Category Archives: Molecular Biology

Forgotten but not gone — take II

The RNA world from whence we sprang strikes again, this time giving us a glimpse into its own internal dynamic.  18 months ago I wrote the following post — which will give you the background to follow the latest (found at the end after the (***)

Life is said to have originated in the RNA world.  We all know about the big 3 important RNAs for the cell, mRNA, ribosomal RNA and transfer RNA.  But just like the water, sewer, power and subway systems under Manhattan, there is another world down there in the cell which doesn’t much get talked about.  These areRNAs, whose primary (and possibly only) function is to interact with other RNAs.

Start with microRNAs (of which we have at least 1,500 as of 12/12).  Their function is to bind to messenger RNA (mRNA) and inhibit translation of the mRNA into protein.  The effects aren’t huge, but they are a more subtle control of protein expression, than the degree of transcription of the gene.

Then there are ceRNAs (competitive endogenous RNAs) which have a large number of binding sites for microRNAs — humans have a variety of them all with horrible acronyms — HULC, PTCSC3 etc. etc. They act as sponges for microRNAs keeping them bound and quiet.

Then there are circular RNAs.  They’d been missed until recently, because typical RNA sequencing methods isolate only RNAs with characteristic tails, and a circular RNA doesn’t have any.  One such is called CiRS7/CDR1) which contain 70 binding sites for one particular microRNA (miR-7).  They are unlike to be trivial.  They are derived from 15% of actively transcribed genes.  They ‘can be’ 10 times as numerous as linear RNAs (like mRNA and everything else) — probably because they are hard to degrade < Science vol. 340 pp. 440 – 441 ’17 >. So some of them are certainly RNA sponges — but all of them?

The latest, and most interesting class are the nonCoding RNAs found in viruses. Some of them function to attack cellular microRNAs and help the virus survive. Herpesvirus saimiri a gamma-herpes virus establishes latency in the T lymphocytes of New World primates, by expressing 7 small nuclear uracil-rich nonCoding RNAs (called HSURs).  They associate with some microRNAs, and rather than blocking their function act as chaperones < Nature vol. 550 pp. 275 – 279 ’17 >.  They HSURs also bind to some mRNAs inhibiting their function — they do this by helping miR-16 bind to their targets — so they are chaperones.  So viral Sm-class RNAs may function as microRNA adaptors.

Do you think for one minute, that the cell isn’t doing something like this.

I have a tendency to think of RNAs as always binding to other RNAs by classic Watson Crick base pairing — this is wrong as a look at any transfer RNA structure will show.  Far more complicated structures may be involved, but we’ve barely started to look.

Then there are the pseudogenes, which may also have a function, which is to be transcribed and sop up microRNAs and other things — I’ve already written about this —  Breast cancer cells think one (PTEN1) is important enough to stop it from being transcribed, even though it can’t be translated into protein.


[ Proc. Natl. Acad. Sci. vol. 116 pp. 7455 – 7464 ’19 ] The work reports a fascinating example of that early world in which the function of one denizen (a circular RNA called cPWWP2A) binds to another denizen of that world (microRNA 579 aka miR-579) acting as a sponge sopping up so it can’t bind to the mRNAs for angiopoetitin1, occludin and SIRT1.

So what you say?  Well it may lead to a way to treat diabetic retinopathy. How did they find cPWWP2A?  They used the Shanghai BIotechnology Company Mouse Circular RNA microArray which measures circular RNAs.  They found that 400 or so that were upregulated in diabetic retinopathy and another 400 or so that were downregulated.  cPWWP2A was on of the 3 top upregulated circular RNAs in diabetic retinopathy.  cPWWP2A comes from (what else?) PWWP2A, a gene coding for a protein which specifically binds the histone protein H2A.Z.

Overexpression of cPWW2PA or inhibition of miR-579 improves retinal vascular dysfunction in experimental diabetes.

So here is all this stuff going on way down there in the RNA world, first interacting with other players in this world and eventually reaching up to the level we thought we knew about and controlling gene expression.  It’s sort of like DOS (Disc Operating System) still being important in Windows.

