notes on experts.
Saturday, December 15, 2018
Screwy Locomotion: the Spirochete
How do spirochete bacteria move?
Getting around isn't easy. Some of our greatest technological advancements have been in locomotion. Taming, then riding, horses; railroads, automobiles, airplanes. Microorganisms have been around for a long time, and while flying may be easy for them, getting through thicker media is not, nor is steering. The classic form of bacterial motion is with an outboard motor- the flagellum. The prototypical bacterium E. coli has several flagella sprinkled around its surface. Each flagellum is slightly helical, thus forming a languid sort of propeller, which if turned along its helical axis, (at roughly 6,000 rpm), can propel the bacterium through watery media. Turning multiple flagella in this same direction (counter-clockwise) encourages them all to entangle coherently and unite into a bundle. It turns out, however, that bacteria can easily switch their motors to the opposite direction, which causes the flagella to separate, and also to flail about, (since for a left-handed helix, this is the "wrong" direction), sending the cell in random directions.
These are the two steering options for most bacteria- forward or flop about. And this choice is made all the time by typical bacteria, which can sense good things in front (keep swimming forward), or sense bad things in front / good things elsewhere (flail about for a second, before resuming swimming). The flagellar base, where the motor resides, uses both ATP and the proton motive force (i.e. protons that were pumped out by cellular respiration, or the breakdown of food). The protons drive the motor, and ATP drives the construction of the flagellum, which is itself a very complicated dance of self-organization, built on the foundation of an extrusion/injection system also used by pathogenic bacteria to inject things into their targets.
But sometimes a bacterium really needs to get somewhere badly, and is faced with viscous fluids, perhaps inside other organisms, or put out by them to defend themselves. One human defense mechanism is a DNA net thrown out by neutrophils, a type of white blood cell. Spirochetes have come up with an ingenious (by evolution, anyway!) solution- the inboard motor. This is not a motor sticking out of the bottom, but a motor fully enclosed within the cell wall of the bacterium.
How can that work? It is an interesting story. Spirochetes, as their name implies, are corkscrews in shape. In mutants lacking flagella, they instead relax to a normal bacterial rod shape. So they have flagella, but these are positioned inside the cell wall, in the periplasmic space. Indeed they form the central axis around which the corkscrew rotates, with one set of (approximately ten) flagella coming from the rear and another set from the front, each ending up around the middle. If each set rotates as hard as it can, they drive their respective ends to counter-rotate, in reaction. If the front motors (of which there are several) turn their flagella counterclockwise, as viewed from the back, they will, in reaction, drive (and bend) the nose into a clockwise orientation. If the back set of motors run clockwise, driving their flagella counterclockwise (also as seen from the back), then the rear part of the bacterium counter-rotates in clockwise fashion, and the coordinate action drives spiral bending and an overall drilling motion forward.
On the other hand, if the motors on the opposite ends of the bacterium go in the same direction, then the flagella induce opposite, instead of coordinate, counter-rotations, and the bacterium doesn't tumble randomly, as normal bacteria do, but contorts and flexes in the middle, with a similar re-orienting effect. This ability incidentally shows the remarkable toughness of these bacteria, considering the lipid bilayer nature of their key protective membranes. These bacteria can also easily reverse direction, by sending both sets of motors in reverse, operating very much like little drills. How this exquisite coordination works has not yet been worked out, however.
One thing that is known, however, is that spirochete motors are massive- almost twice the size of E. coli motors, with special outside hooks to propagate power through the tight turn inside the periplasmic space. It is interesting that these motors can be scaled up in size, with more subunits, and more proton ports for power, as if they were just getting more cylinders in a (fossil fuel-burning) car engine.
All this is in service of getting through messy, gelatinous material. The model for most of this work is the spirochete responsible for Lyme disease. The characteristic red ring seen in that infection is thought to track the progress of the spirochete outward and away from the original tick bite site, in relation to the immune system catching up via inflammation. But such viscous environments are quite common in the organic muck of the biosphere, including biofilms established by other bacteria. So the evolutionary rationale for the superpowers of spirochetes is probably quite ancient.
