Category Archives: Medicine in general

Lactose intolerance and the proteins of the synaptic cleft

What does lactose intolerance have to do with the zillions of proteins happily infesting the synaptic cleft?  Only someone whose mind was warped into very abstract thinking by rooming with philosophy majors in college would see a connection.

The synaptic cleft is of immense theoretical interest to neuroscientists, drug chemists and pharmacologists, and of great practical interest to people affected by neurologic and psychiatric disease either in themselves or someone they know (e.g. just about everyone).

Almost exactly a year ago I wrote a post about a great paper on the proteins of the synaptic cleft by Thomas Sudhof.  You may read the post after the *****

Well Dr. Sudhof is back with another huge review of just how synapses are formed [ Neuron vol. 100 pp. 276 – 293 ’18 ], which covers very similar ground.

It is clear that he’s depressed by the state of the field.  Here are a few quotes

“I believe that we may need to pay more attention to technical details than customary because the pressures on investigators have increased the tendency to publish preliminary results, especially results obtained with new methods whose limitations are not yet clear.”

Translation: a lot of the stuff coming out is junk.

“Given the abundance of papers reporting non-validated protein interactions that cannot possibly be all correct, it seems that confidence in a possible protein-protein interaction requires either isolation of a stable complex or biophysical measurements of interactions using recombinant purified proteins.”

Translation:  Oy vey !

“Pre- or postsynaptic specializations are surprisingly easy to induce by diverse signals. This was first shown in pioneering studies demonstrating that polylysine beads induce formation of presynaptic nerve terminals in cultured neurons and in brain in vivo.” Obviously this means that you have to be very careful when you claim that a given protein or two causes a synapse to form, which researchers have not been.”

Translation not needed.

Then on to the meat of the review.  “An impressive number of candidate synaptic Cell Adhesion Molecules (CAMs) has been described (9 classes are given each with multiple members). For some of these CAMs, compelling data demonstrate their presence in synapses and suggest a functional role in synapses. Others, however, are less well documented. If one looks at the results in total, the overall impression is puzzlement: how do so many CAMs contribute to shaping a synapse?”

Then from 281 – 286 he goes into the various CAMs, showing the extent and variety of proteins found in the synaptic cleft.  Which ones are necessary and what are they doing?  Can they all be important.  There must be some redundancy as knockout of some doesn’t do much.

Here is where lactose tolerance/intolerance comes in to offer succor to the harried investigator.

Bluntly, they must be doing something, and something important,  or they wouldn’t be there.

People with lactose intolerance have nothing wrong with the gene which breaks down lactose.  Babies have no problem with breast milk.  The enzyme (lactase)  produced from the gene is quite normal in all of us.  However 10,000 years ago and earlier, cattle were not domesticated, so there was no dietary reason for a human weaned from the breast to make the enzyme.  Something turned off lactase production — from my reading, it’s not clear what.   The control region (lactase enhancer) for the lactase gene is 14,000 nucleotides upstream from the gene itself.  After domestication of cattle, so that people could digest milk their entire lives a mutation arose changing cytosine to thymine in the enhancer.  The farthest back the mutation has been found is 6.500 years. 3 other mutations are known, which keep the lactase gene expressed past weaning.  They arose independently.  All 4 spread in the population, because back then our ancestors were in a semi-starved state most of the time, and carriers had better nutrition.

How does this offer succor to Dr. Sudhof?  Simply this, here is a mechanism to turn off production of an enzyme our ancestors didn’t need past weaning.  Don’t you think this would be the case for all the proteins found in and around the synapse.  They must be doing something or they wouldn’t be there.  I realize that this is teleology writ large, but evolutionary adaptations make you think this way.


The bouillabaisse of the synaptic cleft

The synaptic cleft is so small ( under 400 Angstroms — 40 nanoMeters ) that it can’t be seen with the light microscope ( the smallest wavelength of visible light 3,900 Angstroms — 390 nanoMeters).  This led to a bruising battle between Cajal and Golgi a just over a century ago over whether the brain was actually made of cells.  Even though Golgi’s work led to the delineation of single neurons he thought the brain was a continuous network.  They both won the Nobel in 1906.

Semifast forward to the mid 60s when I was in medical school.  We finally had the electron microscope, so we could see synapses. They showed up as a small CLEAR spaces (e.g. electrons passed through it easily leaving it white) between neurons.  Neurotransmitters were being discovered at the same time and the synapse was to be the analogy to vacuum tubes, which could pass electricity in just one direction (yes, the transistor although invented hadn’t been used to make anything resembling a computer — the Intel 4004 wasn’t until the 70s).  Of course now we know that information flows back and forth across the synapse, with endocannabinoids (e. g. natural marihuana) being the major retrograde neurotransmitter.

Since there didn’t seem to be anything in the synaptic cleft, neurotransmitters were thought to freely diffuse across it to being to receptors on the other (postsynaptic) side e.g. a free fly zone.

Fast forward to the present to a marvelous (and grueling to read because of the complexity of the subject not the way it’s written) review of just what is in the synaptic cleft [ Cell vol. 171 pp. 745 – 769 ’17 ] (It is likely behind a paywall).  There are over 120 references, and rather than being just a catalogue, the single author Thomas Sudhof extensively discusseswhich experimental work is to be believed (not that Sudhof  is saying the work is fraudulent, but that it can’t be used to extrapolate to the living human brain).  The review is a staggering piece of work for one individual.

The stuff in the synaptic cleft is so diverse, and so intimately involved with itself and the membranes on either side what what is needed for comprehension is not a chemist but a sociologist.  Probably most of the molecules to be discussed are present in such small numbers that the law of mass action doesn’t apply, nor do binding constants which rely on large numbers of ligands and receptors. Not only that, but the binding constants haven’t been been determined for many of the players.

Now for some anatomic detail and numbers.  It is remarkably hard to find just how far laterally the synaptic cleft extends.  Molecular Biology of the Cell ed. 5 p. 1149 has a fairly typical picture with a size marker and it looks to be about 2 microns (20,000 Angstroms, 2,000 nanoMeters) — that’s 314,159,265 square Angstroms (3.14 square microns).  So let’s assume each protein takes up a square 50 Angstroms on a side (2,500 square Angstroms).  That’s room for 125,600 proteins on each side assuming extremely dense packing.  However the density of acetyl choline receptors at the neuromuscular junction is 8,700/square micron, a packing also thought to be extremely dense which would give only 26,100 such proteins in a similarly distributed CNS synapse. So the numbers are at least in the right ball park (meaning they’re within an order of magnitude e.g. within a power of 10) of being correct.

