Do not go gentle into that good night

Cells in the body dying of necroptosis obey Dylan Thomas — “Do not go gentle into that good night” all sorts of inflammation ensues around the cell, and systemically if enough die that way at once.

Cells dying from the first discovered form of programmed cell death e.g. apoptosis disobey.  They die very quietly producing no inflammation, and are quietly munched up by phagocytes.  Just how this happens has been a huge mystery.

Well one way to figure out what is going on looks at a phagocyte before it meets an apoptotic cell and afterwards.  Quite a bit it turns out.  The brute force technique looks at the changes in our 20,000 or so protein coding genes.  They found increased expression in 886 and decreased expression in 966, some 9% of our total.  How do you make sense of that.

This is typical of the brute force approach to any condition (e.g. cancer, infection, vascular disease), and shows you just how hard it is to figure out what is going on from the mass of data produced.

The authors of Nature vol. 580 pp. 130 – 135 ’20 (https://www.nature.com/articles/s41586-020-2121-3.pdf) were far cleverer than that.  What they did was cause a bunch of cells to go apoptotic at once and then the “supernatants and cell pellets from apoptotic cells and live cell controls were subjected to untargeted metabolomic profiling against a library of more than 3,000 biochemical features or compounds.”

Then by a huge amount of work they found 6 metabolites released by the apoptotic cell  which when given together which could switch macrophages (a type of phagocyte)to the non-inflammatory state (e.g. the one above producing all those gene changes).

Then they pared the number of metabolites doing this down to 3 (spermidine, guanosine monophosphate and inosine monophosphate). They call this cocktail of metabolites MEMIX-3.

They get out of the cell dying of apoptosis because the executioner (caspase) chops up a protein channel on the cell surface (pannexin1), allowing the 6 metabolites to escape.  A rather parsimonious suicide note wouldn’t you think.

It gets better. MEMIX-3 obviously is an anti-inflammatory agent, and they showed that it attenuates arthritic symptoms and prevents rejection of a lung transplant.

Brilliant work, and possibly one of great therapeutic import.

A way to end the pandemic

Could infecting people with the four or so coronaviruses that cause the common cold protect them against the new coronavirus causing the pandemic?   The official name for the new virus is SARS-CoV-2, the name for the disease is COVID-19.

“According to Marie-Louise Landry, MD, an infectious disease expert at Yale Medical School and the Director of the Yale Clinical Virology Laboratory, four common human coronaviruses cause 15-30% of common colds”

https://www.health.com/condition/infectious-diseases/coronavirus/coronavirus-symptoms-vs-cold

Now ask yourself how she could make a statement like this.  I’m going to try to get in touch with her tomorrow, but it is very likely that these cold causing coronaviruses are detected by measuring antibodies to them, carried in the blood of people who have been infected by them in the past.

Could one coronavirus (even a benign one) give partial immunity to others?  It’s possible and it’s time to find out.  We could know  in a few weeks.

Assume the test to measure the antibodies to cold coronaviruses exists.  Then measure them in our real, honest to God, modern day heroes on the front lines  — the nurses, docs, EMTs, orderlies, housekeeping, cops, etc. etc.  who are exposed every day to COVID-19.

Every hospital in the country could at least draw blood on them, look to see if antibodies are present and wait.   I doubt that many would refuse the test.

Sadly, it wouldn’t be long before some of them became infected with SARS-CoV-2.  Then investigators couldlook to see if those with the antibodies to the cold causing coronaviruses were protected.

If so, then make a cocktail of the 4 or so coronaviruses and give it to everyone.   It would be Edward Jenner and the cowpox all over again — https://en.wikipedia.org/wiki/Edward_Jenner

Even if the protection was only partial, decreasing the number of susceptible individuals would be enough to slow the pandemic and possibly even stop it.

Do glia think?