Getting around isn't easy. Some of our greatest technological advancements have been in locomotion. Taming, then riding, horses; railroads, automobiles, airplanes. Microorganisms have been around for a long time, and while flying may be easy for them, getting through thicker media is not, nor is steering. The classic form of bacterial motion is with an outboard motor- the flagellum. The prototypical bacterium E. coli has several flagella sprinkled around its surface. Each flagellum is slightly helical, thus forming a languid sort of propeller, which if turned along its helical axis, (at roughly 6,000 rpm), can propel the bacterium through watery media. Turning multiple flagella in this same direction (counter-clockwise) encourages them all to entangle coherently and unite into a bundle. It turns out, however, that bacteria can easily switch their motors to the opposite direction, which causes the flagella to separate, and also to flail about, (since for a left-handed helix, this is the "wrong" direction), sending the cell in random directions.
 |
| A typical bacterium with multiple flagella, which will cooperate in forming a bundle when all turned in the same direction, consonant with their helicity (i.e. counter-clockwise). |
These are the two steering options for most bacteria- forward or flop about. And this choice is made all the time by typical bacteria, which can sense good things in front (keep swimming forward), or sense bad things in front / good things elsewhere (flail about for a second, before resuming swimming). The flagellar base, where the motor resides, uses both ATP and the proton motive force (i.e. protons that were pumped out by cellular respiration, or the breakdown of food). The protons drive the motor, and ATP drives the construction of the flagellum, which is itself a very complicated dance of self-organization, built on the foundation of an extrusion/injection system also used by pathogenic bacteria to inject things into their targets.
Animated video describing how the flagellum and its base are constructed.
But sometimes a bacterium really needs to get somewhere badly, and is faced with viscous fluids, perhaps inside other organisms, or put out by them to defend themselves. One human defense mechanism is a DNA net thrown out by neutrophils, a type of white blood cell. Spirochetes have come up with an ingenious (by evolution, anyway!) solution- the inboard motor. This is not a motor sticking out of the bottom, but a motor fully enclosed within the cell wall of the bacterium.
 |
| Choice of directions (small forward or back arrows) that are dictated by the rotation of the flagella (blue). One set of flagella originate at the rear, and a second set originates at the front. Only if they turn in opposite directions (top two panels) does the spirochete swim coherently, either forward or back. |
How can that work? It is an interesting story. Spirochetes, as their name implies, are corkscrews in shape. In mutants lacking flagella, they instead relax to a normal bacterial rod shape. So they have flagella, but these are positioned inside the cell wall, in the periplasmic space. Indeed they form the central axis around which the corkscrew rotates, with one set of (approximately ten) flagella coming from the rear and another set from the front, each ending up around the middle. If each set rotates as hard as it can, they drive their respective ends to counter-rotate, in reaction. If the front motors (of which there are several) turn their flagella counterclockwise, as viewed from the back, they will, in reaction, drive (and bend) the nose into a clockwise orientation. If the back set of motors run clockwise, driving their flagella counterclockwise (also as seen from the back), then the rear part of the bacterium counter-rotates in clockwise fashion, and the coordinate action drives spiral bending and an overall drilling motion forward.
Video of a non-spirochete bacterium with its flagellum stick to the slide, causing the tail to wag the dog.
Video of spirochete bacteria in motion.
On the other hand, if the motors on the opposite ends of the bacterium go in the same direction, then the flagella induce opposite, instead of coordinate, counter-rotations, and the bacterium doesn't tumble randomly, as normal bacteria do, but contorts and flexes in the middle, with a similar re-orienting effect. This ability incidentally shows the remarkable toughness of these bacteria, considering the lipid bilayer nature of their key protective membranes. These bacteria can also easily reverse direction, by sending both sets of motors in reverse, operating very much like little drills. How this exquisite coordination works has not yet been worked out, however.
 |
| Reconstruction, drawn from electron microscopy, of one end of a spirochete, showing the motor orientations, the sharp hook/base of the flagellum, the membrane and cell wall structure, and one of the signaling proteins (MCP), which transmits a sensory signal to dictate the direction of motor rotation. |
One thing that is known, however, is that spirochete motors are massive- almost twice the size of E. coli motors, with special outside hooks to propagate power through the tight turn inside the periplasmic space. It is interesting that these motors can be scaled up in size, with more subunits, and more proton ports for power, as if they were just getting more cylinders in a (fossil fuel-burning) car engine.
 |
| Structure of the Borrelia flagellar motor, showing the stator (blue), which is attached to the membrane and stabilized against rotation; the rotor (yellow spokes and teal C-ring), and the gateway ATPase complex which unfolds and transmits the structural components (proteins) into the central channel from which they build the machine. |
All this is in service of getting through messy, gelatinous material. The model for most of this work is the spirochete responsible for Lyme disease. The characteristic red ring seen in that infection is thought to track the progress of the spirochete outward and away from the original tick bite site, in relation to the immune system catching up via inflammation. But such viscous environments are quite common in the organic muck of the biosphere, including biofilms established by other bacteria. So the evolutionary rationale for the superpowers of spirochetes is probably quite ancient.