What’s the point?

When you see how many different proteins and different varieties of the same protein reside in the cleft, the numbers for  each individual element is likely to be small, meaning that you can’t use statistical mechanics but must use sociology instead.

The review focuses on the neurExins (I capitalize the E  to help me remember that they are prEsynaptic).  Why?  Because they are the best studied of all the players.  What a piece of work they are.  Humans have 3 genes for them. One of the 3 contains 1,477 amino acids, spread over 1,112,187 basepairs (1.1 megaBases) along with 74 exons.  This means that just over 1/10 of a percent of the gene is actually coding for for the amino acids making it up.  I think it takes energy for RNA polymerase II to stitch the ribonucleotides into the 1.1 megabase pre-mRNA, but I couldn’t (quickly) find out how much per ribonucleotide.  It seems quite wasteful of energy, unless there is some other function to the process which we haven’t figured out yet.

Most of the molecule resides in the synaptic cleft.  There are 6 LNS domains with 3 interspersed EGFlike repeats, a cysteine loop domain, a transmembrane region and a cytoplasmic sequence of 55 amino acids. There are 6 sites for alternative splicing, and because there are two promoters for each of the 3 genes, there is a shorter form (beta neurexin) with less extracellular stuff than the long form (alpha-neurexin).  When all is said and done there are over 1,000 possible variants of the 3 genes.

Unlike olfactory neurons which only express one or two of the nearly 1,000 olfactory receptors, neurons express mutiple isoforms of each, increasing the complexity.

The LNS regions of the neurexins are like immunoglobulins and fill at 60 x 60 x 60 Angstrom box.  Since the synaptic cleft is at most 400 Angstroms long, the alpha -neurexins (if extended) reach all the way across.

Here the neurexins bind to the neuroligins which are always postsynaptic — sorry no mnemonic.  They are simpler in structure, but they are the product of 4 genes, and only about 40 isoforms (due to alternative splicing) are possible. Neuroligns 1, 3 and 4 are found at excitatory synapses, neuroligin 2 is found at inhibitory synapses.  The intracleft part of the neuroligins resembles an important enzyme (acetylcholinesterase) but which is catalytically inactive.  This is where the neurexins.

This is complex enough, but Sudhof notes that the neurexins are hubs interacting with multiple classes of post-synaptic molecules, in addition to the neuroligins — dystroglycan, GABA[A] receptors, calsystenins, latrophilins (of which there are 4).   There are at least 50 post-synaptic cell adhesion molecules — “Few are well understood, although many are described.”

The neurexins have 3 major sites where other things bind, and all sites may be occupied at once.  Just to give you a taste of he complexity involved (before I go on to  larger issues).

The second LNS domain (LNS2)is found only in the alpha-neurexins, and binds to neuroexophilin (of which there are 4) and dystroglycan .

The 6th LNS domain (LNS6) binds to neuroligins, LRRTMs, GABA[A] receptors, cerebellins and latrophilins (of which there are 4)_

The juxtamembrane sequence of the neurexins binds to CA10, CA11 and C1ql.

The cerebellins (of which there are 4) bind to all the neurexins (of a particular splice variety) and interestingly to some postsynaptic glutamic acid receptors.  So there is a direct chain across the synapse from neurexin to cerebellin to ion channel (GLuD1, GLuD2).

There is far more to the review. But here is something I didn’t see there.  People have talked about proton wires — sites on proteins that allow protons to jump from one site to another, and move much faster than they would if they had to bump into everything in solution.  Remember that molecules are moving quite rapidly — water is moving at 590 meters a second at room temperature. Since the synaptic cleft is 40 nanoMeters (40 x 10^-9 meters, it should take only 40 * 10^-9 meters/ 590 meters/second   60 trillionths of a second (60 picoSeconds) to cross, assuming the synapse is a free fly zone — but it isn’t as the review exhaustively shows.

It it possible that the various neurotransmitters at the synapse (glutamic acid, gamma amino butyric acid, etc) bind to the various proteins crossing the cleft to get their target in the postsynaptic membrane (e.g. neurotransmitter wires).  I didn’t see any mention of neurotransmitter binding to  the various proteins in the review.  This may actually be an original idea.

I’d like to put more numbers on many of these things, but they are devilishly hard to find.  Both the neuroligins and neurexins are said to have stalks pushing them out from the membrane, but I can’t find how many amino acids they contain.  It can’t find how much energy it takes to copy the 1.1 megabase neurexin gene in to mRNA (or even how much energy it takes to add one ribonucleotide to an existing mRNA chain).

Another point– proteins have a finite lifetime.  How are they replenished?  We know that there is some synaptic protein synthesis — does the cell body send packages of mRNAs to the synapse to be translated there.  There are at least 50 different proteins mentioned in the review, and don’t forget the thousands of possible isoforms, each of which requires a separate mRNA.

Old Chinese saying — the mountains are high and the emperor is far away. Protein synthesis at the synaptic cleft is probably local.  How what gets made and when is an entirely different problem.

A large part of the review concerns mutations in all these proteins associated with neurologic disease (particularly autism).  This whole area has a long and checkered history.  A high degree of cynicism is needed before believing that any of these mutations are causative.  As a neurologist dealing with epilepsy I saw the whole idea of ion channel mutations causing epilepsy crash and burn — here’s a link —’ve-found-the-mutation-causing-your-disease-not-so-fast-says-this-paper/

Once again, hats off to Dr. Sudhof for what must have been a tremendous amount of work


Who doesn’t want to be smarter?