How much more stuff like this is to be discovered controlling gene expression in us is anyone’s guess


How to treat Alzheimer’s disease

Let’s say you’re an engineer whose wife has early Alzheimer’s disease.  Would you build the following noninvasive device to remove her plaques?  [ Cell vol. 177 pp. 256 – 271 ’19 ] showed that it worked in mice.

Addendum 18 April — A reader requested a better way to get to the paper — Here is the title — “Multisensory Gamma Stimulation Ameliorates Alzheimer’s Associated Pathology and Improves Cognition”.  It is from MIT — here is the person to correspond to  —Correspondence —

The device emits sound and light 40 times a second.  Exposing mice  to this 1 hour a day for a week decreased the number of senile plaques all over the brain (not just in the auditory and visual cortex) and improved their cognition as well.

With apologies to Steinbeck, mice are not men (particularly these mice which carry 5 different mutations which cause Alzheimer’s disease in man).  Animal cognition is not human cognition.  How well do you think Einstein would have done running a maze looking for food?

I had written about the authors’ earlier work and a copy of that post will be found after the ****.

What makes this work exciting is that plaque reduction was seen not only  in the visual cortex (which is pretty much unaffected in Alzheimer’s) but in the hippocampus (which is devastated) and the frontal lobes (also severely affected).  Interestingly, to be effective, both sound and light had to be given simultaneously

Here are the details about the stimuli  —

“Animals were presented with 10 s stimulation blocks interleaved with 10 s baseline periods. Stimulation blocks rotated between auditory-only or auditory and visual stimulation at 20 Hz, 40 Hz, 80 Hz, or with random stimulation (pulses were delivered with randomized inter-pulse intervals determined from a uniform distribution with an average interval of 25 ms). Stimuli blocks were interleaved to ensure the results observed were not due to changes over time in the neuronal response. 10 s long stimulus blocks were used to reduce the influence of onset effects, and to examine neural responses to prolonged rhythmic stimulation. All auditory pulses were 1 ms-long 10 kHz tones. All visual pulses were 50% duty cycle of the stimulation frequency (25 ms, 12.5 ms, or 6.25 ms in length). For combined stimulation, auditory and visual pulses were aligned to the onset of each pulse.”

The device should not require approval by the FDA unless a therapeutic claim is made, and it’s about as noninvasive as it could be.

What could go wrong?  Well a flickering light could trigger seizures in people subject to photic epilepsy (under 1/1,000).

Certainly Claude Shannon who died of Alzheimer’s disease, would have had one built, as would Fields medal winner Daniel Quillen had he not passed away 8 years ago.

Here is the post of 12/16 which has more detail



Will flickering light treat Alzheimer’s disease ?

Big pharma has spent zillions trying to rid the brain of senile plaques, to no avail. A recent paper shows that light flickering at 40 cycles/second (40 Hertz) can do it — this is not a misprint [ Nature vol. 540 pp. 207 – 208, 230 – 235 ’16 ]. As most know the main component of the senile plaque of Alzheimer’s disease is a fragment (called the aBeta peptide) of the amyloid precursor protein (APP).

The most interesting part of the paper showed that just an hour or so of light flickering at 40 Hertz temporarily reduced the amount of Abeta peptide in visual cortex of aged mice. Nothing invasive about that.

Should we try this in people? How harmful could it be? Unfortunately the visual cortex is relatively unaffected in Alzheimer’s disease — the disease starts deep inside the head in the medial temporal lobe, particularly the hippocampus — the link shows just how deep it is -

You might be able to do this through the squamous portion of the temporal bone which is just in front of and above the ear. It’s very thin, and ultrasound probes placed here can ‘see’ blood flowing in arteries in this region. Another way to do it might be a light source placed in the mouth.

The technical aspects of the paper are fascinating and will be described later.

First, what could go wrong?

The work shows that the flickering light activates the scavenger cells of the brain (microglia) and then eat the extracellular plaques. However that may not be a good thing as microglia could attack normal cells. In particular they are important in the remodeling of the dendritic tree (notably dendritic spines) that occurs during experience and learning.

Second, why wouldn’t it work? So much has been spent on trying to remove abeta, that serious doubt exists as to whether excessive extracellular Abeta causes Alzheimer’s and even if it does, would removing it be helpful.

