Just because a receptor is there doesn’t mean it’s doing anything

Normally when we see a receptor on the surface of a cell (such as the receptor for Vascular Endothelial Growth Factor — VEGF) we assume that when its ligand binds, something happens inside the cell. Not always so, says Cell vol. 159 pp. 473 – 474, 584 – 596 ’14. VEGF is crucial in fetal development (inactivation of just one of the two copies of the gene is lethal for the mouse embryo [ PNAS vol. 95 pp. 14389 - 14394 98 ]).

One of the problems in diabetic retinopathy is proliferation of retinal blood vessels. For those who don’t already know — light outside the eye has to pass through all 10 cellular layers of the retina before it hits the photoreceptors which can absorb it. So more vessels in the neuronal layers isn’t good.

The Cell paper shows that in the developing retina, VEGF is depleted near neurons, by neuronal engulfment of the VEGF/VEGF receptor complex and degradation. The complex doesn’t do anything metabolically to the neuron. This prevents misdirected angiogenesis into the neuronal plane.

This turns what we’ve always thought about receptors on the cell surface on its head — they must be doing something inside the cell, when a ligand binds to them. Apparently not always.

Another financial piety bites the dust

If only businesses looked past the next quarterly earnings report all would be well. Productivity and profits would increase if CEOs would think long term. Investors hunger for such thinking.

Well, a large firm with earnings that beat analysts estimate by over 5% did that exactly yesterday and the stock dropped 6% today, Revenue rose 59%, but costs rose 41%.

The head, a brash youngster spoke saying that the ‘company’s long-term goals stretch more than a decade into the future and require “investing aggressively”.

Yes, this happened to Facebook. So much for the long term view

Ebola — an update (25 Oct ’14)

The experiment of nature referred to in a previous post (https://luysii.wordpress.com/2014/10/16/an-experiment-of-nature/) when Amber Vinson, a nurse who had helped care for a fatal case of Ebola, took a commercial flight from Cleveland to Dallas the day she became symptomatic with Ebola is almost over. She was diagnosed 14 October, the day she took the flight, and so far no one on the flight has become ill (presumably the 100+ or so are under surveillance).

However, another experiment of Nature has just begun. An M. D. who’d been in Africa treating Ebola victims was diagnosed with it on the 23rd. He had returned to NYC from Africa 14 October and had been up and about in the city. According to the Times he began to feel sluggish the evening of the 21st, went all over the city on the 22nd, including a 3 mile jog on the west side, and noted a mild temperature (100.3 not 103 as initially reported) the morning of the 23rd — reported it immediately and was hospitalized the same day. New York City chastened by the disastrous response to the first case in Texas, sent 3 guys in Hazmat suits to his apartment to pick the doctor up, according to the NYT of 26 October. Some contacts, such as his fiancee are easy to trace, the people he rode with on the subway are not.

The incubation period is said to be no more than 21 days, so neither experiment of nature is truly over. From this case we now know the incubation period can be as short as 7 – 9 days.

As noted in the previous post — The genome of Ebola is RNA which mutates much more rapidly than DNA genomes. It does this so quickly that at death from AIDS (another RNA virus), there are so many viral variants present that the infecting ensemble is called a quasiSpecies. With a large population infected in Africa there is more Ebola virus extant than at any time in the past.

We have a small handle on just how fast the virus is mutating [ Science vol. 345 pp. 1369 - 1372 '14 (12 Sep '14) ]. This is a report of 98 virus genomes from 78 patients from Sierra Leone (all this year). The Ebola genome contains 18,959 to 18,961 nucleotides and codes for at least 7 proteins. Compared to all previously known Ebola genome sequences, the virus from Sierra Leone contains 341 fixed changes (e.g. the changes were present in every virus they sequenced). The changes were present in all 7 proteins.

It isn’t clear (to me) from reading the paper how much variation in the viral genome there is (1) in a given individual (2) between individuals. Note that all samples were obtained from late May to early June this year, so the work is a good baseline.

Why is this scary? Because, as is typical for a virus with a genome made of RNA, Ebola is mutating rapidly. This means that we can’t be sure that its incubation characteristics, or its ability to spread from human to human will remain constant.