I’ve never met anyone (even future Nobel laureates) who didn’t wish they were smarter.  So cognitive training should do the trick.  Right?  Not so fast.  In a very well written (and even funny in parts) article in PNAS vol 115 pp. 9897 – 9904 ’18 titled “How to play 20 questions with nature and lose: Reflections on 100 years of brain training research” all the pitfalls of setting up a study to prove or disprove the benefits of cognitive training are laid out.  The paper is worth reading for anyone considered any sort of manipulation to change human behavior it (including medication which is why drug chemists should be interested in it).  You can read it for free at

This didn’t work for someone —

Try this one —

An enormous number of pitfalls of the work already done on the efficacy of cognitive training are laid out, far too numerous to summarize here.

I’ve written about one such pitfall (expectancy effects) earlier — here it is

Science proves cognitive training will raise your IQ 5 – 10 points

Who among you doesn’t want to be smarter? A placebo controlled study with 25 people in each group showed that cognitive training raised IQ 5 – 10 points [ Proc. Natl. Acad. Sci. vol. 113 pp. 7470 – 7474 ’16 ].

You know that there has to be a catch and there is. The catch points to a problem with every placebo controlled trial ever done, particularly those with drugs, so drug chemists pay attention.

What was the placebo? It was the way subjects are recruited for these studies. Of 19 previous studies in the literature, 17 recruited patients using terms like ‘cognition’ or ‘brain training’, so the authors put out two ads for subjects.

Here are the two ads they used

Ad #1

Brain Training and Cognitive Enhancement
Numerous studies of ahown that working memory training can increase fluid intelligences (several references cited)
Participate in a study today !
EMail for more information

Ad #2

EMail Today and Participate in a study
Need SONA credits? (I have no idea what they are)
Sign up for a study today and earn up to 5 credits
Participate in a study today !

I might mention that the two ads were identical in total size, font sizes, coloration used etc. etc.

” Two individual difference metrics regarding beliefs about cognition and intelligence were also collected as potential moderators. The researchers who interacted with participants were blind to the goal of the experiment and to the experimental condition”  Not bad. Not bad at all.

The results: those recruited with ad #1 showed the increase in IQ, those recruited with ad #2 showed no improvement.

It was an expectancy effect. Those who thought intelligence could be raised by training, showed the greatest IQ improvement.   Every sick patient wants to get better, and any drug trial simply must mention what it is for, the risks and rewards, so this effect is impossible to avoid. It probably explains the high placebo response rate for migraine and depression (over 30% usually).

What is really impressive (to me at least) is that the improvement was not in a subjective rating scale (such as is used for depression), but in something as objective as it gets. IQ questions have a right and wrong answers. You can argue about whether they ‘really’ measure intelligence, but they measure what they measure and fluid intelligence is one of them.

Medicine is full of fads and fashions, sugar is poison, fat is bad (no it’s good) etc. etc. and this is true in spades for treatments, particularly those touted in the press. Next time you’re in a supermarket, look at the various nostrums mentioned in the magazines at the checkout stand.

When I first started out in practice, one particular headache remedy was getting great results. The rationale behind it seemed bizarre, so I asked a very smart  old GP about it — his advice — “use it while it works”. Rest in peace, Herb

Non-patent trolling

A conversation with a son who is in high tech brought up what a blister on the body politic patent trolling is  I told him that I’m having trouble simply trying to give an idea away.  The idea is basically that some cases of chronic fatigue syndrome are due to senescent cells.  There is a simple way to look for this — measure a master transcription factor for cellular senescence (p16INK^4a) in blood cells.  If correct, a rational therapy for CFS (senolytics) is immediately at hand.  I’ve shopped this around, and someone at Stanford involved with CFS claims that he will test it.  I’ve heard nothing so far.  The idea is free for the taking.  Therapy for CFS essentially  helps patients live their symptoms rather than diminishing them or attacking the underlying problem.

Since I”m going to Venice for 2 weeks to celebrate my wife’s birthday, there won’t be any new posts for a while — so here is the idea as presented in two posts from my blog — take it and run with it.  The patients are waiting.

Not a great way to end 2017

Not a great way to end 2017

2017 ended with a rejection of the following letter to PNAS.

As a clinical neurologist with a long standing interest in muscular dystrophy(1), I was referred many patients who turned out to have chronic fatigue syndrome (CFS) . Medicine, then and now, has no effective treatment for CFS.

A paper (2) cited In an excellent review of cellular senescence (3) was able to correlate an intracellular marker of senescence (p16^INK4a) with the degree of fatigue experienced by patients undergoing chemotherapy for breast cancer. Chemotherapy induces cellular senescence, and the fatigue was thought to come from the various cytokines secreted by senescent cells (Senescence Associated Secretory Phenotype—SASP) It seems logical to me to test CFS patients for p16^INK4a (4).
I suggested this to the senior author; however, he was nominated as head of the National Cancer Institute just 9 days later. There the matter rested until the paper of Montoya et al. (5) appeared in July. I looked up the 74 individual elements of the SASP and found that 9 were among the 17 cytokines whose levels correlated with the degree of fatigue in CFS. However, this is not statistically significant as Montoya looked at 51 cytokines altogether.

In October, an article(6) on the possibility of killing senescent cells to prevent aging contained a statement that Judith Campisi’s group (which has done much of the work on SASP) had identified “hundreds of proteins involved in SASPs”. (These results have not yet been published.) It is certainly possible that many more of Montoya’s 17 cytokines are among them.

If this is the case, a rational therapy for CFS is immediately apparent; namely, the senolytics, a class of drugs which kills senescent cells. A few senolytics are currently available clinically and many more are under development as a way to attack the aging process (6).

If Montoya still has cells from the patients in the study, measuring p16^INK4a could prove or disprove the idea. However, any oncology service could do the test. If the idea proves correct, then there would be a way to treat the debilitating fatigue of both chemotherapy and CFS—not to mention the many more medical conditions in which severe fatigue is found.
Chemotherapy is a systemic process, producing senescent cells everywhere, which is why DeMaria (2) was able to use circulating blood cells to measure p16^INK4a. It is possible that the senescent cells producing SASP in CFS are confined to one tissue; in which case testing blood for p16^INK4a would fail. (That would be similar to pheochromocytoma cells, in which a few localized cells produce major systemic effects.)

Although senolytics might provide symptomatic treatment (something worthwhile having since medicine presently has nothing for the CFS patient), we’d still be in the dark about what initially caused the cells to become senescent. But this would be research well worth pursuing.