Now for some fascinating detail on the paper (for the cognoscenti)

They used a mouse model of Alzheimer’s disease (the 5XFAD mouse). This poor creature has 3 different mutations associated with Alzheimer’s disease in the amyloid precursor protein (APP) — these are the Swedish (K670B), Florida (I716V) and London (V717I). If that wasn’t enough there are two Alzheimer associated mutations in one of the enzymes that processes the APP into Abeta (M146L, L286V) — using the single letter amino acid code – Then the whole mess is put under control of a promoter particularly active in mice (the Thy1 promoter). This results in high expression of the two mutant proteins.

So the poor mice get lots of senile plaques (particularly in the hippocampus) at an early age.

The first experiment was even more complicated, as a way was found to put channelrhodopsin into a set of hippocampal interneurons (this is optogenetics and hardly simple). Exposing the channel to light causes it to open the membrane to depolarize and the neuron to fire. Then fiberoptics were used to stimulate these neurons at 40 Hertz and the effects on the plaques were noted. Clearly a lot of work and the authors (and grad students) deserve our thanks.

Light at 8 Hertz did nothing to the plaques. I couldn’t find what other stimulation frequencies were used (assuming they were tried).

It would be wonderful if something so simple could help these people.

For other ideas about Alzheimer’s using physics rather than chemistry please see —

Apologies to Hamlet

Apologies to Shakespeare and Hamlet.  Serotonin does “more things in heaven and Earth, Horatio, than are dreamt of in your philosophy.”  How about chemically modifying histones?We all know about serotonin and depression (or at least we think we know).  Block serotonin reuptake by the releasing neuron and bingo you’ve  cured depression (sometimes).  Do not ask the lecturer which of the 15 known serotonin receptors in the brain the increased serotonin actually binds to and what effects the increased levels produce after binding (and which are important for the alleviation of depression).The two body organs producing the most serotonin are the brain and the gut.  Chemical modification of proteins by serotonin has been known for 10 years.  The enzyme responsible is transglutaminase2, it takes the NH2 group of serotonin and replaces the NH2 of glutamine with it — forming an isopeptide bond.

Interestingly, the serotonylation of histones is quite specific.  Only glutamine #5 on histone H3 is modified this way.  For the reaction to occur lysine #4 on histone H3 must be trimethylated (H3K4Me3) — now you can begin to see the combinatorial possibilities of the various histone modifications known.  Over 130 post-ranslational modifications of histones were known by 2013 [ Cell vol. 155 p. 42 ’13 ].

The H3K4Me3Q5Ser is enriched in euchromatin and correlates with permissive gene expression.  Changing glutamine #5 to something else so it can’t be serotonylated changes the transcription pattern, and deficits in cellular differentiation.  You can read more about it in Nature vol. 567 pp. 464 – 465, 535 – 539 ’19 ]

Yet another mechanism of gene regulation

A snippet of RNA from an intron in a gene can bind to an upstream regulatory element forming a triple helix and shut off transcription of the gene.  Rather amazing don’t you think?  Yet exactly was found in a far from obscure gene, the beta globin gene of hemoglobin on chromosome #11 [ Proc. Natl. Acad. Sci. vol. 116 pp. 6130 – 6139 ’19 ].

We’re talking large segments of DNA.  There are five genes for the beta subunit of hemoglobin located from 5′ to 3′ as epsilon, gammaG, gammaA, delta and beta.  The first 4 are expressed during fetal development.  Beta globin is the one found in our red blood cells.  The regulatory element controlling all 5 is found FIFTY kiloBases upstream from the beginning (5′ end) of beta globin.

The regulatory region is called the locus control region (LCR)and stretches over 20+ kiloBases.  It has 7 sites where transcription factors bind (called hypersensitive sites HS1 — HS7).  The hypersensitivity comes from the fact the chromosome is relative ‘open’ at these places and not compacted, so that an enzyme (DNAase I) can break the chromosome.

So after the beta globin gene is transcribed, the introns are spliced out, and the RNA from the second intron binds to HS2 forming a triple helix and displacing transcription factors bound there (USF2, GATA1, TAL1) which recruit RNA polymerase II (Pol II)  In the normal course of events the whole mess would then march around the genome and eventually hit the promoter of beta globin (at least 50 kiloBases away) and turn on transcription.

