The Grandmother cell comes to the neural net

We really don’t know how concepts are stored in the brain (or where) despite a lot of work.  Sure, if you destroy an area of the brain, you have a neurologic deficit, but if you pull out the plug of a lamp it does dark, but that the plug isn’t making the light. There has been a huge amount of human experimentation on subjects awaiting resection of an epileptic focus (to find and remove it).  Patients can be awake while all this is going on as the brain has no pain endings.

Is the concept of grandmother stored diffusely among many different neurons (or even glial cells) or is there a single cell storing it, so that if you lose that cell you lose the concept of grandmother.  Certainly the epilepsy work has found some very specific cells. In one fantastic (unreplicatable) paper, a neuron was found that responded to 7 different images of Jennifer Aniston, but not to other pictures of actresses (Julia Roberts) or even pictures of Aniston with another actor. [ Nature vol. 435 pp. 1102 – 1107, 2005 ].  Another neuron would respond only to another actress (Halle Berry).  The direction of presentation of the actress didn’t matter — face on, side view, etc. etc.

Nearly the same thing has been found with neural nets [ Proc. Natl. Acad. Sci. vol. 117 pp. 30071 – 30078 ’20 ].  A trained network contains units responding to high level visual concepts which aren’t explicitly labeled in the training data.  This paper was able to activate and deactivate any unit in the net to see where things were stored.

In a network with 512 output units, removing the 20 most important units for each class to be identified reduced accuracy to chance (53%).  Removing the 492 least important units only reduces class accuracy by 4%.

Not quite a grandmother cell, but getting close.

A letter to the PNAS editor which will never be published

“Starting out the year 2021 by looking back at the year 2020 might seem like an exercise in masochism, given the horrific loss of life, the untold economic hardships, the resurgence of white supremacy across the country“

As a good friend and college and grad school classmate of Nick Cozzarelli who edited PNAS for 10 years, I find this statement by the current PNAS editor — May Berenbaum, unhelpful, unscientific and frankly appalling. Had Nick not been taken from us far too soon in 2006 by Burkitt’s lymphoma, he’d likely be editing PNAS still. Does the editor’s statement rank with any of Nick’s work on DNA gyrase or DNA topology?

It is an exercise in the religion of political correctness, showing adherence to its current catechism, for political correctness and wokeness is nothing but a religion for the secular.  In our town expressions of faith abound on front lawns complete with statues of the virgin and signs proclaiming “we believe in science’. There really is no difference.

How a hack like Berenbaum got to be editor is beyond me, given the women scientists of great stature around (Doudna, Ghez, Randall).

Is Nick — https://en.wikipedia.org/wiki/Nicholas_R._Cozzarelli — an example of white supremacy? Nick’s father was an immigrant shoemaker from Jersey City and Nick worked his way through Princeton waiting on tables in commons.

Hydrogen bonding — again, again

I’ve been thinking about hydrogen bonding ever since my senior thesis in 1959. Although its’ role in the protein alpha helix had been known since ’51 and in the DNA double helix since ’53, little did we realize at the time just how important it would be for the workings of the cell. So I was lucky Dr. Schleyer put me at an IR spectrometer and had me make a bunch of compounds, to look for hydrogen bonding of OH, NH and SH to the pi electrons of the benzene ring. I had to make a few of them, which involved getting a (CH2)n chain between the benzene ring and the hydrogen donor. Just imagine the benzene as the body of a scorpion and the (CH2) groups as the length of the tail.  The SH compounds were particularly nasty, and people would look at their shoes when I’d walk into the eating club. Naturally the college yearbook screwed things up and titled my thesis “Studies in Hydrogen Bombing”, to which my parents’ friends would say — he looks like such a nice young man, why was he doing that?

At any rate I’m going to talk about a recent paper [ Science vol. 371 pp. 160 – 164 ’21 ] on the nature of the bond in the F H F – anion.  It’s going to be pretty hard core stuff with relatively little explanatory material. You’ve either been previously exposed to this stuff or you haven’t.  So this post is for the cognoscenti.  Hold on, it’s going to be wild ride.

In conventional hydrogen bonds, the donor (D) atom is separated from the Acceptor atom (A) by 2.7 Angstroms or more, and the hydrogen nucleus is found closer to A where the potential energy minimum is found.