- EPI has a comprehensive solution for righting the inequality ship.
- John Dingle also has a solution.
- "Entitlements" are OK- on the importance of social insurance. Remember, the military is always insolvent, from a budgetary perspective.
- Sleazebags to the end.
- On the types of epilepsy.
- A persistent cycle of resource extraction, incumbent interests, regressive politics, and non-development. Let's not go there ourselves.
- A lesson in jazz.
Saturday, September 1, 2018
Striptease by HIV
How the virus disrobes is an important part of its life cycle, secrets of which are still being uncovered.
For such a tiny entity, HIV-1 has a very complicated life. Its study has generated numerous drugs that interfere with key life events, and have brought it under control in most developed areas of the world. But there is a lot left to learn. While its fusion with target cell membranes and eventual replication have received the most study, less is known about what happens in between.
The virus has several layers. On the outside is a membrane coat, designed to protect the virus in moist environments, but more imporantly to expose surface proteins that seek out T-cells specifically, and then to fuse with their membranes, thus entering them. Inside the lipid membrane and its supporting proteins is the capsid, a protein coat that protects the HIV genome from attack by internal T-cell defenses like RNases, helicases, RNA sensing proteins, interferons, and their various downstream effects. This capsid finally lets go if its cargo at the nucleus, where the viral genome, now transformed into double stranded DNA, integrates itself into the cell's genome.
Innermost is the genome, composed of RNA, not DNA. Packaged alongside it are key enzymes integrase, RNase H, and reverse transcriptase. Once HIV enters the cell, and meets the rich mother-lode of ATP and other nucleotides, including deoxy-ribonucleotides that are the building blocks of DNA, the reverse transcriptase can begin its work. It copies the genome in a complex sequence of starts within the linear RNA, hops to the other end of the RNA, continuation, and finally termination. All the while, the RNase digests the original RNA genome, in a remarkable process of self-transmutation. It is thought that synthesis of the complementary DNA strand only begins after the virus rides the cell's light rail system (microtubules) to the nucleus, where it leaves its capsid behind.
Some recent papers shed a bit of light on the nature of the HIV capsid, which is more interesting than previously understood. It was not previously clear whether the capsid uncoats at the membrane during original entry, or only at the nucleus, or even inside the nucleus. But one paper shows quite definitively that the capsid remains intact through the first strand of DNA synthesis, thus through most or all of the virus's trip from the outside membrane the nucleus. This work required rather difficult assays for capsid integrity, judged by the inclusion of fluorescent protein GFP into the viral genome. Most of this protein escaped during viral fusion with the membrane, since most of the free volume of the virus is outside the capsid. But a small portion remained visible as long as the capsid remained intact.
Another paper took a closer look at the stability and composition of the capsid. It is known to be composed of roughly 1500 copies of the viral CA protein, in multimeric (hexameric and pentameric) rosettes. The structure above shows that at the center of these rosettes is a small pore, big enough to let in ATP- suggesting clearly that the deoxynucleotides needed for reverse transcription can enter even while the capsid is intact, providing the virus with the best of both worlds- protection from the cell's various specific antiviral defenses, but also food for its replication. In the lab, these capsids have been remarkably unstable, however, leading some to believe that the virus uncoats soon after it enters the cell. But these authors find that a chemical that is obscure, but common in cells, IP6, is shaped just right, and negatively charged just right, to sit in these rosette pores and stabilize the whole structure, extending the lifespan of capsids in the test tube to hours instead of minutes.
Two mysteries remain- first is how deoxyribonucleotides get in if IP6 is blocking the pores and stabilizing the structure. The likely answer is simply that these small molecules are not plugs. They have more stochastic behavior, jumping on and off frequently enough to allow other small molecules occasional access. The second question is what finally causes the capsid to unravel at the nucleus and release its now-DNA genome to join that of the cell. It is thought that these capsids are too large to go through the nuclear pore, so they may dock in some fashion and only transmit their contents, probably using special signals that mimick one of the many other proteins and molecules that are regularly imported into the nucleus. That process is currently unknown, and may be another avenue of viral inhibition and drug development.