Anyone intrigued by the idea should feel free to go ahead and test it. I am a retired neurologist with no academic affiliation, lacking the means to test it.

1 Robinson, L (1979) Split genes and musclar dystrophy. Muscle Nerve 2: 458 – 464

2. He S, Sharpless N (2017) Senescence in Health and Disease. Cell 170: 1000 – 1011

3. Demaria M, et al. (2014) Cellular senescence promotes adverse effects of chemotherapy and cancer relapse. Cancer Discov. 7: 165 – 176


5. Montoya JG, et al., (2017) Cytokine signature associated with disease severity in chronic fatigue syndrome patients, Proc Natl Acad Sci USA 114: E7150-E7158

6. Scudellari M, (2017) To stay young, kill zombie cells Nature 551: 448 – 450

Is a rational treatment for chronic fatigue syndrome at hand?

If an idea of mine is correct, it is possible that some patients with chronic fatigue syndrome (CFS) can be treated with specific medications based on the results of a few blood tests. This is precision medicine at its finest.  The data to test this idea has already been acquired, and nothing further needs to be done except to analyze it.

Athough the initial impetus for the idea happened only 3 months ago, there have been enough twists and turns that the best way explanation is by a timeline.

First some background:

As a neurologist I saw a lot of people who were chronically tired and fatigued, because neurologists deal with muscle weakness and diseases like myasthenia gravis which are associated with fatigue.  Once I ruled out neuromuscular disease as a cause, I had nothing to offer then (nor did medicine).  Some of these patients were undoubtedly neurotic, but there was little question in my mind that many others had something wrong that medicine just hadn’t figured out yet — not that it hasn’t been trying.

Infections of almost any sort are associated with fatigue, most probably caused by components of the inflammatory response.  Anyone who’s gone through mononucleosis knows this.    The long search for an infectious cause of chronic fatigue syndrome (CFS) has had its ups and downs — particularly downs — see

At worst many people with these symptoms are written off as crazy; at best, diagnosed as depressed  and given antidepressants.  The fact that many of those given antidepressants feel better is far from conclusive, since most patients with chronic illnesses are somewhat depressed.

The 1 June 2017 Cell had a long and interesting review of cellular senescence by Norman Sharpless [ vol. 169 pp. 1000 – 1011 ].  Here is some background about the entity.  If you are familiar with senescent cell biology skip to the paragraph marked **** below

Cells die in a variety of ways.  Some are killed (by infections, heat, toxins).  This is called necrosis. Others voluntarily commit suicide (this is called apoptosis).   Sometimes a cell under stress undergoes cellular senescence, a state in which it doesn’t die, but doesn’t reproduce either.  Such cells have a variety of biochemical characteristics — they are resistant to apoptosis, they express molecules which prevent them from proliferating and — most importantly — they secrete a variety of proinflammatory molecules collectively called the Senescence Associated Secretory Phenotype — SASP).

At first the very existence of the senescent state was questioned, but exist it does.  What is it good for?  Theories abound, one being that mutation is one cause of stress, and stopping mutated cells from proliferating prevents cancer. However, senescent cells are found during fetal life; and they are almost certainly important in wound healing.  They are known to accumulate the older you get and some think they cause aging.

Many stresses induce cellular senescence of which mutation is but one.  The one of interest to us is chemotherapy for cancer, something obviously good as a cancer cell turned senescent has stopped proliferating.   If you know anyone who has undergone chemotherapy, you know that fatigue is almost invariable.


One biochemical characteristic of the senescent cell is increased levels of a protein called p16^INK4a, which helps stop cellular proliferation.  While p16^INK4a can easily be measured in tissue biopsies, tissue biopsies are inherently invasive. Fortunately, p16^INK4a can also be measured in circulating blood cells.

What caught my eye in the Cell paper was a reference to a paper about cancer [ Cancer Discov. vol. 7 pp. 165 – 176 ’17 ] by M. Demaria, in which the levels of p16^INK4a correlated with the degree of fatigue after chemotherapy.  The more p16^INK4a in the blood cells the greater the fatigue.

I may have been the only reader of both papers with clinical experience wth chronic fatigue syndrome.  It is extremely difficult to objectively measure a subjective complaint such as fatigue.

As an example of the difficulty in correlating subjective complaints with objective findings, consider the nearly uniform complaint of difficulty thinking in depression, with how such patients actually perform on cognitive tests — e. g. there is  little if any correlation between complaints and actual performance — here’s a current reference — Scientific Reports 7, Article number: 3901(2017) —  doi:10.1038/s41598-017-04353.

If the results of the Cancer paper could be replicated, p16^INK4 would be the first objective measure of a patient’s individual sense of fatigue.

So I wrote both authors, suggesting that the p16^INK4a test be run on a collection of chronic fatigue syndrome (CFS) patients. Both authors replied quickly, but thought the problem would be acquiring patients.  Demaria said that Sharpless had a lab all set up to do the test.

Then fate (in the form of Donald Trump) supervened.  A mere 9 days after the Cell issue appeared, Sharpless was nominated to be the head of the National Cancer Institute by President Trump.  This meant Dr. Sharpless had far bigger fish to fry, and he would have to sever all connection with his lab because of conflict of interest considerations.

I also contacted a patient organization for chronic fatigue syndrome without much success.  Their science advisor never responded.

There matters stood until 22 August when a paper and an editorial about it came out [ Proc. Natl. Acad. Sci. vol. 114 pp. 8914 – 8916, E7150 – E7158 ’17 ].  The paper represented a tremendous amount of data (and work).  The blood levels of 51 cytokines (measures of inflammation) and adipokines (hormones released by fat) were measured in both 192 patients with CFS (which can only be defined by symptoms) and 293 healthy controls matched for age and gender.

In this paper, levels of 17 of the 51 cytokines correlated with severity of CFS. This is a striking similarity with the way the p16^INK4 levels correlated with the degree of fatigue after chemotherapy).  So I looked up the individual elements of the SASP (which can be found in Annu Rev Pathol. 21010; 5: 99–118.)  There are 74 of them. I wondered how many of the 51 cytokines measured in the PNAS paper were in the SASP.  This is trickier than it sounds as many cytokines have far more than one name.  The bottom line is that 20 SASPs are in the 51 cytokines measured in the paper.