Do glia think Dr. Gonatas?  This was part of an exchange between G. Milton Shy, head of neurology at Penn, and Nick Gonatas a brilliant neuropathologist who worked with Shy as the two of them described new disease after new disease in the 60s ( myotubular (centronuclear) myopathy, nemaline myopathy, mitochondrial myopathy and oculopharyngeal muscular dystrophy).

Gonatas was claiming that a small glial tumor caused a marked behavioral disturbance, and Shy was demurring.  Just after I graduated, the Texas Tower shooting brought the question back up in force — https://en.wikipedia.org/wiki/University_of_Texas_tower_shooting.

A recent paper [ Neuron vol. 105 pp. 954 – 956, 1036 – 1047 ’20] gives good evidence that glia are more than the janitors and the maintenance crew of the brain.

Glia cover most synapses (so neurotransmitter there doesn’t leak out, I thought) giving rise to the term tripartite synapse (presynaptic terminal + postsynaptic membrane + glial covering).

Here’s what they studied.  The cerebral cortex projects some of its axons (which use glutamic acid as a neurotransmitter) to a much studied nucleus in animals (the nucleus accumbens).  This is synapse #1. The same nucleus gets a projection of axons from the brainstem ventral tegmental area (VTA) which uses dopamine as a neurotransmitter.  However, the astrocytes (a type of glia) covering synapse #1 have the D1 dopamine receptor (there are 5 different dopamine receptors) on them.  It isn’t clear if the dopamine neurons actually synapse (synapse #2) on the astrocytes, or whether the dopamine  just leaks out of the synaptic cleft to the covering glia.

Optogenetic stimulation of the VTA dopamine neurons results in an elevation of calcium in the astrocytes (a sign of stimulation). Chemogenetic activation of these astrocytes depresses the presynaptic  terminals of the neurons projecting the nucleus accumbens  from the cerebral cortex .  How does this work?  Stimulated astrocytes release ATP or its produce adenosine.  This binds to the A1 purinergic receptor on the presynaptic terminal of the cortical projection.

So what?

The following sure sounds like the astrocyte here is critical to brain function.  Activation of the astrocyte D1 receptor contributes to the locomotor hyperactivity seen after an injection of amphetamine.

Dopamine is intimately involved in reward, psychosis, learning and other processes (antipsychotics and drugs for hyperactivity manipulate it).  That the humble astrocyte is involved in dopamine action takes it out of the maintenance crew and puts it in to management.

A final note about Dr. Shy.  He was a brilliant and compelling teacher, and instead of the usual 1% of a medical school class going into neurology, some 5% of ours did.  In 1967 he ascended to the chair of the pinnacle of American Neurology at the time, Columbia University.  Sadly, he died the month he assumed the chair.  Scuttlebut has it that he misdiagnosed his own heart attack as ‘indigestion’ and was found dead in his chair.

Decoys and the Strategic Defense Initiative (SDI)

It will take a detour through history to understand how lung cells try to defeat MRSA (Methicillin Resistant Staph. Aureus), a very nasty bug.

Back in 1983 President Reagan proposed building an antiMissile defense system, which would shoot down Russian InterContinental Ballistic Missiles (ICBMs) aimed at us.  Almost every scientist of note said it was impossible technically, because even if you could shoot down one (which they didn’t think you could), the Russians would send multiple decoy ICBMs without warheads.  It was an enormously expensive project and one the Russians had no hope of matching.  People still argue whether their attempt to match the US caused the Russians  to collapse — https://history.howstuffworks.com/history-vs-myth/who-won-cold-war1.htm — although collapse they did being overextended in Afghanistan (as we’ve been for 20 years).

But that’s exactly what A549 cells (derived from lung epithelium) do to fake out MRSA according to Nature vol. 579 pp. 260 – 264 ’20.  One of the reasons MRSA is so nasty is that it secretes a protein (alpha toxin) which forms holes in cells it binds to.  Well alpha toxin has a target it must bind to cause trouble, otherwise it would form holes in everything including itself.  The target is an enzyme on the surface of the cell called ADAM10, which is a protease found on the cell membrane.