Producing the paper, required lots of collaboration between people in the USA and Africa, so there are 58 co-authors of the paper. Showing just how bad the disease is five of the fifty-eight co-authors died of Ebola. R. I. P. Mohamed Fullah, Mbalu Fonnie, Alex Moigboi, Alice Kovoma, S. Humarr Khan.

Who said this?

“You have to take care of all the sectors in —- as much as you can,” he said, “and if it’s entirely a numbers game and numeric representation, then obviously you would be talking to half of the people in —– who earn less than $1,800 a month.”

The present system serves to “insulate candidates from popular pressure to create a welfare state, and would allow the city government to follow more business-friendly policies.”

Clue: It is not a Republican dinosaur or the Koch brothers.

No it’s the Beijing-appointed leader of Hong Kong, Leung Chun-ying as reported 2 days ago in the New York Times — http://www.nytimes.com/2014/10/21/world/asia/leung-chun-ying-hong-kong-china-protests.html?_r=0

Amazing isn’t it? Well, perhaps not. In March 2013 my wife and I saw Bentley dealerships in Beijing. In the Causeway Bay area of Hong Kong, there appeared to be one high end jewelry store (Cartier, etc. etc.) per block.

What’s a fellow-traveller to do?

Watching electrons being pushed

Would any organic chemist like to watch electrons moving around in a molecule? Is the Pope Catholic? Attosecond laser pulses permit this [ Science vol. 346 pp. 336 - 339 '14 ]. An attosecond is 10^-18 seconds. The characteristic vibrational motion of atoms in chemical bonds occurs at the femtosecond scale (10^-15 seconds). An electron takes 150 attoseconds to orbit a hydrogen atom [ Nature vol. 449 p. 997 '07 ]. Of course this is macroscopic thinking at the quantum level, a particular type of doublethink indulged in by chemists all the time — http://luysii.wordpress.com/2009/12/10/doublethink-and-angular-momentum-why-chemists-must-be-adept-at-it/.

The technique involves something called pump probe spectroscopy. Here was the state of play 15 years ago — [ Science vol. 283 pp. 1467 - 1468 '99 ] Using lasers it is possible to blast in a short duration (picoseconds 10^-12 to femtoseconds 10^-15) pulse of energy (pump pulse ) at one frequency (usually ultraviolet so one type of bond can be excited) and then to measure absorption at another frequency (usually infrared) a short duration later (to measure vibrational energy). This allows you to monitor the formation and decay of reactive intermediates produced by the pump (as the time between pump and probe is varied systematically).

Time has marched on and we now have lasers capable of producing attosecond pulses of electromagnetic energy (e.g. light).

A single optical cycle of visible light of 6000 Angstrom wavelength lasts 2 femtoseconds. To see this just multiply the reciprocal of the speed of light (3 * 10^8 meters/second) by the wavelength (6 * 10^3 *10^-10). To get down to the attosecond range you must use light of a shorter wavelength (e.g. the ultraviolet or vacuum ultraviolet).

The paper didn’t play around with toy molecules like hydrogen. They blasted phenylalanine with UV light. Here’s what they said “Here, we present experimental evidence of ultrafast charge dynamics in the amino acid phenylalanine after prompt ionization induced by isolated attosecond pulses. A probe pulse then produced a doubly charged molecular fragment by ejection of a second electron, and charge migration manifested itself as a sub-4.5-fs oscillation in the yield of this fragment as a function of pump-probe delay. Numerical simulations of the temporal evolution of the electronic wave packet created by the attosecond pulse strongly support the interpretation of the experimental data in terms of charge migration resulting from ultrafast electron dynamics preceding nuclear rearrangement.”

OK, they didn’t actually see the electron dynamics but calculated it to explain their results. It’s the Born Oppenheimer approximation writ large.

You are unlikely to be able to try this at home. It’s more physics than I know, but here’s the experimental setup. ” In our experiments, we used a two-color, pump-probe technique. Charge dynamics were initiated by isolated XUV sub-300-as pulses, with photon energy in the spectral range between 15 and 35 eV and probed by 4-fs, waveform-controlled visible/near infrared (VIS/NIR, central photon energy of 1.77 eV) pulses (see supplementary materials).”