For such a tiny entity, HIV-1 has a very complicated life. Its study has generated numerous drugs that interfere with key life events, and have brought it under control in most developed areas of the world. But there is a lot left to learn. While its fusion with target cell membranes and eventual replication have received the most study, less is known about what happens in between.
 |
| The layers of HIV. Outside is a membrane, which features proteins like gp120 that binds to T-cell receptors. |
The virus has several layers. On the outside is a membrane coat, designed to protect the virus in moist environments, but more imporantly to expose surface proteins that seek out T-cells specifically, and then to fuse with their membranes, thus entering them. Inside the lipid membrane and its supporting proteins is the capsid, a protein coat that protects the HIV genome from attack by internal T-cell defenses like RNases, helicases, RNA sensing proteins, interferons, and their various downstream effects. This capsid finally lets go if its cargo at the nucleus, where the viral genome, now transformed into double stranded DNA, integrates itself into the cell's genome.
 |
| Electron micrograph of HIV particles. The capsid sits inside the membrane coat, all of which is about 120 nm in diameter. |
Innermost is the genome, composed of RNA, not DNA. Packaged alongside it are key enzymes integrase, RNase H, and reverse transcriptase. Once HIV enters the cell, and meets the rich mother-lode of ATP and other nucleotides, including deoxy-ribonucleotides that are the building blocks of DNA, the reverse transcriptase can begin its work. It copies the genome in a complex sequence of starts within the linear RNA, hops to the other end of the RNA, continuation, and finally termination. All the while, the RNase digests the original RNA genome, in a remarkable process of self-transmutation. It is thought that synthesis of the complementary DNA strand only begins after the virus rides the cell's light rail system (microtubules) to the nucleus, where it leaves its capsid behind.
Some recent papers shed a bit of light on the nature of the HIV capsid, which is more interesting than previously understood. It was not previously clear whether the capsid uncoats at the membrane during original entry, or only at the nucleus, or even inside the nucleus. But one paper shows quite definitively that the capsid remains intact through the first strand of DNA synthesis, thus through most or all of the virus's trip from the outside membrane the nucleus. This work required rather difficult assays for capsid integrity, judged by the inclusion of fluorescent protein GFP into the viral genome. Most of this protein escaped during viral fusion with the membrane, since most of the free volume of the virus is outside the capsid. But a small portion remained visible as long as the capsid remained intact.
 |
| One hexamer of the capsid protein CA. At the center is pore, accommodating a very small molecule of ATP, in yellow/orange. The surrounding blue parts of the protein are positively charged, ideally suited to attract nucleotides and the IP6 stabilizing molecule. |
Another paper took a closer look at the stability and composition of the capsid. It is known to be composed of roughly 1500 copies of the viral CA protein, in multimeric (hexameric and pentameric) rosettes. The structure above shows that at the center of these rosettes is a small pore, big enough to let in ATP- suggesting clearly that the deoxynucleotides needed for reverse transcription can enter even while the capsid is intact, providing the virus with the best of both worlds- protection from the cell's various specific antiviral defenses, but also food for its replication. In the lab, these capsids have been remarkably unstable, however, leading some to believe that the virus uncoats soon after it enters the cell. But these authors find that a chemical that is obscure, but common in cells, IP6, is shaped just right, and negatively charged just right, to sit in these rosette pores and stabilize the whole structure, extending the lifespan of capsids in the test tube to hours instead of minutes.
Two mysteries remain- first is how deoxyribonucleotides get in if IP6 is blocking the pores and stabilizing the structure. The likely answer is simply that these small molecules are not plugs. They have more stochastic behavior, jumping on and off frequently enough to allow other small molecules occasional access. The second question is what finally causes the capsid to unravel at the nucleus and release its now-DNA genome to join that of the cell. It is thought that these capsids are too large to go through the nuclear pore, so they may dock in some fashion and only transmit their contents, probably using special signals that mimick one of the many other proteins and molecules that are regularly imported into the nucleus. That process is currently unknown, and may be another avenue of viral inhibition and drug development.
- We are all replicants.
- Carbon emissions not only keep going up, but up at an ever-increasing rate.
- Textbook regulatory capture. And in record time!
- Remember Bernie Madoff?
- China may be starting to wean off of coal .. or not.
Saturday, August 18, 2018
Blood Tests For Cancer
"Liquid biopsies" for cancer are coming to the clinic.
Cancer remains the winner in the war on cancer. New molecularly-driven precision treatments have improved outcomes for a few types of cancer, and the reduction in smoking has provided substantial improvements in death rates, but the overall statistics remain grim, most treatments are dreadful, and early detection is more a mirage than reality. One promising, though still experimental, area of progress is in detecting cancers using blood samples.
Early detection has been a holy grail, with enormous resources devoted to mammography and PSA tests, among much else, which have turned out to be of marginal utility, or far less than touted. I do not believe there is currently any cancer for which a reliable medical test of any kind provides detection before symptoms or manual / visible detection is possible. After the various reliable and unreliable methods of detection, assessment of cancers involves biopsy, which is far more invasive and disruptive than it sounds, piercing the putative site / organ with a large sampling needle which can cause permanent damage. Biopsy should be regarded as a full surgical procedure in its own right.