This seems to be yet another mechanism of gene regulation.  Just how widespread this is, isn’t known, but most protein coding genes have introns.  Stay tuned.

Molecular biology is fascinating

Another research idea yours for the taking

How many of our 20,000 or so protein coding genes are essential for human existence?  There is a way to find out with no human experimentation whatsoever.  Even better, probably all the data is out there.  Looking at it the right way, finding and collating it is where you come in.  Be warned, it would be a lot of work.

Previous work [ Science vol. 350 pp. 1028 – 1029, 1092 – 1096, 1096 – 1101 ’15 ] came up with the idea that only 2,000 or so of our protein coding genes were truly essential.  The authors cleverly looked at a ‘near haploid’ chronic myelogenous leukemia cell line (KBM7).  Then because only one copy of a gene was present, they systematically knocked out gene after gene using CRISPR and looked at viability.

Similar work in yeast stated that only 1,000 of its 6,000 protein coding genes were essential.

But this is single cell stuff.  What about living breathing people?

Where is this data?  How should it be interrogated?  See if you can figure it out before reading further.

Probably more has been done since Science vol. 337 pp. 64 – 69 ’12 sequenced just the portion of our genome coding for proteins (the exomes) in 1,351 Europeans and 1,088 Africans.  Each individual had 35 premature termination codons, meaning that the gene likely didn’t produce a functional protein.  The average person also had 13,595 single nucleotide polymorphisms (from the standard genome), and probably some of them a less than functional protein.

Do you see how you could use this sort of thing to find out which genes are essential to our existence?

People sequence exomes because it’s easy and because the exome accounts for only 2% of our genome.

My guess is that probably a million exomes have been sequenced thus far, if not more.

So all you have to do is look at all million exome sequences and all 20,000 protein coding genes, and see —

In one of the Sherlock Holmes stories the following dialog appears

Gregory (Scotland Yard): “Is there any other point to which you would wish to draw my attention?”
Holmes: “To the curious incident of the dog in the night-time.”
Gregory: “The dog did nothing in the night-time.”
Holmes: “That was the curious incident.”

The curious incident would be a gene which never (or rarely) had a premature termination codon in the 1,000,000 or so exomes.  That would imply that it was essential for the existence of a living breathing human being.

Cute !  Well I’m a retired neurologist with no academic affiliation — take the idea and run with it.

Addendum 31 Mar ’19 – I received the following comment from Bryan

You may be interested in reading this pre-print on the topic:
Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes

To which I replied
    • Bryan– thanks for the link. It was a good enough idea that the people at the Broad Institute had thought of it and carried it out. As people in grad school used to say when they got scooped on a paper — at least we were thinking well.

      It was hard to tell from reading the preprint whether there were genes with no pLoF (predicted loss of function) proving them essential. They do say that the 678 genes essential for human cell viability (characterized by CRISPR screening were ‘depleted’ for pLoF.


Does gamma-secretase have sex with its substrates?

This is a family blog (for the most part), so discretion is advised in reading further.   Billions have been spent trying to inhibit gamma-secretase.  Over 150 different mutations have been associated with familial Alzheimer’s disease.  The more we know about the way it works, the better.

A recent very impressive paper from China did just that [ Science vol. 363 pp. 690- 691, 701 eaaw0930 pp. 1 –> 8 ’19 ].

Gamma secretase is actually a combination of 4 proteins (presenilin1, nicastrin, APH1 (anterior pharynx defect) and PEN-2 (presenilin enhancer 2). It is embedded in membranes and has at least 19 transmembrane segments.  It cleaves a variety of proteins spanning membranes (e.g it hydrolyzes a peptide bond — which is just an amide).  The big deal is that cleavage occurs in the hydrophobic interior of the membrane rather than in the cytoplasm where there is plenty of water around.

Gamma secretase cleaves at least 20 different proteins this way, not just the amyloid precursor protein, one of whose cleavage products is the Abeta peptide making up a large component of the senile plaque of Alzheimer’s disease.