The incredible information economy of frameshifting

Her fox and dog ate our pet rat

H erf oxa ndd oga teo urp etr at

He rfo xan ddo gat eou rpe tra t

The last two lines make no sense at all, but (neglecting the spaces) they have identical letter sequences.

Here are similar sequences of nucleotides making up the genetic code as transcribed into RNA




Again, in our genome there are no spaces between the triplets. But all the triplets you see are meaningful in the sense that they each code for one of the twenty amino acids (except for TAA which says stop). ATG codes for methionine (the purists will note that all the T’s should be U). I’m too lazy to look the rest up, but the ribosome doesn’t care, and will happily translate all 3 sequences into the sequential amino acids of a protein.

Both sets of sequences have undergone (reading) frame shifts.

A previous post https://luysii.wordpress.com/2014/10/13/the-bach-fugue-of-the-genome/ marveled about how something too small even to be called a virus coded for a protein whose amino acids were read in two different frames.

Frameshifting is used by viruses to get more mileage out of their genomes. Why? There is only so much DNA you can pack into the protein coat (capsids) of a virus.

[ Proc. Natl. Acad. Sci. vol. 111 pp. 14675 - 14680 '14 ] Usually DNA density in cell nuclei or bacteria is 5 – 10% of volume. However, in viral capsids it is 55% of volume. The pressure inside the viral capsid can reach ten atmospheres. Ejection is therefore rapid (60,000 basepairs/second).

The AIDS virus (HIV1) relies on frame shifting of its genome to produce viable virus. The genes for two important proteins (gag and pol) have 240 nucleotides (80 amino acids) in common. Frameshifting occurs to allow the 240 nucleotides to be read by the cell’s ribosomes in two different frames (not at once). Granted that there are 61 3 nucleotide combinations to code for only 20 amino acids, so some redundancy is built in, but the 80 amino acids coded by the two frames are usually quite different.

That the gag and pol proteins function at all is miraculous.

The phenomenon is turning out to be more widespread. [ Proc. Natl. Acad. Sci. vol. 111 pp. E4342 - E4349 '14 ] KSHV (Kaposi’s Sarcoma HerpesVirus) causes (what else?) Kaposi’s sarcoma, a tumor quite rare until people with AIDS started developing it (due to their lousy immune system being unable to contend with the virus). Open reading frame 73 (ORF73) codes for a major latency associated nuclear antigen 1 (LANA1). It has 3 domains a basic amino terminal region, an acidic central repeat region (divisible into CR1, CR2 and CR3) and another basic carboxy terminal region. LANA1 is involved in maintaning KSHV episomes, regulation of viral latency, transcriptional regulation of viral and cellular genes.

LANA1 is made of multiple high and lower molecular weight isoforms — e.g. a LANA ladder band pattern seen in immunoblotting.

This work shows that LANA1 (and also Epstein Barr Nuclear antigen 1` ) undergo highly efficient +1 and -2 programmed frameshifting, to generate previously undescribed alternative reading frame proteins in their repeat regions. Programmed frameshifting to generate multiple proteins from one RNA sequence can increase coding capacity, without increasing the size of the viral capsid.

The presence of similar repeat sequences in human genes (such as huntingtin — the defective gene in Huntington’s chorea) implies that we should look for frame shifting translation in ourselves as well as in viruses. In the case of mutant huntingtin frame shifting in the abnormally expanded CAG tracts rproduces proteins containing polyAlanine or polySerineArginine tracts.

Well G, A , T and C are the 1’s and 0’s of the way genetic information is stored in our genomic computer. It really isn’t surprising that the genome can be read in alternate frames. In the old days, textual information in bytes had parity bits to make sure the 1’s and 0’s were read in the correct frame. There is nothing like that in our genome (except for the 3 stop codons).