Both of these problems could be alleviated with effective blood tests for cancer presence, type, and progression. A significant development in the research field over the last decade or two has been the realization that cancers shed material constantly. Cells are sloughed off in live and dead form, and DNA from tumors is generally in circulation. One corollary is that metastasis is more a matter of these cells finding a congenial home than of their dispersal from their primary source. A second is that blood tests can detect these DNAs and cells on a routine basis.
The root method for doing so is PCR- that revolutionary method in molecular biology that harnesses DNA replication to amplify nucleic acids exponentially, allowing detection of infinitesimal amounts. One of the papers under review in this post claims that a single molecule of cancer cell DNA can be detected in 5 ml of blood. This is astonishing, but also puts bounds on the ultimate utility of this method, since they also say that less than half of grade 1 cancers provide even such a tiny signal. It turns out that, as one might expect, earlier and smaller cancers shed less material than later ones do.
This landmark paper tests patients with many different types of cancer to evaluate the possibility of a relatively blood test for certain known cancer mutations. They find that brain cancers are particularly poorly represented- their shed materials are likely to be confined due to the blood-brain barrier system, plus the glymphatic system. But other cancers are quite amenable to blood testing, at least when in an advanced state. This would at least be a boon to recurrence tracking, and treatment monitoring, for which (repeated) biopsy is either impractical or impossible.
For early screening, blood testing is not, as of this paper in 2014, truly reliable. On the other hand, it finds half of stage 1 cancers, which otherwise might not be found at all, raising the question of how such a cancer should diagnosed and found if a blood test finds, for example, that a common mutation (for example, in the gene TP53) is found to be afoot in a patient. Such mutations, which drive many different cancers, could come from virtually any organ. Some more sleuthing would be in order.
One such approach came up recently, in studies of regulatory markings on DNA, which some call "epigentic" marks. C nucleosides in DNA can be methylated and then derivitized from there to 5-hydroxymethyl 5-formyl, 5-carboxyl, and finally identified by the DNA repair pathway and excised / replaced. Typically, methylation is a repressive signal, part of the cellular machinery that turns off gene expression. In contrast, 5-hydroxymethy modified C residues seems to be associated with higher gene expression. At any rate, both modifications are dramatically reduced in cancer cells, and their patterns can be informative about the cancer's tissue of origin and prognosis/stage. There is even the possibility that the relative positions of 5-methyl-C and 5-hydroxymethyl-C in very small segments of DNA (detected by FRET, no less) could be informative on these issues, though that is more esoteric.
So far, these methods are plumbing the blood samples for specific DNA mutations in specific genes known to drive cancer, and thus have high specificity, but limited utility as general screening tools for patients who have not yet been diagnosed and could have any (or several) of thousands of different mutations. To do that, a far larger panel of genes needs to be assayed, possibly even whole genome sequencing, with an unbiased analysis of their mutations. But that begs the question of how to separate the cancer-derived DNA from all the other junk floating around in a blood sample. Methylation marks may be biased in cancer-derived DNA in useful ways, but they do not have categorically different characteristics usable for separating the wheat from the chaff. This is the big problem right now in cancer blood testing. On a practical level, it will start being used for already-diagnosed patients, to track their treatment and relapse. The cancer selection problem will likely be solved in a brute-force way by sequencing everything in the blood sample and sifting through that data using a growing catalog of cancer-causing mutations. But if some mark or characteristic can be found that is specific to cancer DNA, then general and convenient cancer screening via blood tests will come much sooner.
Cancer remains the winner in the war on cancer. New molecularly-driven precision treatments have improved outcomes for a few types of cancer, and the reduction in smoking has provided substantial improvements in death rates, but the overall statistics remain grim, most treatments are dreadful, and early detection is more a mirage than reality. One promising, though still experimental, area of progress is in detecting cancers using blood samples.
 |
| Cancer trends in the US, overall. |
Early detection has been a holy grail, with enormous resources devoted to mammography and PSA tests, among much else, which have turned out to be of marginal utility, or far less than touted. I do not believe there is currently any cancer for which a reliable medical test of any kind provides detection before symptoms or manual / visible detection is possible. After the various reliable and unreliable methods of detection, assessment of cancers involves biopsy, which is far more invasive and disruptive than it sounds, piercing the putative site / organ with a large sampling needle which can cause permanent damage. Biopsy should be regarded as a full surgical procedure in its own right.