You may not have thought of it, but when you diet, your cells eat themselves, rather than just sloughing of the cells in the fat you don’t like (love handles, double chin etc. etc.).  Wouldn’t that be nice though.  The process is called autophagy, in which membranes appear, surround small bits of each cell and them fuse with the lysosome, which breaks the contents down into metabolically useful material (sugars, fats, amino acids).  Some 41 different proteins are involved called ATG’s (for AuTophagy Gene).

But the autophagy genes can also be used to secrete stuff to the outside of the cell, and in fact that’s how the lung cells beat MRSA, they secrete zillions of little vesicles called exosomes (an entirely interesting newly discovered story, to be covered at another time), containing the target of alpha toxin — ADAM10.  Clever no?  The authors were so excited they invented a new word for it the defensosome. The ATG involved is called ATG16L1.  Previously the function of ATG16L1 appeared well defined, conjugating phosphatidylethanolamine to LC3, a ubiquitinLike molecule to form the autophagosome.  That’s probably nomenclature overload, but it’s worthwhile getting an appreciation of the complicated things going on inside our cells.

 

Some sanity (and hope) about the Wuhan flu

There is an excellent article in the following link,  https://news.harvard.edu/gazette/story/2020/03/public-urged-to-ramp-up-social-distancing-increase-coronavirus-tests/

Epidemiology Professor Marc Lipsitch, head of the Center for Communicable Disease Dynamics at the Harvard T.H. Chan School of Public Health, called the pace of coronavirus testing in the U.S. “utterly inadequate” and “a debacle” that robs public health officials of information crucial to devising an effective response and protecting health care workers. Testing has been so slow that no one knows the extent of the U.S. epidemic, though scientists guess at somewhere between “tens of thousands and hundreds of thousands” of cases.

Let’s say that is true that 100,000 people in the USA have already been infected.  What does that mean?  It means that most people with it aren’t very sick (we don’t have 100K people in ICUs on respirators).  This is the hope anyway

The CDC monopolized testing for Corona virus for a while and then distributed faulty test kits (while you may not like Trump, this isn’t his fault). So we have no way of knowing what’s out there.

That’s information gap #1 — we don’t know how many people are actively infected with the virus

Information gap #2 is even worse, and very likely longer to be corrected.  We don’t know how many people are out there who have fought the infection off and are no longer sick.  This will require a test for the antibodies to the virus they’ve developed (something that doesn’t happen right away during the acute infection although that’s where it begins).  Such information  is not available now, and likely won’t be for a month or two.

Addendum: Not everyone reading this knows what an antibody is so this is in response to a few questions.  Antibodies are proteins made by your body in response to an invading organism (which kill it).  This is why your kids have measles, mumps and rubella vaccines — they give the kids proteins from the viruses (not the whole organism), and your kids react as if infected by the intact virus (which is why they often seem to have the flu after vaccination), producing antibodies to those proteins, so when the real thing comes sneaking around, your kid’s antibodies recognize it and fight it off.  Having an antibody to the Wuhan flu is perfect evidence that you’ve been infected with it (even if the virus is long gone).  They aren’t something you’d have normally.  It isn’t simple to develop and validate a test for anti-Wuhan antibodies which why I think it will be a while before we have a test we can trust.

This will tell us just how much to worry about the virus.  Most reading this are too young  to remember the polio epidemic (which also put people on respirators). https://en.wikipedia.org/wiki/Polio  After antibody tests finally became available it was found that only one out of every one hundred people infected developed paralysis.

 

How can it be like that?

The following quote is from an old book on LISP programming (Let’s Talk LISP) by Laurent Siklossy.“Remember, if you don’t understand it right away, don’t worry. You never learn anything, you only get used to it.”

Unlike quantum mechanics, where Feynman warned never to ask ‘how can it be like that’, those of us in any area of biology should always  be asking ourselves that question.  Despite studying the brain and its neurons for years and years and years, here’s a question I should have asked myself (but didn’t, and as far as I can tell no one has until this paper [ Proc. Natl. Acad. Sci. vol. 117 pp. 4368 – 4374 ’20 ] ).