What is truly suprising it that reading in alternate frame produces ‘meaningful’ proteins. This gets us into philosophical waters. Clearly

Erf oxa ndd oga teo urp etr at

Rfo xan ddo gat eou rpe tra t

aren’t meaningful to us. Yet gag and pol are quite meaningful (even life and death meaningful) to the AIDS virus. So meaningful in the biologic sense, means able to function in the larger context of the cell. That really is the case for linguistic meaning. You have to know a lot about the world (and speak English) for the word cat to be meaningful to you. So meaning can never be defined by the word itself. Probably the same is true for concepts as well, but I’ll leave that to the philosophers, or any who choose to comment on this.

An experiment of nature

Yesterday’s post https://luysii.wordpress.com/2014/10/15/ebola/ concerned the fact that 2 nurses taking care of a patient in Texas had been infected (presumably even after taking all the recommended precautions). Given that, I was concerned about the possibility of airborne spread.

Bryan wrote in to say the following:

“It seems doubtful airborne spread was involved. Remember, the Texas patient was initially sent home after showing symptoms, yet none of his family members were infected. Only those health workers directly involved in his care (and thus exposed to infected bodily fluids) have been infected, consistent with the idea that the disease can be transmitted only though contact with infected bodily fluids.”

I certainly hope he is right.

In something right out a novel, the possibility of airborne spread is now going to be empirically tested, as one of the two infected nurses flew to Cleveland, and then back to Texas in the 24 hours prior to her diagnosis. She apparently had a slight fever on boarding. So 100+ people were in a confined space with her for a few hours.

It’s why I don’t read fiction — reality is far more fantastic than anything writers can produce.

One more bizarre development. Here in Massachusetts, legislators today are scheduled to hear about the readiness of the state’s hospitals to handle Ebola. Amazingly, they will only get input from hospital CEOs. No nurses, thank you. Naturally the nurses are pissed as they should be (and so should you if you live in the state). If there were ever a time to hear from boots on the ground about Ebola readiness, it is now.

Addendum 17 Oct ’14

The Obama administration has just appointed a former chief of staff for former vice-president Gore and present vice-president Biden as the “Ebola czar”. Presumably, not for his medical expertise but for his ability to coordinate various governmental agencies, which was hardly the problem in the CDC’s response to the Texas cases. Hopefully, this will not be another case of “Brownie, you’re doing a heck of a job,” but I’m not optimistic — http://en.wikipedia.org/wiki/Michael_D._Brown

Now for some molecular biology. The genome of Ebola is RNA which mutates much more rapidly than DNA genomes. It does this so quickly that at death from AIDS (another RNA virus), there are so many viral variants present that the infecting ensemble is called a quasiSpecies. With a large population infected in Africa there is more Ebola virus extant than at any time in the past. There is some reason to hope that natural selection for a more transmissible form of Ebola in the large infected human population will not occur (the AIDS virus hasn’t become more infectious over the years). This is only a hope.


This morning (15 October) it was announced that a second health care worker at the Texas hospital where an ebola patient died has ‘tested positive’ for it. If ebola can spread in a hospital environment where presumably precautions were taken, once it gets out into the populace at large it can spread much faster. This had to be human to human transmission — no other animal vector is involved (as it probably is in Africa).

How does it spread? We don’t know, but the two Texas cases probably imply that airborne spread is possible.

What to do?

In our case it means not getting into a confined space with over 100 people we don’t know from all over the world for an 8 – 16 hour period (e.g. an international flight). Have you ever been on a flight where no one had a cold?

For the USA, it should mean banning all flights from endemic countries. This has been the case in the past. My cousin’s wife has a lot of relatives in Brazil, because the people on the boat had lots of pink eye, and the boat was simply turned away over 100 years ago.

It should mean caring for Ebola patients in specialized facilities where only they are cared for –e.g. not in a general hospital since we don’t know how it spreads.

The greatest way to spread the disease (the Hajj — millions of people from all over the world crowded together for days followed by worldwide dispersal) has mercifully just ended before the disease escaped Africa to any extent.

Will ISIS or Al-Qaeda try to bring Ebola to the USA? Of course.

We live in a society where children have supervised play dates, and where walking unattended to school is almost considered child abuse. What will happen to such a risk-averse society when there is actual risk to going out to (the mall, the school, to work)?