So it looks like this D – H . .. A

The D-H bond isn’t normal, but is stretched  and weakened.  This means that it takes less energy to stretch it meaning that it absorbs infrared radiation at a lower frequency (higher wavelength) — red shift if you will. 

Such is what we were looking for and we found it comparing 

Benzene (CH2)n OH vibrations to butanol, pentanol, hexanol, etc etc. cyclohexane (CH2)n OH.

As the D – A distance shrinks there is ultimately a flat bottomed single well potential, where H becomes a confined particle (but still delocalized) betwen D and A.

The vibrations of protons in hydrogen bonds deviate markedly from the classic quantum harmonic oscillator beloved by physicists.  Here the energy levels on solving the classic H psi = E psi equation of quantum mechanics are evenly spaced (see Lancaster & Blundell “Quantum Field Theory” p. 20.)

However in real molecules, as you ascend the vibrational ladder, conventional hydrogen bonds show a decrease in the difference between energy levels (positive anharmonicity).  By contrast, when proton confinement dictates the potential shape in short hydrogen bonds (when D and A are close together, mimicking the particle in a box model in quantum mechanics) the spacing between states increases (negative anharmonicity).

The present work shows that in FHF- the proton motion is superharmonic — https://en.wikipedia.org/wiki/Subharmonic_function — which they don’t describe very well. 

When the F F distance gets below 2.4 Angstroms, covalent bonding starts to become a notable contributor to the short hydrogen bond, and the authors actually have evidence that there is overlap in FHF- between the 3s orbital of H and the 2 Pz orbitals of the donor and the acceptor atoms, yielding a stabilization of the resulting molecular orbital. 

Is that cool or what.  The bond sits right on the borderland between a covalent bond and a hydrogen bond, taking on aspects of both. 

 

Moonlighting molecules

Just when you thought you knew what your protein did, it goes off and does something completely different (and unexpected). This is called moonlighting, and is yet another reason drug discovery is hard. You can never be sure that your target is doing only what you think it’s doing.

Today’s example is PACAP, a neuromodulator/neurotransmitter made by neurons. Who knew that PACAP can and does act as an antibiotic when the brain is infected. [ Proc. Natl. Acad. Sci. vol. 118 e1917623117 ’21 ] does (PNAS no longer pages its journals, as last year’s total was over 33,000 !).   PACAP is a member of the vasoactive intestinal polypeptide, secretin, glucagon family of neuropeptides (mammals have over 100 neuropeptides according to the paper).

PACAP stands for Pituitary Adenylate Cyclase Activating Polypeptide. It comes in two forms containing 27 or 38 amino acids, both cleaved from a 176 amino acid precursor. There are 3 receptors for PACAP, all G Protein Coupled Receptors (GPCRs). A zillion functions have been ascribed to it, setting the circadian clock, protecting granule cells of the cerebellum. Outside the nervous system it is produced by immune cells in response to inflammatory conditions and antigenic stimulation. It is one of the most conserved neuropeptides throughout the course of evolution. Now we probably know why.

Showing how hard protein chemistry really is, PACAP is structurally similar to cathelicidin LL-37 an antimicrobial peptide, despite having less than 5% amino acid sequences in common. PACAP is cationic. Different sides of the protein have different characteristics, with one side being highly positively charged, and the other being hydrophobic (e.g. the protein is amphipathic). This is typical of antimicrobial peptides, and perturbation of microbial membranes by inducing negative Gaussian curvature probably explains its antibacterial activity.

In mouse models of Staph Aureus or Candida infections, PACAP is induced ‘up to’ 50 fold in the brain (or spleen or kidney) where it kills the bugs. Yet another reason drug discovery is so hard. We are mucking about in a system we barely understand.

There are many other examples of moonlighting proteins. Probably the best known is cytochrome c which is is a heme protein localized in the compartment between the inner and outer mitochondrial membranes where it functions to transfer electrons between complex III and complex IV of the respiratory chain. Oxidation and reduction of the iron atom in the heme along with movement along the mitochondrial intermembrane space allows it to schlep electrons between complexes of the respiratory chain.

All well and good. But cytochrome c also can tell a cell to commit suicide (apoptosis) when mitochondria are sufficiently damaged that cytochrome c can escape the intermembrane space. Who’d a thunk it?

How many more players are there in the cell (whose function we think we know) that are sneaking around — doing more things in heaven and Earth, Horatio, than are dreamt of in your philosophy?