It’s a simple enough question.  How does a neuron know what receptor to put at a given synapse, given that all neurons in the CNS have both excitatory and inhibitory synapses on them. Had you ever thought about that?  I hadn’t.

Remember many synapses are far away from the cell body.  Putting a GABA receptor at a glutamic acid synapse would be less than useful.

The paper used a rather bizarre system to at least try to answer the question.  Vertebrate muscle cells respond to acetyl choline.  The authors bathed embryonic skeletal muscle cells (before innervation) with glutamic acid, and sure enough glutamic acid receptors appeared.

There’s a lot in the paper about transcription factors and mechanism, which is probably irrelevant to the CNS (muscle nuclei underly the neuromuscular junction).   Even if you send receptors for many different neurotransmitters everywhere in a neuron, how is the correct one inserted and the rest not at a given synapse.

I’d never thought of this.  Had you?

 

When is the AIDs virus really dead?

When should we regard an AIDs virus lurking in the genome of a white blood cell as dead (or at least harmless).  Such proviruses are called defective, and commonly formed, because the process of reverse transcription (of RNA into DNA) is quite error prone.

Most would say an HIV1 provirus in the genome is dead  if can’t reproduce and get outside the cell carrying it.  Not so fast says Proc. Natl. Acad. Sci. vol. 117 pp. 3704 – 3710 ’20.  They show that such defective proviruses can be transcribed into RNA and these RNAs can produce proteins (when translated).

There is some evidence for this as the Nef protein of HIV1 can be detected in cells and plasma even when HAART (Highly Active Anti Retroviral Therapy) has knocked plasma viremia down to a level of under   50 copies/milliLiter.

How could this cause trouble ? Easy.  This would be chronically stimulating the immune system and in effect wearing it out.

This is very new stuff, and the fate of white cells containing replication incompetent proviruses which are still producing proteins isn’t known (but I’m sure this isn’t far off).

The ubiquitin wars

Ubiquitin used to be simple.  All it had to do was form an amide between its carboxy terminal glycine and the epsilon amino group of lysine of a target protein, and bingo — the protein was targeted for degradation by the proteasome.

Before proceeding, it’s worth thinking why this sort of thing doesn’t happen more often, by which I mean amide formation between carboxyl groups on aspartic and glutamic acid on one protein and lysines on the surface of another.  That’s where the 3 amino acids are likely to be found, because they are charged at physiological pH, meaning they cost energy (and probably entropy) to put into the relatively hydrophobic interior of a protein where there isn’t a lot of water around to hide their charges.   Also, every noncyclic protein (which is just about all of them) has a carboxy terminal amino acid — why don’t they link up spontaneously to the lysines on the surface of other proteins?

Well, ubiquitin does NOT link up spontaneously.  It has a suite of enzymes to do so.  Like a double play in baseball, 3 enzymes are involved, which move ubiquitin to E1 (the shortstop) to E2 (the second baseman) to E3 (the first baseman).  We have over 600 E3 enzymes, 40 E2s and 9 E1s.  650/20,000 protein coding genes is a significant number — and the 600 E3s are likely there to provide specificity to just what protein gets linked to.

Addendum 21 Feb — Silly me, I should have added in the nearly 100 genes coding for proteins that remove attached ubiquitins (e.g. the deubiquitinases).

A few more fun facts and then down to business.  First ubiquitin is so stable that boiling water doesn’t denature it [ Science vol. 365 pp. 502 – 505 ’19 ].  Second ubiquitin can link to itself, as it contains 7 lysines at amino acids 6, 11, 27, 28, 33, 48 and 63 of the 72 amino acids contained in the protein.

Polyubiquitin chains are often made up of multiple ubiquitin monomers with lengths up to 10 [ Nature. vol. 462 pp. 615 – 619 ’09  2009 ] meaning that there could be a lot of different ones ( 7^10 = 282,475,249.  However chains found in nature seem to use just one type of link, e.g. linking the carboxyl group of one ubiquitin to just one of the 7 lysines over and over, forming a rather monotonous polymer.