If the fatigue of CFS is due to senescent cells and the SASPs  they release, then they should be over-represented in the 17 of the 51 cytokines correlating with symptom severity.  Well they are; 9 out of the 17 are SASP.  However although suggestive, this increase is not statistically significant (according to my consultants on Math Stack Exchange).

After wrote I him about the new work, Dr. Sharpless noted that CFS is almost certainly a heterogeneous condition. As a clinician with decades of experience, I’ve certainly did see some of the more larcenous members of our society who used any subjective diagnosis to be compensated, as well as a variety of individuals who just wanted to withdraw from society, for whatever reason. They are undoubtedly contaminating the sample in the paper. Dr. Sharpless thought the idea, while interesting, would be very difficult to test.

But it wouldn’t at all.  Not with the immense amount of data in the PNAS paper.

Here’s how. Take each of the 9 SASPs and see how their levels correlate with the other 16 (in each of the 192 CSF patients). If they correlate better with SASPs than with nonSASPs, than this would be evidence for senescent cells being the cause some cases of CFS. In particular, patients with a high level of any of the 9 SASPs should be studied for such correlations.  Doing so should weed out some of the heterogeneity of the 192 patients in the sample.

This is why the idea is testable and, even better, falsifiable, making it a scientific hypothesis (a la Karl Popper).  The data to refute it is in the possession of the authors of the paper.

Suppose the idea turns out to be correct and that some patients with CFS are in fact that way because, for whatever reason, they have a lot of senescent cells releasing SASPs.

This would mean that it would be time to start trials of senolyic drugs which destroy senescent cells on the group with elevated SASPs. Fortunately, a few senolytics are currently inc linical use.  This would be precision medicine at its finest.

Being able to alleviate the symptoms of CFS would be worthwhile in itself, but SASP levels could also be run on all sorts of conditions associated with fatigue, most notably infection. This might lead to symptomatic treatment at least.  Having gone through mono in med school, I would have loved to have been able to take something to keep me from falling asleep all the time.

The chemical ingenuity of the AIDs virus

Pop quiz:  You are a virus with under 10,000 nucleotides in your genome.  To make the capsid enclosing your genome, you need to make 250 hexamers of a particular protein.  How do you do it?


Give up?


You grab a cellular metabolite with a mass under 1,000 Daltons to bind the 6 monomers together.  The metabolite occurs at fairly substantial concentrations (for a metabolite) of 10 – 40 microMolar.

What is the metabolite?

Give up?


It has nearly perfect 6 fold symmetry.


Still give up?

[ Nature vol. 560 pp. 509 – 512 ’18 ] says that it’s inositol hexakisphosphate (IP6)  — nomenclature explained at the end.

Although IP6 looks like a sugar (with 6 CHOH groups forming a 6 membered ring), it is not a typical one because it is not an acetal (no oxygen in the ring).  All 6 hydroxyls of IP6 are phosphorylated.  They bind to two lysines on a short (21 amino acids) alpha helix found in the protein (Gag which has 500 amino acids).  That’s how IP6 binds the 6 Gag proteins together. The paper has great pictures.

It is likely that IP6 is use by other cellular proteins to form hexamers (but the paper doesn’t discuss this).

IP6 is quite symmetric, and 5 of the 6 phosphorylated hydroxyls can be equatorial, so this is likely the energetically favored conformation, given the bulk (and mass) of the phosphate group.

I think that the AIDS virus definitely has more chemical smarts than we do.  Humility is definitely in order.

Nomenclature note:  We’re all used to ATP (Adenosine TriPhosphate) and ADP (Adenosine DiPhosphate) — here all 3 or 2 phosphates form a chain.  Each of the 6 hydroxyls of inositol can be singly phosphorylated, leading to inositol bis, tris, tetrakis, pentakis, hexakis phosphates.  Phosphate chains can form on them as well, so IP7 and IP8 are known (heptakis?, Octakis??)

When the dissociation constant doesn’t tell you what you want to know

Drug chemists spend a lot of time getting their drugs to bind tightly to their chosen target.  Kd’s (dissociation constants) are measured with care –  But Kd’s are only  a marker for the biologic effects that are the real reason for the drug.  That’s why it was shocking to find that Kd’s don’t seem to matter in a very important and very well studied system.

It’s not the small molecule ligand protein receptor most drug chemists deal with, it’s the goings on at the immunologic synapse between antigen presenting cell and T lymphocyte (a much larger ligand target interface — 1,000 – 2,000 Angstroms^2 — than the usual site of drug/protein binding).   A peptide fragment lies down in a groove on the Major Histocompatibility Complex (pMHC) where it is presented to the T lymphoCyte Receptor (TCR) — another protein complex.  The hope is that an immune response to the parent protein of the peptide fragment will occur.


However, the Kd’s (affinities)of strong (e.g. producing an immune response) peptide agonist ligands and those producing not much (e.g. weak) are similar and at times overlapping.  High affinity yet nonStimulatory interactions occur with high frequency in the human T cell repertoire [ Cell vol. 174 pp. 672 – 687 ’18 ].  The authors  determined the structure of both weak and strong ligands bound to the TCR.  One particular TCR had virtually the same structure when bound to strong and weak agonist ligands. When studied in two dimensional membranes, the dwell time of ligand with receptor didn’t distinguish strong from weak antigens (surprising).

In general the Kds  pMHC/TCR  are quite low — not in the nanoMolar range beloved by drug chemists (and found in antigen/antibody binding), but 1000 times weaker in the micromolar range.  So [ Proc. Natl. Acad. Sci. vol. 115 pp. E7369 – E7378 ’18 ] cleverly added an extra few amino acids which they call molecular velcro, to boost the affinity x 10 (actually this decreases Kd tenfold).

One rationale for the weak binding is that it facilitates scanning by the TCR of  the pMHC  repertoire allowing the TCR to choose the best.  So they added the velcro, expecting the repertoire to be less diverse (since the binding was tighter).  It was just the same. Again the Kd didn’t seem to matter.