Both of these problems could be alleviated with effective blood tests for cancer presence, type, and progression. A significant development in the research field over the last decade or two has been the realization that cancers shed material constantly. Cells are sloughed off in live and dead form, and DNA from tumors is generally in circulation. One corollary is that metastasis is more a matter of these cells finding a congenial home than of their dispersal from their primary source. A second is that blood tests can detect these DNAs and cells on a routine basis.
The root method for doing so is PCR- that revolutionary method in molecular biology that harnesses DNA replication to amplify nucleic acids exponentially, allowing detection of infinitesimal amounts. One of the papers under review in this post claims that a single molecule of cancer cell DNA can be detected in 5 ml of blood. This is astonishing, but also puts bounds on the ultimate utility of this method, since they also say that less than half of grade 1 cancers provide even such a tiny signal. It turns out that, as one might expect, earlier and smaller cancers shed less material than later ones do.
 |
| Early stage cancers are hard to detect, but not impossible. The lowest Y-axis levels correspond to one molecule in the sample. |
This landmark paper tests patients with many different types of cancer to evaluate the possibility of a relatively blood test for certain known cancer mutations. They find that brain cancers are particularly poorly represented- their shed materials are likely to be confined due to the blood-brain barrier system, plus the glymphatic system. But other cancers are quite amenable to blood testing, at least when in an advanced state. This would at least be a boon to recurrence tracking, and treatment monitoring, for which (repeated) biopsy is either impractical or impossible.
 |
| Which cancers give usable blood-born DNA samples? |
"... 47% of patients with stage I cancers of any type had detectable ctDNA, whereas the fraction of patients with detectable ctDNA was 55, 69, and 82% for patients with stage II, III, and IV cancers, respectively."
For early screening, blood testing is not, as of this paper in 2014, truly reliable. On the other hand, it finds half of stage 1 cancers, which otherwise might not be found at all, raising the question of how such a cancer should diagnosed and found if a blood test finds, for example, that a common mutation (for example, in the gene TP53) is found to be afoot in a patient. Such mutations, which drive many different cancers, could come from virtually any organ. Some more sleuthing would be in order.
One such approach came up recently, in studies of regulatory markings on DNA, which some call "epigentic" marks. C nucleosides in DNA can be methylated and then derivitized from there to 5-hydroxymethyl 5-formyl, 5-carboxyl, and finally identified by the DNA repair pathway and excised / replaced. Typically, methylation is a repressive signal, part of the cellular machinery that turns off gene expression. In contrast, 5-hydroxymethy modified C residues seems to be associated with higher gene expression. At any rate, both modifications are dramatically reduced in cancer cells, and their patterns can be informative about the cancer's tissue of origin and prognosis/stage. There is even the possibility that the relative positions of 5-methyl-C and 5-hydroxymethyl-C in very small segments of DNA (detected by FRET, no less) could be informative on these issues, though that is more esoteric.
So far, these methods are plumbing the blood samples for specific DNA mutations in specific genes known to drive cancer, and thus have high specificity, but limited utility as general screening tools for patients who have not yet been diagnosed and could have any (or several) of thousands of different mutations. To do that, a far larger panel of genes needs to be assayed, possibly even whole genome sequencing, with an unbiased analysis of their mutations. But that begs the question of how to separate the cancer-derived DNA from all the other junk floating around in a blood sample. Methylation marks may be biased in cancer-derived DNA in useful ways, but they do not have categorically different characteristics usable for separating the wheat from the chaff. This is the big problem right now in cancer blood testing. On a practical level, it will start being used for already-diagnosed patients, to track their treatment and relapse. The cancer selection problem will likely be solved in a brute-force way by sequencing everything in the blood sample and sifting through that data using a growing catalog of cancer-causing mutations. But if some mark or characteristic can be found that is specific to cancer DNA, then general and convenient cancer screening via blood tests will come much sooner.
- Rules are for little people.
- Such as immigrants. Who are Christians, right?
- Does Amazon need to be split up?
- Go ahead and stretch ... every day.
- Skateboard therapy.
- More bullying and sleaze.
- Studies in groveling and sycophancy. What causes this temperament?
- Remember Martha Mitchell?
- The Taliban is winning.
- Meanwhile, Japan has a problem of not asking questions.
- Capitalism is so not working.
- Aretha on piano.
- In California, old-money landlords make out like bandits.
Saturday, May 12, 2018
The Biology of Fluoride
Fluoride has no biological functions, other than the need to get rid of it.