Life at the edge of foldability

Insulin has contains 51 amino acids, split into two chains held together by disulfide bonds. The two chains come from a single gene and a single mRNA. Clearly a lot of processing is required. There is an A chain containing 21 amino acids and a B chain containing 30.

Mutations of phenylalanine at position #24 on the B chain results in MODY (Maturity Onset Diabetes of the Young) in which not enough insulin is made. Every known vertebrate insulin contains phenylalanine at B#24.

A fascinating paper [ Proc. Natl. Acad. Sci. vol. 117 pp. 29618 – 29628 ’20 ] explains why.

The reason is that having phenylalanine at B#24 appears to be crucial in folding of the insulin into its final form. We have 20 amino acids, and changing phenylalanine at B#24 to any of the other 19 amino acids results in poor insulin production.

Well we can now make any protein this long by automated peptide synthesis. Which amino acid is closest in shape and structure to phenylalanine? Tyrosine clearly. So the authors made insulin with tyrosine at B#24 (outside the cell).

Guess what — insulin synthesized (outside the cells) B#24 tyrosine bound to the insulin receptor almost as well (20 fold less well), but in terms of biological activity there was no difference. The 3 dimensional structures of B#24 tyrosine and B#24 phenylalanine were nearly identical.

The problem was in the processing of the parent protein (proinsulin) with something other than B#24 phenylalanine to insulin, which involves breaking the chain and forming 3 disulfide bonds between 6 cysteines. So it isn’t structures which evolution is conserving by B#24 phenylalanine but the ability to be processed and folded correctly.

Time to let the authors speak for themselves “Our results suggest that sequences required for insulin’s bioactivity (similar in all vertebrates) are frozen at the edge of nonfoldability.”

 

 

Feeling mentally soggy? It could be that your brain has shrunk what with it being winter and all

I wouldn’t have believed that part of the brain can shrink by 40% and then regrow, but exactly that happens to the Etruscan Shrew in the winter. So maybe it’s not you or even COVID19.

It’s a fascinating critter — the world’s smallest mammal, tipping the scales at 1.8 grams (about 6 aspirins). It has a very rapid metabolic rate, eating twice its weight daily. Things get tight in the winter so it shrinks its brain. Remember that even in big and sluggish us, as we sit there reading (or writing) this, our brain is receiving 20% of our cardiac output, despite being 3% or so of our body weight. For more about the Shrew see https://en.wikipedia.org/wiki/Etruscan_shrew

For more detail see https://www.pnas.org/content/pnas/117/50/32136.full.pdf.

What’s really exciting is that the number of neurons increases in the shrew’s brain come summer. Since it’s a mammal, we’re not talking about lizards regrowing limbs, but something evolutionarily close to us. For more detail see https://www.pnas.org/content/pnas/117/50/32136.full.pdf.

There are actually some conditions with reversible cerebral atrophy, and as a neurologist I made sure to look for them.

Here they are

l. Alcoholism

2. Adrenal corticosteroids (exogenous or endogenous)

3. Malnutrition in kids

4. Depakene (valproic acid)

5. Anorexia nervosa.

 

A non-coercive way to get people to accept vaccination for the pandemic virus

Many people are afraid of being vaccinated (for anything, not just the pandemic flu). Yelling at them won’t help. Calling them stupid won’t help. You can’t pass a law to coerce them, but here’s a law that would likely convince them that it is a good idea.

Can you think of it?

I’m not sure if congress could do it, or whether it would have to be done state by state.

Just require all death certificates for people dying with COVID-19 to state whether they’d been vaccinated or not. Certainly now all of the deaths will be in unvaccinated people, but as time passes (say 3 – 6 months) and 95% of them remain in the unvaccinated (as studies of the vaccine have shown) and 1/3 to half of the population is vaccinated, people will take notice.

I don’t know any legislators, but maybe you do, and you should suggest it to them.

Good riddance 2020

It’s no good being right if nobody listens. It was clear to me in late January that we would be in a pandemic as my post of 27 January 2020 below will show. The press in those early months did not cover itself in glory. Here’s a link to a bunch of press headlines — forget that the site is a bit dicey — I remember reading many of these at the time — https://twitter.com/dbongino/status/1245341299320016897. Why the CDC didn’t figure this out is anyone’s guess. The problem wasn’t lack of staff, but lack of brains. We’re all playing the price.