The Bach Fugue of the Genome

There are more things in heaven and earth, Horatio,
Than are dreamt of in your philosophy.
– Hamlet (1.5.167-8), Hamlet to Horatio

Just when you thought we’d figured out what genomes could do, the virusoid of rice yellow mottle virus performs a feat of dense coding I’d have thought impossible. The following work requires a fairly sophisticated understanding of molecular biology which the articles in “Molecular Biology Survival Guide for Chemists” might provide the background. Give it a shot. This is fascinating stuff. If the following seems incomprehensible, start with –https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ and then follow the links forward.

Virusoids are single stranded circular RNAs which are dependent on a virus for replication. They are distinct from viroids because viroids need nothing else to replicate. Neither the virusoid or the viroid were thought to code for protein (until now). They are usually found inside the protein shells of plant viruses.

[ Proc. Natl. Acad. Sci. vol. 111 pp. 14542 - 14547 '14 ] Viroids and virusoids (viroid like satellite RNAs) are small (220 – 450 nucleotide) covalently closed circular RNAs. They are the smallest known replicating circular RNA pathogens. They replicate via a rolling circle mechanism to produce larger concatemers which are then processed into monomeric forms by a self-splicing hammerhead ribozyme, or by cellular enzymes.

The rice yellow mottle virus (RYMV) contains a virusoid which is a covalently closed circular RNA of a mere 220 nucleotides. A 16 kiloDalton basic protein is made from it. How can this be? Figure the average molecular mass of an amino acid at 100 Daltons, and 3 codons per amino acid. This means that 220 can code for 73 amino acids at most (e.g. for a 7 – 8 kiloDalton protein).

So far the RYMV virusoid is the only RNA of viroids and virusoids which actually codes for a protein. The virusoid sequence contains an internal ribosome entry site (IRES) of the following form UGAUGA. Intiation starts at the AUG, and since 220 isn’t an integral multiple of 3 (the size of amino acid codons), it continues replicating in another reading frame until it gets to one of the UGAs (termination codons) in UGAUGA or UGAUGA. Termination codons can be ignored (leaky codons) to obtain larger read through proteins. So this virusoid is a circular RNA with no NONcoding sequences which codes for a protein in either 2 or 3 of the 3 possible reading frames. Notice that UGAUGA contains UGA in both of the alternate reading frames ! So it is likely that the same nucleotide is being read 2 or 3 ways. Amazing ! ! !

It isn’t clear what function the virusoid protein performs for the virus when the virus has infected a cell. Perhaps there aren’t any, and the only function of the protein is to help the virusoid continue existence inside the virus.

Talk about information density. The RYMV virusoid is the Bach Fugue of the genome. Bach sometimes inverts the fugue theme, and sometimes plays it backwards (a musical palindrome if you will).

It is unfortunate that more people don’t understand the details of molecular biology so they can appreciate mechanisms of this elegance. Whether you think understanding it is an esthetic experience, is up to you. I do. To me, this resembles the esthetic experience that mathematics offers.

A while back I wrote a post, wondering if the USA was acquiring brains from the MidEast upheavals, the way we did from Europe because of WWII. Here’s the link https://luysii.wordpress.com/2014/09/28/maryam-mirzakhani/.

Clearly Canada has done just that. Here are the authors of the PNAS paper above and their affiliations. Way to go Canada !

Mounir Georges AbouHaidar
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and
Srividhya Venkataraman
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and
Ashkan Golshani
bBiology Department, Carleton University, Ottawa, ON, Canada K1S 5B6
Bolin Liu
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and
Tauqeer Ahmad
aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3B2; and

The Silence is Deafening

A while back I wrote a post concerning a devastating paper which said that papers concerning the default mode of brain activity (as seen by functional magnetic resonance imaging { fMRI } ) had failed to make sure that the subjects were actually awake during the study (and most of them weren’t). The post is copied here after the ****

Here’s a paper from July ’14 [ Proc. Natl. Acad. Sci. vol. 111 pp. 10341 - 10346 '14 ] Functional brain networks are typically mapped in a time averaged sense, based on the assumption that functional connections remain stationary in the resting brain. Typically resting state fMRI (default network == rsfMRI) is sampled at a resolution of 2 seconds or slower.