Fluorine is the smallest and most reactive halogen, a relative of chlorine, bromine, and iodine. Chloride is ubiquitous in salts and in the ionic milieu of our bodies, and iodine has found a central role in metabolism in virtually all species. Even bromine has various biological roles, though mostly in microorganisms. Fluorine, however, has found no role at all, despite being relatively common- more abundant in rocks than chlorine, let alone bromine or iodine. Its only part to play is as a noxious ion to get rid of. And all organisms have ways to get rid of it, via both active and passive trasporters. More on that below.
Humans, in their wisdom, however, have found some remarkable uses for fluorine. Modern chemistry uses it a great deal, to make very tough chemicals like Teflon, Lipitor, and Lexapro. Virtually nothing displaces fluorine from carbon bonds, so its compounds, while very useful, also end up as rather persistent waste products. More interesting, however, is our practice of ingesting fluoride (the ionic form of fluorine) in small amounts for oral health. This has been a subject of tremendous controversy and conspiracy theorizing for decades. But the benefits couldn't be more clear- teeth are much tougher from trace topical exposure to fluorine, which works its way into the crystal structure of enamel.
However, ingesting fluoride is another matter. It is reputed to cause kidney problems at higher concentrations, but there is very little epidemiology to support claims that these risks start at low concentrations.. anywhere near the levels used in drinking water supplementation. Similar to observations of bone deformities and tooth fluorosis, the syndrome of too much fluoride common in geologic regions with excess fluoride, there would have been observations of rampant kidney or other disorders. But that doesn't seem to be the case, other than very sketchy reports. At any rate, the therapeutic dose of fluoride put in water supplies is about 30 micromolar, while the newest regulations in the US establish a conservative cap of about 100 micromolar, in light of the lack of any use for higher concentrations, and the occasional problems from higher natural exposure, top which the artificial amount adds.
So we can't do much with fluorine, biologically speaking. Indeed it is generally toxic, messing with the phosphate chemistry that is central to all life. How do we get rid of it? There are three mechanisms, overall. In animals, our kidneys take care of it, using clever ion transport to excrete excess fluoride. (Recall that the first step of the kidney's work is to remove all the small solutes from blood plasma, and then later to selectively bring back the important things we want, like sugar, some salts, lactate, water, etc. The remainder that is not actively re-absorbed includes such oddities as fluoride.)
But other organisms that live directly in the soup, i.e. microorganisms, all need to take specific and active measures on the cellular level against fluoride. An important point in this chemistry is that fluoride has a significant acid-base preference. HF forms at relatively high pH (pKa of 3.4, much higher than HCl, which stays ionic to pH 1 and below, an oddity of fluoride's chemistry), which means that in moderately acidic environments, external HF can easily form and diffuse into cells as an uncharged entity, and there, under more neutral conditions, dissociate and be trapped as F- ions. This leads to chronic over-loading of cells with F-, (up to 30X over external levels), which can be remedied by a protein channel (second mechanism) that lets these ions back out passively, while not letting out other ions such as the closely related chloride. The third mechanism, exclusively used by bacteria, is active antiport, (H+/F-), using the stored energy of the proton gradient (high outside, low inside) to drive F- excretion.
A recent paper described the simple passive transporters that are ubiquitously used for fluoride export in microbes. They are odd in that it takes two proteins to form a functional transporter. The channel through which the F- ion passes is on the surface between the two proteins, in a symmetric structure that forms two channels (above). In eukayotic microbes like yeast cells, two such genes have become fused to form one gene encoding a protein that retains dimeric symmetry, but one of whose channels has become non-functional / vestigial.
The channel is, understandably, very tight, with intensive coordination all along the way, particularly with uncharged phenylalanines which provide an unusual side-ways polar coordination that is proposed to make the channel particularly specific to F-, vs Cl-. And it is very selective- over 10,000-fold selective for F- vs Cl-. Replacing these phenylalanines with the hydrophobic amino acid isoleucine reduces F- transport to negligible levels. It would have been interesting to ask what a less bulky and less hydrophobic replacement like glycine or threonine does to the channel's activity, perhaps making it significantly less selective, while still functional.
Fluorine is the smallest and most reactive halogen, a relative of chlorine, bromine, and iodine. Chloride is ubiquitous in salts and in the ionic milieu of our bodies, and iodine has found a central role in metabolism in virtually all species. Even bromine has various biological roles, though mostly in microorganisms. Fluorine, however, has found no role at all, despite being relatively common- more abundant in rocks than chlorine, let alone bromine or iodine. Its only part to play is as a noxious ion to get rid of. And all organisms have ways to get rid of it, via both active and passive trasporters. More on that below.