Even more interesting, the first paper noted that productive TCR/pMHC bonds had catch bonds — e.g. bonds which get stronger the more you pull on them. The authors were actually able to measure the phenomenon. Catch bonds been shown to exist in a variety of systems (white cells sticking to blood vessel lining, bacterial adhesion), but their actual mechanism is still under debate.  The great thing about this paper (p. 682) is molecular dynamics simulation showed the conformational changes which occurred during catch bond formation in one case..   They even have videos.  Impressive.

This sort of thing is totally foreign to all solution chemistry, as there is no way to pull on a bond in solution.  Optical tweezers allow you to pull and stretch molecules (if you can attach them to large styrofoam balls).

Will acyclovir be a treatment for Alzheimer’s ?

When I was a first year medical student my aunt died of probable acute herpes simplex encephalitis at Columbia University Hospital in New York City.  That was 55 years ago and her daughters (teenagers at the time) still bear the scars.  Later, as a neurologist I treated it, and after 1977, when acyclovir, which effectively treats herpes encephalitis came out, I would always wonder if acyclovir would have saved her.

The drug is simplicity itself.  It’s just guanosine ( with two of the carbons of the ribose missing.  Herpesviruses have an enzyme which forms the triphosphate incorporating it into its DNA killing the virus.  Well, actually we have the same enzyme, but the virus’s enzyme is 3,000,000 times more efficient than ours, so acyclovir is relatively nontoxic to us.  People with compromised renal function shouldn’t take it.

What does this have to do with Alzheimer’s disease?  The senile plaque of Alzheimers is mostly the aBeta peptide (39 – 43 amino acids) from the amyloid precursor protein (APP).  This has been known for years, and my notes on various papers about over the years contain 150,000 characters or so.

Even so, there’s a lot we don’t understand about APP and the abeta peptide — e.g. what are they doing for us?  You can knockout the APP gene in mice and they appear normal and fertile.  The paper cited below notes that APP has been present in various species for the past 400,000,000 years of evolutionary time remaining pretty much unchanged throughout, so it is probably doing something useful

A recent paper in Neuron (vol. 99 pp. 56 – 63 ’18) noted that aBeta is actually an antimicrobial peptide.  When exposed to herpes simplex it binds to glycoproteins on its surface and then  oligomerizes forming amyloid (just like in the senile plaque) trapping the virus.  Abeta will protect mice against herpes simplex 1 (HSV1) encephalitis.  Even more important — infection of the mice with HSV1 induced abeta production in their brains.

People have been claiming infections as the cause of just about every neurodegeneration since I’ve been a neurologist, and papers have been written about HSV1 and Alzheimer’s.

Which brings me to the second paper (ibid. pp. 64 – 82) that looked for the viral RNAs and DNAs in over 900 or so brains, some with and some without Alzheimer’s.  They didn’t find HSV but they found two other herpes viruses known to infect man (HHV6, HHV7 — which cause roseola infantum).  Humans are subject to infection with 8 different herpes virus (Epstein Barr — mononucleosis, H. Zoster — chickenpox etc. etc.).   Just about everyone of us has herpes virus in latent form in the trigeminal ganglion — which gets sensory information from our faces.

So could some sort of indolent herpesvirus infection be triggering abeta peptide production as a defense with the senile plaque as a byproduct?  That being the case, given the minimal benefits of any therapy we have for Alzheimer’s disease so far, why not try acyclovir (Zovirax) on Alzheimer’s.

I find it remarkable that neither paper mentioned this possibility, or even discussed any of the antivirals active against herpesviruses.

Marshall McLuhan rides again

Marshall McLuhan famously said “the medium is the message”. Who knew he was talking about molecular biology?  But he was, if you think of the process of transcription of DNA into various forms of RNA as the medium and the products of transcription as the message.  That’s exactly what this paper [ Cell vol. 171 pp. 103 – 119 ’17 ] says.

T cells are a type of immune cell formed in the thymus.  One of the important transcription factors which turns on expression of the genes which make a T cell a Tell is called Bcl11b.  Early in T cell development it is sequestered away near the nuclear membrane in highly compacted DNA. Remember that you must compress your 1 meter of DNA down by 100,000fold to have it fit in the nucleus which is 1/100,000th of a meter (10 microns).

What turns it on?  Transcription of nonCoding (for protein) RNA calledThymoD.  From my reading of the paper, ThymoD doesn’t do anything, but just the act of opening up compacted DNA near the nuclear membrane produced by transcribing ThymoD is enough to cause this part of the genome to move into the center of the nucleus where the gene for Bcl11b can be transcribed into RNA.

There’s a lot more to the paper,  but that’s the message if you will.  It’s the act of transcription rather than what is being transcribed which is important.

Here’s more about McLuhan —

If some of the terms used here are unfamiliar — look at the following post and follow the links as far as you need to.

Well that was an old post.  Here’s another example [ Cell vol. 173 pp. 1318 – 1319, 1398 – 1412 ’18 ] It concerns a gene called PVT1 (Plasmacytoma Variant Translocation 1) found 25 years ago.  It was the first gene coding for a long nonCoding (for proteins RNA (lncRNA) found as a recurrent breakpoint in Burkitt’s lymphoma, which sadly took a friend (Nick Cozzarelli) far too young as (he edited PNAS for 10 years).

So PVT1 is involved in cancer.  The translocation turns on expression of the myc oncogene, something that has been studied out the gazoo and we’re still not sure of how it causes cancer. I’ve got 60,000 characters of notes on the damn thing, but as someone said 6 years ago “Whatever the latest trend in cancer biology — cell cycle, cell growth, apoptosis, metabolism, cancer stem cells, microRNAs, angiogenesis, inflammation — Myc is there regulating most of the key genes”

We do know that the lncRNA coded by PVT1 in some way stabilizes the myc protein [ Nature vol. 512 pp. 82 – 87 ’14 ].  However the cell experiments knocked out the lncRNA of PVT1 and myc expression was still turned on.

PVT1 resides 53 kiloBases away from myc on chromosome #8.  That’s about 17% of the diameter of the average nucleus (10 microns) if the DNA is stretched out into the B-DNA form seen in all the textbooks.  Since each base is 3.3 Angstroms thick that’s 175,000 Angstroms 17,500 nanoMeters 1.7 microns.  You can get an idea of how compacted DNA is in the nucleus when you realize that there are 3,200,000,000/53,000 = 60,000 such segments in the genome all packed into a sphere 10 microns in diameter.