On to the interesting paper, namely the ubiquitin wars inside a macrophage invaded by TB [ Nature vol. 577 pp. 682 – 688 ’20 ]  Ubiquitin initially was thought to be a tag marking a protein for destruction.  It’s much more complicated than that.  A host E3 ubiquitin ligase (ANAPC2, a core subunit of the anaphase promoting complex/cyclosome) promotes the attachment of lysine #11 linked ubiquitin chains to lysine #76 of the TB protein Rv0222.  In some way this helps Rv022 to suppress the expression of proinflammatory cytokines.

We do know that the ubiquitination of Rv022 facilitates in some way the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6 (Tumor necrosis factor Receptor Associated Family member 6) preventing the its ubiquitination and activation.  Of interest is the fact that TRAF6 itself is an E3 ubiquitin ligase which acts on many proteins.

Now to continue and show the further complexity of what’s going on inside our cells.  Autophosphorylated IRAK leaves the TLR (Toll Like Receptor) signaling complex forming a complex with TRAF6 resulting in the oligomerization of TRAF6.  Somehow this activates TAK1, a member of the MAP3 kinase family and this leads to the activation of the family of IKappaB kinases which phosphorylate IKappaB leading to its proteolysis.  Once IKappaB is removed from NFKappaB, translation of NFKappaB to the nucleus occurs where it turns on transcription of cytokines and other proinflammatory genes.

It is really amazing when you think of all the checks and balances going on down there.  How crude our weapons against inflammation are now, compared to what we might have when we know all the mechanisms behind it.

The Wuhan flu epidemic in China has likely peaked

Could the Wuhan flu epidemic in China be peaking, or am I indulging in wishful thinking because of a son, daughter-in-law and two grandkids living in Hong Kong?  Possibly but here’s why.

The South China Morning post (https://www.scmp.com) keeps a total of the cases of the flu in China and worldwide.  The figures change throughout the day, but they don’t vary too much during the US day (Chinese night).

Looking at the totals in the US evening

From 2 Feb to 3 Feb there were just under 3,000 new cases

From 3 Feb to 4 Feb there were just over 3,000 new cases

From 4 Feb to 5 Feb there just under 4,000 new cases (3891)

From 5 Feb to 6 Feb there were slightly fewer newer case (3789)

From 6 Feb to 7 Feb there were definitely fewer new cases (3143)

 

Even though the totals are horrible — 31,161 cases in China (worldwide 31,482) with 636 worldwide deaths as of 5AM Eastern Standard time  7 Feb (USA), this is the second time in the China epidemic that there have been fewer new cases (and more importantly with a significant reduction from yesterday’s increase). This means we are likely at or over the peak of spreading of the epidemic (although the number of cases will continue to increase).

The number of new cases probably doesn’t contain any false positives, because of the thoroughness of the way they’ve been checked.

Caveat — only those lucky enough to make it into a hospital get checked, and stories I’ve read about how crowded they are and the long waits for care and admission, means that the number of cases are likely much higher.

Note that I include both the Chinese totals, and those worldwide as when the disease spreads worldwide (as it has) the total number of new cases will continue to increase (even if the Chinese new cases drops).

Having flown back and forth to Hong Kong several times, you couldn’t ask for a better way to spread the flu than cooping up 100+ people packed cheek by jowl in the steerage section of a large airplane for 16 – 18 hours.   This is particularly true since we now know asymptomatic people can spread the disease.

A very smart friend asked me ‘Why the excitement since influenza in the USA causes many more deaths each year’.  Well from the Center for Disease Control (CDC) we have the following — https://www.cdc.gov/flu/about/burden/index.htmlhttps://www.cdc.gov/flu/about/burden/index.html

“CDC estimates that influenza has resulted in between 9 million – 45 million illnesses, between 140,000 – 810,000 hospitalizations and between 12,000 – 61,000 deaths annually since 2010.”