To get near gamma secretase, another enzyme must first cleave APP in another place so one extramembrane fragment is short.  Why so the rest of the protein can fit under a loop between two transmembrane helices of nicastrin.  This is elegance itself, so the gamma secretase doesn’t go around chopping up the myriad of extracellular proteins we have.

The 19 or so transmembrane helices of the 4 gamma secretase proteins form a horseshoe, into which migrates the transmembrane segment of the protein to be cleaved (once it can fit under the nicastrin loop).

So why is discretion advised before reading further?  Because the actual mechanism of cleavage involves intimate coupling of the proteins.    One of the transmembrane helices of presenilin1 unfolds to form two beta strands, and the transmembrane helix of the target protein unfolds to form one beta strand, the two strands pair up forming a beta sheet, and then the aspartic acid at the active site of gamma secretase cleaves the target (deflowers it if you will).  Is this sexual or what?

All in all another tribute to ingenuity (and possibly the prurience) of the blind watchmaker. What an elegant mechanism.

Have a look at the pictures in the Science article, but I think it is under a paywall.

Proline rides again !

Proline is a kinky amino acid.  Kinky in the sense that it is only one of the twenty with a fixed configuration of its alpha carbon because of the ring (which may be why there is more of it in organisms living at high temperature) and kinky in the sense that when present in alpha helices it produces a kink.  The previous post shows how it is used to schlep the body weight’s worth of ATP we make each day out of our mitochondria —

Well here it is in one of the marijuana receptors (CB1).  Binding of delta9 THC in the 7 transmembrane alpha helix bundles of the G Protein Coupled Receptor (GPCR) causes an alteration in the kink allowing transmembrane helix 6 (TM6) to move outward toward the cytoplasm, creating a cavity on the intracellular side, where the G protein trimer can bind.

You can read much more about this in an exquisite paper [ Cell vol. 176 pp. 448 – 458 `19 ] describing the CB1 receptor bound to a synthetic ligand 20 times more potent that delta-9 tetrahydrocannabinol (delta9 THC).  It is a cryoEM study which used 177,000 projections to come up with a 3 Angstrom resolution structure of CB1 bound to MBDB-FUBINACA in complex with its G protein trimer.  They had to use a single chain variable fragment (scFv6) along with a positive allosteric modulator (PAM) called ZCZ-011 to stabilize the complex.

MBDB-FUBINACA is a story in itself.  It is presently the fentanyl of synthetic cannabinoids, which “has been linked to thousands of hospitalizations and numerous fatalities”  [ New England Journal of Medicine vol. 376 pp. 235 – 242 ’17 ].  I’m surprised I’ve never heard of it — have you? But then I’ve been retired from clinical practice for some time. Perhaps the mainstream press, pushing marihuana legalization as it has been, kept it quiet, or more likely there have been no further episodes of mass intoxication from the AMB-FUBINACA (aka the zombie drug) since 2017.

I’ve never knowingly used marihuana.  Frankly it scares me — for why please see —

There are 4 molecular switches buried in GPCRs [ Current Med. Chem. vol. 19 pp. 1090 – 1109 ’12 ]

1. The ionic lock switch between the D/E R Y sequence at the cytoplasmic end of TM3 and E286 at the cytoplasmic end of TM6 (single letter amino acid code used) –

2. TM3 – TM7 lock switch.  In rhodopsin it is between the protonated Schiff base of lysine and a glutamic acid and it broken on light activation,.=

3. Toggle switch linked with the n P x x Y motif in TM7 (x stands for any amino acid) — much more about this later in the post.

4. Transmission switch — produced by agonist binding, the outward movement of TM6 to to ligand binding creating a hole fo the G protein to bind to the receptor on the cytoplasmic side.

So why did I call the Cell paper exquisite?  Because of the molecular detail it provides about just how MDMB FUBINACA activates CB1.  Here’s the structure of AB-FUBINACA —   Both look like drugs designed by a committee.  They both have a para-iodophenyl group, an amide, and a fused indole ring with an extra nitrogen (imidazole ring — I never could keep heterocyclic nomenclature straight).    MDMB has a methyl ester (in place of the amide) and a tertiary butyl group (in place of the isoPropyl group).