Humans, in their wisdom, however, have found some remarkable uses for fluorine. Modern chemistry uses it a great deal, to make very tough chemicals like Teflon, Lipitor, and Lexapro. Virtually nothing displaces fluorine from carbon bonds, so its compounds, while very useful, also end up as rather persistent waste products. More interesting, however, is our practice of ingesting fluoride (the ionic form of fluorine) in small amounts for oral health. This has been a subject of tremendous controversy and conspiracy theorizing for decades. But the benefits couldn't be more clear- teeth are much tougher from trace topical exposure to fluorine, which works its way into the crystal structure of enamel.
However, ingesting fluoride is another matter. It is reputed to cause kidney problems at higher concentrations, but there is very little epidemiology to support claims that these risks start at low concentrations.. anywhere near the levels used in drinking water supplementation. Similar to observations of bone deformities and tooth fluorosis, the syndrome of too much fluoride common in geologic regions with excess fluoride, there would have been observations of rampant kidney or other disorders. But that doesn't seem to be the case, other than very sketchy reports. At any rate, the therapeutic dose of fluoride put in water supplies is about 30 micromolar, while the newest regulations in the US establish a conservative cap of about 100 micromolar, in light of the lack of any use for higher concentrations, and the occasional problems from higher natural exposure, top which the artificial amount adds.
So we can't do much with fluorine, biologically speaking. Indeed it is generally toxic, messing with the phosphate chemistry that is central to all life. How do we get rid of it? There are three mechanisms, overall. In animals, our kidneys take care of it, using clever ion transport to excrete excess fluoride. (Recall that the first step of the kidney's work is to remove all the small solutes from blood plasma, and then later to selectively bring back the important things we want, like sugar, some salts, lactate, water, etc. The remainder that is not actively re-absorbed includes such oddities as fluoride.)
But other organisms that live directly in the soup, i.e. microorganisms, all need to take specific and active measures on the cellular level against fluoride. An important point in this chemistry is that fluoride has a significant acid-base preference. HF forms at relatively high pH (pKa of 3.4, much higher than HCl, which stays ionic to pH 1 and below, an oddity of fluoride's chemistry), which means that in moderately acidic environments, external HF can easily form and diffuse into cells as an uncharged entity, and there, under more neutral conditions, dissociate and be trapped as F- ions. This leads to chronic over-loading of cells with F-, (up to 30X over external levels), which can be remedied by a protein channel (second mechanism) that lets these ions back out passively, while not letting out other ions such as the closely related chloride. The third mechanism, exclusively used by bacteria, is active antiport, (H+/F-), using the stored energy of the proton gradient (high outside, low inside) to drive F- excretion.
 |
| Structure of two copies of E. coli-derived Fluc, a passive fluoride channel/exporter. The proteins are blue and yellow, respectively, the membrane represented by black lines, and the fluoride ions are modeled as gray or red balls. Given the symmetry of the proteins and their passive role, the orientation (up/down) makes no difference. The channel is formed at the interfaces between the two proteins. |
A recent paper described the simple passive transporters that are ubiquitously used for fluoride export in microbes. They are odd in that it takes two proteins to form a functional transporter. The channel through which the F- ion passes is on the surface between the two proteins, in a symmetric structure that forms two channels (above). In eukayotic microbes like yeast cells, two such genes have become fused to form one gene encoding a protein that retains dimeric symmetry, but one of whose channels has become non-functional / vestigial.
 |
| A close-up view of one of the channels, showing some of the key individual amino acids that coordinate / bind to the fluoride ion as it travels along. This close physical and electrostatic coordination insures that nothing that is not fluorine can get through. Notably, part of the job is done by uncharged phenylalanine residues, (blue and orange ring structures), which are usually regarded as hydrophobic, but have a slight face/edge polarization that can be exploited by strong ions |
The channel is, understandably, very tight, with intensive coordination all along the way, particularly with uncharged phenylalanines which provide an unusual side-ways polar coordination that is proposed to make the channel particularly specific to F-, vs Cl-. And it is very selective- over 10,000-fold selective for F- vs Cl-. Replacing these phenylalanines with the hydrophobic amino acid isoleucine reduces F- transport to negligible levels. It would have been interesting to ask what a less bulky and less hydrophobic replacement like glycine or threonine does to the channel's activity, perhaps making it significantly less selective, while still functional.
- Unfit to serve on a sewer board, cont. Climate, shimate.
- Desperate US military and allies have lost ground.
- Antitrust should not be just about consumers.
- Property is monopoly.
- Tourism makes up 8% of carbon emissions.
- Lowdown on money laundering.
- Driving remains dangerous.
- Some non-profits are not so non-profitable.
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