What to do about the Wuhan flu

This was published 27 Jan ’20.  Nothing has been altered (other than this).

What to do about the Wuhan flu?  The short answer is to lay in a month or two of dried food and drink, and have plenty of bottled water around.

The long answer depends on whether the new corona virus (called 2019-nCOV) becomes a pandemic and if the (symptomatic) case fatality rate continues at 3.5% (based on 80 deaths in 2,800 cases as of yesterday).

With a son, Chinese daughter in law and two grandchildren living in Hong Kong, I’ve followed the outbreak ever since hearing of it 1 January.

The best and most current source of info about the outbreak is the South China Morning Post — https://www.scmp.com.  It is in English and is not a government mouth piece.

Here’s the bad news

(1) As of a few days ago the virus had been found in 29/31 Chinese provinces.  This means that confining the virus to China is nearly impossible — how do you cut off a billion or so people from the rest of the world?

(2) Here’s more from today

• Hong Kong University  faculty of medicine dean Gabriel Leung says research shows self-sustaining human-to-human transmission is already happening in all major mainland cities.   Here’s a link
• https://www.scmp.com/news/hong-kong/health-environment/article/3047813/china-coronavirus-hong-kong-medical-experts-call
•  Why is this significant?  You have to know how docs operate.  When I wanted information about some issue or disease, I’d call a doc whose opinion and background I respected.  It is likely that Leung made this statement after calling med school deans he personally knew in major mainland cities.

(3) There is no treatment, in the sense of stopping the virus in its tracks.  All we have is supportive care, oxygen rest, medication for fever, bronchodilators.  This is true for the vast majority of viruses.  Remember the joke that modern medical science can cure a cold in 14 days, but otherwise it takes two weeks.

(4) We know that you don’t have to be clinically ill to transmit the disease.  Screening new arrivals for fever is well and good but that won’t totally prevent spread.

(5) Some individuals are what is called ‘superspreaders’ — one individual infected 15 hospital personnel.

(6) I wouldn’t hope for a specific treatment any time soon — look how long it took to get any treatment for AIDS, despite the huge amount of resources devoted to it.

Here is some good news. It is quite possible that there are many more cases out there with people who were either asymptomatic or  just mildly ill.  The classic example is polio, in which for every case with paralysis there were 99 cases with mild GI illness or nothing at all.

This will need to wait until we can test people for antibodies to 2019-nCOV to find out how many people have had it.  This is probably at least a month away

Vaccines (if they can be made) are even more months away.  We’ll just have to hunker down and hope for the best.

Why lay in dried food ?– in a pandemic people will panic and clear out all food they can get their hands on.  There were pictures of empty bins in a Wuhan food market last week.

People are getting serious about it.  From Reuters -“U.S. President Donald Trump offered China whatever help it needed on Monday”.  It would be nice to have some of our people from the Center for Disease Control over there. Hopefully the Chinese won’t be too proud to accept the offer.

Addendum 28 Jan — apparently the US (in the form of the CDC) is begging China to let them help out — sad — why should they have to beg?  Apparently the first overture was 3 weeks ago ! ! ! ! — https://www.scmp.com/news/china/article/3047967/china-coronavirus-washington-asks-beijing-permission-send-health-team

The past year

The past year was exactly what practicing clinical neurology from ’67 -’00 was like. Fascinating intellectual material along with impotence in the face of horrible suffering

Force in physics is very different from the way we think of it

I’m very lucky (and honored) that a friend asked me to read and comment on the galleys of a his book. He’s trying to explain some very advanced physics to laypeople (e.g. me). So he starts with force fields, gravitational, magnetic etc. etc. The physicist’s idea of force is so far from the way we usually think of it. Exert enough force long enough and you get tired, but the gravitational force never does, despite moving planets stars and whole galaxies around.

Then there’s the idea that the force is there all the time whether or not it’s doing something a la Star Wars. Even worse is the fact that force can push things around despite going through empty space where there’s nothing to push on, action at a distance if you will.

You’ve in good company if the idea bothers you. It bothered Isaac Newton who basically invented action at a distance. Here he is in a letter to a friend.

“That gravity should be innate inherent & {essential} to matter so that one body may act upon another at a distance through a vacuum without the mediation of any thing else by & through which their action or force {may} be conveyed from one to another is to me so great an absurdity that I beleive no man who has in philosophical matters any competent faculty of thinking can ever fall into it. “

So physicists invented the ether which was physical, and allowed objects to push each other around by pushing on the ether between them. 