I don’t have time to look up how Pfizer came up with it.  The FUBINACAs do not resemble delta9 THC at all —

The pictures in the paper show how the hydrophobic aromatic side chains of FIVE phenylalanines and 2 tryptophans create a nice oily space for delta9 THC and MBDB-FUBINACA to bind.

F200 (phenylAlanine 200) and W356 are the toggle twin switch which stabilize the inactive conformation of CB1.  The rotation of F200 to interact with the imidazole of FUBINACA, allows W356 to rotate outward, changing the kink produced the the proline #358  in TM6 allowing the helix to straighten and rotate outward toward the cytoplasm, creating a cavity for the G protein to bind to.

Definitely a tour de force for the blind watchman.

Let’s hear it for the blind watchmaker

The blind watchmaker had a lot of foresight in choosing to use a rather  funky looking amino acid (proline) resembling none of the others.  A lot of kindness was also shown to structural molecular biologists by two of the watchmaker’s henchmen – Burkholderia gladioli and the common daisy.

All appear in a fascinating paper [ Cell vol. 176 pp. 435 – 447  ’19 ] in which the structure and better the mechanism of action of the mitochondrial ADP/ATP translocase, a molecule of some interest since our mitochondria make our body weight of ATP each day and need some way to get it out into the cytoplasm where it is used.

The molecule has quite a job to do, getting the rather large ATP molecule out to the intermembrane space (and thence out to the cytoplasm) without allowing protons to sneak out with it, since it is the proton gradient which is used to power ATP synthase the exquisite machine which makes ATP.   This is quite a trick as no chemical moiety is as small as a proton.

The translocase has two states — one in which it is open to the mitochondrial matrix (called the m-state) and another in which it is open (eventually) to the cytoplasm — called the c-state. In the m-state the cytoplasmic portion is shut, and in the c-state the membrane portion is shut.

The rather wierd looking molecule bongkrekic acid  made by Burkholderia gladioli binds to the translocase fixing it in the m-state.  Atractyloside, made by daisies binds to the molecule fixing it in the c-state.  They made life much easier for the structural biologist and cryoEMographers who wrote the paper.

Proline comes in because when placed in an alpha helix, proline’s 5 membered ring structure fixes the alpha carbon so that it is essentially inflexible, meaning that it can’t get into the conformation that the other 19 amino acids can get into when an alpha helix is formed.  Translation — proline is a helix breaker, forming a kink in the helix.

The translocase contains 3 modules of 100 amino acids each of which has 2 alpha helices, one of them containing a proline causing a kink in the helix.  The prolines are in the middle of the helix.  The ATP channel is formed by the 6 helices.

Essentially in the middle of the membrane, the kinked alpha helices form a pivot (fulcrum), so the helices rock back and forth, opening one side while simultaneously shutting the other, permitting ATP to bind near the fulcrum without letting anything else through, when the pivot shifts   — out goes the ATP (without letting protons sneak past).

There is far more beautiful protein chemistry on display.  There is a conserved signature motif Proline x Aspartic acid/Glutamic acid X X Lysine/Arginine at the carboxy terminal end of one of the helices of each other 3 modules — this forms a salt bridge shutting the channel on the matrix side.  Glycine and other small amino acids (alanine) allow close packing of the helices on the cytoplasmic side.

It is unfortunate that the most of humanity doesn’t have the background to appreciate the elegance and beauty of Nature’s solution to the problem.  I say Nature rather than God to be scientifically correct, but it’s elegant chemistry like this that makes it hard for me to accept that it arose by the machinations of a blind watchmaker.

Science fiction for the cognoscenti – III — not all the background you need will be explained

Now that every team in the NFL has its own molecular biologist and antiVirologist, you might be interested in knowing how it all started.  Like most technologies affecting our lives it had a military origin.

The escape of the Taiwanese pacifist virus started it all –

The technology of infectious gene transfer by recombinant adeno-associated virus  (AAV) was well advanced long before there were garage molecular biologists.

The NFL wars began with the New England Patriots, (who else?), they of deflategate and other nefarious ways to win.

Tom Brady was getting all set to win superbowl LVII in 2023 at age 45 when the first counterattack was successful.