However the human connectome project (HCP) has high-quality rsfMRI data at subsecond resolution (using multiband accelerated echo planar imaging. This work used a sliding window approach mapping the evolution of functional brain networks over a continuous 15 minute interval at subsecond resolution in 10 people. I wrote the lead author 21 July ’14 to ask how he knew the subjects weren’t asleep during this time.

No response. The silence is deafening.

Another more recent paper [ Proc. Natl. Acad. Sci. vol. 111 pp. 14259–14264 '14 ] had interesting things to say about brain maturation in attention deficit disorder/ hyperactivity — here’s the summary

It was proposed that individuals with attention-deficit/hyperactivity disorder (ADHD) exhibit delays in brain maturation. In the last decade, resting state functional imaging has enabled detailed investigation of neural connectivity patterns and has revealed that the human brain is functionally organized into large-scale connectivity networks. In this study, we demonstrate that the developing relationships between default mode network (DMN) and task positive networks (TPNs) exhibit significant and specific maturational lag in ADHD. Previous research has found that individuals with ADHD exhibit abnormalities in DMN–TPN relationships. Our results provide strong initial evidence that these alterations arise from delays in typical maturational patterns. Our results invite further investigation into the neurobiological mechanisms in ADHD that produce delays in development of large-scale networks.

I wrote the lead author a few days ago to ask how he knew the subjects weren’t asleep during this time.

No response. The silence is deafening.


If you Google “default mode network” you get 32 million hits in under a second. This is what the brain is doing when we’re sitting quietly not carrying out some task. If you don’t know how we measure it using functional mMRI skip to the #### and then come back. I’m not a fan of functional MRI (fMRI), the pictures it produces are beautiful and seductive, and unfortunately not terribly repeatable.

If [ Neuron vol. 82 pp. 695 - 705 '14 ] is true than all the work on the default network should be repeated.


Because they found that less than half of 71 subjects studied were stably awake after 5 minutes in the scanner. E.g. they were actually asleep part of the time.

How can they say this?

They used Polysomnography — which simultaneously measures tons of things — eye movements, oxygen saturation, EEG, muscle tone, respiration pulse; the gold standard for sleep studies on the patients while in the MRI scanner.

You don’t have to be a neuroscientist to know that cognition is rather different in wake and sleep.



There are now noninvasive methods to study brain activity in man. The most prominent one is called BOLD, and is based on the fact that blood flow increases way past what is needed with increased brain activity. This was actually noted by Wilder Penfield operating on the brain for epilepsy in the 30s. When the patient had a seizure on the operating table (they could keep things under control by partially paralyzing the patient with curare) the veins in the area producing the seizure turned red. Recall that oxygenated blood is red while the deoxygenated blood in veins is darker and somewhat blue. This implied that more blood was getting to the convulsing area than it could use.

BOLD depends on slight differences in the way oxygenated hemoglobin and deoxygenated hemoglobin interact with the magnetic field used in magnetic resonance imaging (MRI). The technique has had a rather checkered history, because very small differences must be measured, and there is lots of manipulation of the raw data (never seen in papers) to be done. 10 years ago functional magnetic imaging (fMRI) was called pseudocolor phrenology.

Some sort of task or sensory stimulus is given and the parts of the brain showing increased hemoglobin + oxygen are mapped out. As a neurologist, I was naturally interested in this work. Very quickly, I smelled a rat. The authors of all the papers always seemed to confirm their initial hunch about which areas of the brain were involved in whatever they were studying. Science just isn’t like that. Look at any issue of Nature or Science and see how many results were unexpected. Results were largely unreproducible. It got so bad that an article in Science 2 August ’02 p. 749 stated that neuroimaging (e.g. functional MRI) has a reputation for producing “pretty pictures” but not replicable data. It has been characterized as pseudocolor phrenology (or words to that effect).

What was going on? The data was never actually shown, just the authors’ manipulation of it. Acquiring the data is quite tricky — the slightest head movement alters the MRI pattern. Also the difference in NMR signal between hemoglobin without oxygen and hemoglobin with oxygen is small (only 1 – 2%). Since the technique involves subtracting two data sets for the same brain region, this doubles the error.


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