But action at a distance without one atom pushing on the next etc. etc. is exactly what an incredible paper found [ Proc. Natl. Acad. Sci. vol. 117 pp. 25445 – 25454 ’20 ].

Allostery is an abstract concept in protein chemistry, far removed from everyday life. Far removed except if you like to breathe, or have ever used a benzodiazepine (Valium, Librium, Halcion, Ativan, Klonopin, Xanax) for anything. Breathing? Really? Yes — Hemoglobin, the red in red blood cells is really 4 separate proteins bound to each other. Each of the four can bind one oxygen molecule. Binding of oxygen to one of the 4 proteins produces a subtle change in the structure of the other 3, making it easier for another oxygen to bind. This produces another subtle change in structure of the other making it easier for a third oxygen to bind. Etc. 

This is what allostery is, binding of molecule to one part of a protein causing changes in structure all over the protein. 

Neurologists are familiar with the benzodiazepines, using them to stop continuous seizure activity (status epilepticus), treat anxiety (Xanax), or seizures (Klonopin). They all work the same way, binding to a complex of 5 proteins called the GABA receptor, which when it binds Gamma Amino Butyric Acid (GABA) in one place causes negative ions to flow into the neuron, inhibiting it from firing. The benzodiazepines bind to a completely different site, making the receptor more likely to open when it binds GABA. 

The assumption about all allostery is that something binds in one place, pushing the atoms around, which push on other atoms which push on other atoms, until the desired effect is produced. This is the opposite of action at a distance, where an effect is produced without the necessity of physical contact.

The paper studied TetR, a protein containing 203 amino acids. If you’ve ever thought about it, almost all the antibiotics we have come from bacteria, which they use on other bacteria. Since we still have bacteria around, the survivors must have developed a way to resist antibiotics, and they’ve been doing this long before we appeared on the scene. 

TetR helps bacteria resist tetracycline, an antibiotic produced by bacteria. When tetracycline binds to TetR it causes other parts of the protein to change so it binds DNA causing the bacterium, among other things, to make a pump which moves tetracyline out of the cell. Notice that site where tetracycline binds on TetR is not the business end where TetR binds DNA, just as where the benzodiazepines bind the GABA receptor is not where the ion channel is. 

This post is long enough already without describing the cleverness which allowed the authors to do the following. They were able to make TetRs containing every possible mutation of all 203 positions. How many is that — 203 x 19 = 3838 different proteins. Why 19? Because we have 20 amino acids, so there are 19 possible distinct changes at each of the 203 positions in TetR.  

Some of the mutants didn’t bind to DNA, implying they were non-functional. The 3 dimensional structure of TetR is known, and they chose 5 of nonfunctional mutants. Interestingly these were distributed all over the protein. 

Then, for each of the 5 mutants they made another 3838 mutants, to see if a mutation in another position would make the mutant functional again. You can see what a tremendous amount of work this was. 

Here is where it gets really interesting. The restoring mutant (revertants if you want to get fancy) were all over the protein and up to 40 – 50 Angstroms away from the site of the dead mutation. Recall that 1 Angstrom is the size of a hydrogen atom, a turn of the alpha helix is 5.4 Angstroms and contains 3.5 amino acids per turn.The revertant mutants weren’t close to the part of the protein binding tetracycline or the part binding to DNA. 

Even worse the authors couldn’t find a contiguous path of atom pushing atom pushing atom, to explain why TetR was able to bind DNA again. So there you have it — allosteric action at a distance.

There is much more in the paper, but after all the work they did it’s time to let the authors speak for themselves. “Several important insights emerged from these results. First, TetR exhibits a high degree of allosteric plasticity evidenced by the ease of disrupting and restoring function through several mutational paths. This suggests the functional landscape of al- lostery is dense with fitness peaks, unlike binding or catalysis where fitness peaks are sparse. Second, allosterically coupled residues may not lie along the shortest path linking allosteric and active sites but can occur over long distances “

But there is still more to think about, particularly for drug development. Normally, in developing a drug for X, we have a particular site on a particular protein as a target, say the site on a neurotransmitter receptor where a neurotransmitter binds. But the work shows that sites far removed from the actual target might have the same effect