That’s quite a range (5 fold) for number of illnesses and number of deaths, but the ratio is the same.  So what is the mortality of the flu we have in the USA?  Well 12,000/9,000,000 = .0013 or .13% which is 20 – 30 times less than the current known mortality rate of 2 – 3% for Wuhan flu.

Remember the figures are for symptomatic diagnosed cases.  There may well be many more cases with minimal or no symptoms.  Remember for every case of paralytic poliomyelitis, there were 99 infections where that didn’t happen.  Hopefully soon, we’ll have a way to test for human antibodies to Wuhan flu with specific antibodies (not all antibodies are specific) and we’ll find out the true prevalence of Wuhan flu infection.

Could the Wuhan flu epidemic in China be peaking?

Could the Wuhan flu epidemic in China be peaking, or am I indulging in wishful thinking because of a son, daughter-in-law and two grandkids living in Hong Kong?  Possibly but here’s why.

The South China Morning post (https://www.scmp.com) keeps a total of the cases of the flu in China and worldwide.  The figures change throughout the day, but they don’t vary too much during the US day (Chinese night).

Looking at the totals in the US evening

From 2 Feb to 3 Feb there were just under 3,000 new cases

From 3 Feb to 4 Feb there were just over 3,000 new cases

From 4 Feb to 5 Feb there just under 4,000 new cases (3891)

From 5 Feb to 6 Feb there were slightly fewer newer case (3789)

Addendum 6 Feb (USA) 9:20 PM 7 Feb China  (10:20 AM) — the news is good — the total number of new cases from 6 Feb to 7 Feb in China dropped significantly (967).  Hopefully this is accurate, and not due to some reporting glitch, or suppression of the numbers by directive from above.  If so, the epidemic has peaked, and all people have to do is stay indoors and not infect others.   Let’s hope this holds up.  Tomorrow’s count will be very important.   End Addendum 6 Feb 

Even though the totals are horrible — 28,396 cases worldwide with 566 deaths the morning of 6 Feb (USA), this is the first time in the China epidemic that there have been fewer new cases. This means we may be at the peak of epidemic spreading (although the number of cases will continue to increase).

The number of new cases probably doesn’t contain any false positives, because of the thoroughness of the way they’ve been checked.

Caveat — only those lucky enough to make it into a hospital get checked, and stories I’ve read about how crowded they are and the long waits for care and admission, means that the number of cases are likely much higher.

Note that I include only the Chinese totals, as when the disease spreads worldwide (as it has) the total number of new cases will continue to increase (even if the Chinese new cases drops).

Having flown back and forth to Hong Kong several times, you couldn’t ask for a better way to spread the flu than cooping up 100+ people packed cheek by jowl in the steerage section of a large airplane for 16 – 18 hours.   This is particularly true since we now know asymptomatic people can spread the disease.

A very smart friend asked me ‘Why the excitement since influenza in the USA causes many more deaths each year’.  Well from the Center for Disease Control (CDC) we have the following — https://www.cdc.gov/flu/about/burden/index.htmlhttps://www.cdc.gov/flu/about/burden/index.html

“CDC estimates that influenza has resulted in between 9 million – 45 million illnesses, between 140,000 – 810,000 hospitalizations and between 12,000 – 61,000 deaths annually since 2010.”

That’s quite a range (5 fold) for number of illnesses and number of deaths, but the ratio is the same.  So what is the mortality of the flu we have in the USA?  Well 12,000/9,000,000 = .0013 or .13% which is 20 – 30 times less than the current known mortality rate of 2 – 3% for Wuhan flu.

Remember the figures are for symptomatic diagnosed cases.  There may well be many more cases with minimal or no symptoms.  Remember for every case of paralytic poliomyelitis, there were 99 infections where that didn’t happen.  Hopefully soon, we’ll have a way to test for human antibodies to Wuhan flu with specific antibodies (not all antibodies are specific) and we’ll find out the true prevalence of Wuhan flu infection.