To cut to the chase, within the PVT1 gene there are at least 4 enhancers (use the link above to find what all the terms to be used actually mean).  Briefly enhancers are what promoters bind to to help turn on the transcription of the genes in DNA into RNA (messenger and otherwise).  This means that the promoter of PVT1 binds one or more of the enhancers, preventing the promoter of the myc oncogene from binding.

Just how they know that there are 4 enhancers in PVT1 is a story in itself.  They cut various parts of the PVT1 gene (which itself has 306,721 basepairs) out, and place it in front of a reporter gene and see if transcription increases.

The actual repressor of myc is the promoter of PVT1 according to the paper (it binds to the enhancers present in the gene body preventing the myc promoter from doing so).  Things may be a bit more complicated as the PVT1 gene also codes for a cluster of 7 microRNAs and what they do isn’t explained in the paper.

So it’s as if the sardonic sense of humor of ‘nature’, ‘evolution’, ‘God’, (call it what you will) has set molecular biologists off on a wild goose chase, looking at the structure of the gene product (the lncRNA) to determine the function of the gene, when actually it’s the promoter in front of the gene and the enhancers within which are performing the function.

The mechanism may be more widespread, as 4/36 lncRNA promoters silenced by CRISPR techniques subsequently activated genes in a 1 megaBase window (possibly by the same mechanism as PVT1 and myc).

Where does McLuhan come in?  The cell paper also notes that lncRNA gene promoters are more evolutionarily conserved than their gene bodies.  So it’s the medium (promoter, enhancer) is the message once again (rather than what we thought the message was).


The other uses of amyloid (not all bad)

Neurologists and drug chemists pretty much view amyloid as a bad thing.  It is the major component of the senile plaque of Alzheimer’s disease, and when deposited in nerve causes amyloidotic polyneuropathy.  A recent paper and editorial casts amyloid in a different light [ Cell vol. 173 pp. 1068 – 1070, 1244 – 2253 ’18 ].  However if amyloid is so bad why do cytomegalovirus, herpes simplex viruses and E. Coli make proteins to prevent a type of amyloid from forming.

Cell death isn’t what it used to be.  Back in the day, they just died when things didn’t go well.  Now we know there are a variety of ways that cells die, and all of them have rather specific mechanisms.  Apoptosis (aka programmed cell death) is a mechanism of cell death used widely during embryonic development.  It allows the cell to die very quietly without causing inflammation.  Necroptosis is entirely different, it is another type of programmed cell death, designed to cause inflammation — bringing the immune system in to attack invading pathogens.

Two proteins (Receptor Interacting Protein Kinase 1 — RIPK1, and RIPK3) bind to each other forming amyloid, that looks for all the world like typical amyloid –it binds Congo Red, shows crossBeta diffraction and has a filamentous appearance.  Fascinating chemistry aside, the amyloid formed is crucial for necroptosis to occur, which is why various bugs try to prevent it.

The paper above describes the structure of the amyloid formed — unusual in itself, because until now amyloid was thought to involve the aggregation of a single protein.

The proteins are large: RIPK1 contains 671 amino acids, and RIPK3 contains 518.  They  both contain RHIMs (Receptor interacting protein Homotypic Interaction Motifs) which are fairly large themselves (amino acids 496 – 583 of RIPK1 and 388 – 518 of RIPK3).  Yet the amyloid the two proteins form use a very small stretches (amino acids 532 – 543 from RIPK1 and 451 – 462 from RIPK3).  How the rest of these large proteins pack around the beta strands of the 11 amino acid stretches isn’t discussed in the paper.  Even within these stretches, it is two consensus tetrapeptides (IQIG from RIPK1, and VQVG from RIPK3) that do most of the binding.

Even if you assume that I (Isoleucine) Q (glutamine) G (glycine) V (valine) occur at a frequency of 5%, in our proteome of 20,000 proteins assuming a length of amino acids IQIG and VQVG should occur 10 times each.  This may explain why 300/20,000 of our proteins contain a 100 amino acid  segment called BRICHOS which acts as a chaperone preventing amyloid formation. For details see —

Just another reason to take up the research idea in the link and find out just what other things amyloid is doing within our cells in the course of their normal functioning.


How Badly are Thy Genomes, Oh Humanity — take II

With apologies to Numbers 24:5, “How goodly are thy tents, Oh Jacob” —  a recent paper shows how shockingly error ridden our genomes actually are [ Science vol. 360 pp. 327 – 331  ’18 ].  I’d written about this in 2012 (see the end), but technology has marched on.  Back then only the parts of the genome coding for protein (the exome) were sequenced.  The present work did whole genome sequencing (WGS) to a mean coverage of 40+ (e.g. they sequenced the other 98 percent of the genome).

The authors were studying families in which one or more children had autism spectrum disorder to find genome abnormalities which might have caused the ASD. They were looking for structural variants (SVs) by which they mean ” biallelic deletion, tandem duplications, inversions, four classes of complex SV, and four families of mobile element insertions”

Why?  Because studying proteins alone doesn’t tell you how they are controlled.  That’s in the DNA surrounding them.  Structural variants are more likely to affect control elements than the proteins themselves.

Showing how technology has marched on they determined the whole genomes of 9274 subjects from 2600 families affected by ASD.

The absolutely mindboggling point in the article is the following direct quote “An average of 3746 SVs were detected per individual”.  That’s simply incredible (assuming the above isn’t a misprint).

Here’s the older post

How Badly Are Thy Genomes, Oh Humanity

With apologies to Numbers 24:5, “How goodly are thy tents, Oh Jacob” —  a recent paper shows how shockingly error ridden our genomes actually are [ Science vol. 337 pp. 64 – 69 ’12 ].  The authors sequenced roughly three quarters of the genes coding for proteins in some 2,439 people — e.g. 15,585 protein coding genes.  This left 98% of the genome untouched, primarily because we really don’t know what it does or how it does it, despite the fact that it controls, when, where and how much of each protein is made.  So they basically looked at the bricks from which we are built (the proteins) and not the plans (the 98%).

The news is not very good.  The subjects came from two groups: 1,351 Europeans and 1,088 Africans (the latter, because genetic diversity is far higher among Africans as that’s where humanity arose, and where mutations have had the longest time to accumulate).