His wife, the beautiful Gisele, hated the idea of him playing so long, being very worried about dementia pugilista from all the head trauma.  Tom had agreed to yearly PET scans with Pittsburgh compound B, an uncharged derivative of thioflavin T which gets through the blood brain barrier and which stains senile plaques.  They showed no evidence of plaques (although plenty of demented people don’t have them) so he kept on playing behind Belichick’s not so secret weapon — 400 pound linemen.  Even though he’d lost a step or two, his eye and arm were still good and the linemen gave him plenty of time to throw.

Football players have always been bulking up.  Even the early experience with extra testosterone (which causes testicular atrophy in high doses) didn’t dissuade them.  Newer anabolic steroids had somewhat fewer testicular effects.  Eventually players took to using HCG to help normalize things, but some testicular atrophy was a price they were willing to pay.  The cheerleaders felt a lot safer around those using them.

So how did the Patriots have 400 pound linemen when no one else did?  The answer goes back to Piedmontese and Belgian Blue cattle which were bred for their large muscles.  They turned out to have inactivating mutations in the gene for myostatin, a protein which causes muscles to stop growing.

Boston isn’t known as the home of biotech for nothing, and Belichick contracted with an as yet un-named biotech firm (their depositions having been sealed by the court) to come up with a small molecule (compound M) absorbable through the skin which inhibited myostatin.

No one caught on why Belicheck had separate showers installed for the lineman and defensive backs, but they had to use them and got  dosed that way.  Testing for performance enhancing drugs was always negative. The linemen loved it, as their testicles grew back to normal size.  The cheerleaders didn’t.

So there the Patriots were, about to play the Arizona Cardinals, a team only winning 3 games in 2018 in superbowl LVII. No one understood how the Cardinals turned around and how they got those very slippery running backs.

No one, except the molecular biologist they hired.  But that’s for next time.

Another way to study Alzheimer’s

Until I read the paper PLOS Genet. 14, e1007791 (2018)., I thought that this was a sure way to win Nobel prize.  It’s still pretty interesting.  The abstract in Science was misleading, implying that there was an APOE4 variant which was actually protective against Alzheimer’s disease. That would have been fantastic, as it would provide a clue as to just what the APOE4 allele was doing to increase the risk of Alzheimer’s disease.

A huge amount of work has been done on APOE4.   Googling produced 433,000 results (0.46 seconds).  Theories abound but we still don’t know.

The authors studied Blacks and Puerto Ricans and found that if you inherited the APOE4 allele from an African source (rather than a European source), your chance of developing Alzheimer’s disease was significantly less.  A total of 1,766 African American and 220 Puerto Rican individuals with late-onset Alzheimer disease, and 3,730 African American and 169 Puerto Rican cognitively healthy individuals (> 65 years) participated in the study.

The numbers: ApoE ε4 alleles on an African background conferred a lower risk than those with a European ancestral background, regardless of population (Puerto Rican: OR = 1.26 on African background, OR = 4.49 on European; African American: OR = 2.34 on African background, OR = 3.05 on European background).

Note that the ORs are still up for Alzheimer’s if you have APOE4, but the differences are significant and certainly real given the size of the study.

The authors think it’s the area around the APOE  gene, rather than the total genetic background (African vs. European etc. etc.)

It still might be worth doing the following.  Take skin fibroblasts from all four types of people (Puerto Ricans with APOE4 on African background, Puerto Ricans with APOE4 on European background, Blacks with APOE4 on African background, APOE4 on a European background).

Make induced pluripotent stem cells (iPSCs) from them (the technology to do so is quite advanced). Differentiate these iPSCs into neurons  and others into glia (technology quite available).  Study protein and mRNA expression, epigenetic modifications in neurons and glia from all 4 groups.  This might tell you just what APOE4 was doing in high and lower risk people, and possibly might give a clue as to how it was increasing Alzheimer’s risk.

My hopes were really up, because the abstract in Science implied that APOE4 in Blacks and Puerto Ricans was actually absolutely rather than relatively protective, which would have given us some serious clues to Alzheimer pathogenesis, when APOE4 protective cells were contrasted with APOE4 increased risk cells.

Oh well.