The news is not very good. First, some background.

Recall that each nucleotide is one of four possibilities (A, T, G, C), and that each 3 nucleotides therefore has 4^3 = 64 possibilities.  61/64 combinations code for amino acids which, since we have only 20 gives a certain redundancy of the famed genetic code.   The other 3 combinations code for no amino acid (usually) and tell the machinery making proteins to stop.  Although crucial to our existence, these are called nonsense codons.

The genetic code is therefore 3fold degenerate (on average).  However, some amino acids are coded for by just 1 combination of 3 nucleotides while others are coded by as many as 6.  So some single nucleotide variants (SNVs) leave the amino acid coded for the same (these are the synonymous SNVs), while others change the amino acid (nonSynonymous SNVs), and possibly protein function.

Ask some one with sickle cell anemia how much trouble just one nonSynonymous SNV can cause — it’s only 1 amino acid out of 147.  Even worse, ask someone with cystic fibrosis where just one of 1,480 amino acids is missing.

Here’s the bad news.  In the population as a whole, they found 500,000 single nucleotide variants (SNVs).  If you’re still not sure what is meant by this, the 5 articles in should be all the background you need.

More than 400,000 of the variants were previously unknown.  Also more than 400,000 of them were found either in Africans or Europeans but not both.  If you divide 500,000 by 2,439 you get 205 variants per person.  However, SNVs are far more common than that, and each individual contains an average of 14,000.

Well, how many of the 500,000 or so SNVs they found are nonSynonymous? One would think about 1/3 statistically.  However, They found more than half 292,125/500,000 — nearly 60% — were nonSynonymous.

It get’s worse: 6,165 of the nonSynonymous variants are nonSense codons.  This means that the protein coded for by such a gene, terminates prematurely, meaning that it can terminate anywhere.  On average one would expect that half of these nonsense codons result in a protein of less than half the normal length.   This would very likely obliterate whatever function the protein had.

Obviously, they couldn’t test all 500,000 SNVs to see how they affected protein function (and we really only have a decent idea of what half our 20,000 or so proteins are doing).  They had to guess.  They came up with a figure of 2 – 4% of the 14,000 SNVs being functionally significant — That’s 280 – 560 significant mutations per individual.

Clearly, despite the horrible examples of cystic fibrosis and sickle cell anemia above, most of these can’t be doing very much, because these were normal people being studied.

There are all sorts of implications of this work.  One is the subject of a future post — how hard this diversity makes drug discovery.  Another reiterates the Tolstoy theme mentioned earlier about the genetic defects causing schizophrenia and autism — ““Happy families are all alike; every unhappy family is unhappy in its own way”.  Thus beginneth Anna Karenina.

For details please see  and

A third is that this shows that the 1000 fold expansion of the human population has pretty much obviated much natural selection eliminating these variants.  I’ll leave it to the geneticists to figure out what this means for the eventual survival of the species, as these mutants continue to accumulate.

The paper is fascinating, and sure to change our conception of what a ‘normal’ genome actually is.  Nonetheless, all they did was follow Yogi Berra’s dictum — “You can observe a lot by watching.”   It certainly wasn’t creative or ingenious in any sense.  Sometimes grunt work like this wins the day.

A research idea yours for the taking

Why would the gene for a protein contain a part which could form amyloid (the major component of the senile plaque of Alzheimer’s disease) and another part to prevent its formation. Therein lies a research idea, requiring no grant money, and free for you to pursue since I’ll be 80 this month and have no academic affiliation.

Bri2 (aka Integral TransMembrane protein 2B — ITM2B) is such a protein.  It is described in [ Proc. Natl. Acad. Sci. vol. 115 pp. E2752 – E2761 ’18 ]

As a former neurologist I was interested in the paper because two different mutations in the stop codon for Bri2 cause 2 familial forms of Alzheimer’s disease  Familial British Dementia (FBD) and Familial Danish Dementia (FDD).   So the mutated protein is longer at the carboxy terminal end.  And it is the extra amino acids which form the amyloid.

Lots of our proteins form amyloid when mutated, mutations in transthyretin cause familial amyloidotic polyneuropathy.  Amylin (Islet Amyloid Polypeptide — IAPP) is one of the most proficient amyloid formers.  Yet amylin is a protein found in the beta cell of the pancreas which releases insulin (actually in the same secretory granule containing insulin).

This is where Bri2 is thought to come in. It is also found in the pancreas.   Bri2 contains a 100 amino acid motif called BRICHOS  in its 266 amino acids which acts as a chaperone to prevent IAPP from forming amyloid (as it does in the pancreas of 90% of type II diabetics).

Even more interesting is the fact that the BRICHOS domain is found in 300 human genes, grouped into 12 distinct protein families.

Do these proteins also have segments which can form amyloid?  Are they like the amyloid in Bri2, in segments of the gene which can only be expressed if a stop codon is read through.  Nothing in the cell is perfect and how often readthrough occurs at stop codons isn’t known completely, but work is being done — Nucleic Acids Res. 2014 Aug 18; 42(14): 8928–8938.

I find it remarkable that the cause and the cure of a disease is found in the same protein.

Here’s the research proposal for you.  Look at the other 300 human genes containing the BRICHOS motif (itself just a beta sheet with alpha helices on either side) and see how many have sequences which can form amyloid.  There should be programs which predict the likelihood of an amino acid sequence forming amyloid.

It’s very hard to avoid teleology when thinking about cellular biochemistry and physiology.  It’s back to Aristotle where everything has a purpose and a design.  Clearly BRICHOS is being used for something or evolution/nature/natural selection/the creator would have long ago gotten rid of it.  Things that aren’t used tend to disappear in evolutionary time — witness the blind fish living in caves in Mexico that have essentially lost their eyes. The BRICHOS domain clearly hasn’t disappeared being present in over 1% of our proteins.

Suppose that many of the BRICHOS containing proteins have potential amyloid segments.  That would imply (to me at least) that the amyloid isn’t just junk that causes disease, but something with a cellular function. Finding out just what the function is would occupy several research groups for a long time.   This is also where you come in.  It may not pan out, but pathbreaking research is always a gamble when it isn’t stamp collecting.