Saturday, August 27, 2016

What Does it Mean to be Supernatural?







A more modern trinity, participating in another archetypal savior story.


Saturday, August 20, 2016

Donuts and Zippers for DNA Repair

How the protein RAD52 conducts homologous search and rescue of damaged DNA. And what we can do with that knowledge.

Our bodies are full of wonders. Despite being about fifty years into the age of molecular biology, we are still only scratching the surface of the complexity that lies within. Remember that when you hear that someone has found a gene "for" a disease. That gene and the protein it encodes have a lengthy and intricate story to tell about what it does in the normal organism, how it got to that point in evolution, and maybe a multitude of ways in which mutations might drive it off track. Linking it to one disease is only a small part of a much larger tale.

BRCA2 is an example of this: a pre-eminent gene "for" breast cancer. But what does it do? The point of its existence is hardly to make us miserable. No, it participates in DNA repair, and thus some mutations in its sequence, or full deletions, can leave cells open to a cascade of further mutations, which can lead to cancer.

Recent work has shown that BRCA2 is synthetically lethal with a better understood protein called RAD52. Being synthetically lethal (in mice in this case, where genetics can be done) means that a mutation or deletion of either one of the genes is not lethal by itself, but the combination of both *is lethal. Such an observation implies that the two proteins participate in the same process, at least partially. One can "cover" for defects in the other one, in a pinch. But take away both, and the organism can not survive.

So BRCA2 and RAD52 do similar things, which is helpful to know, since RAD52 is actually better understood. Yeast cells, which are even easier to study than mice, have RAD52 but no BRCA2, which seems to have arisen more recently in evolution. We'll get back to BRCA2 briefly below, but I will focus on RAD52- a protein that conducts that amazing process that is homology-driven DNA repair.

Homologous DNA repair happens when a mutation is detected, such as a double-strand break, a missing nucleotide (and consequent single strand break), or even just where a mis-matching nucleotide was inserted by the DNA polymerase by mistake. Perhaps the sequence goes CAGCT on one strand, and GGTTG on the other, antiparallel strand. There is a G:T pair in here that is an error- not part of the official Watson-Crick regime!

A mismatch happens (orange).

What was the right sequence? The sequence of this duplex does not tell you, unfortunately, anymore. In some cases, there are chemical marks like methylation that can be used to tell the difference between the newly synthesized strand and the other one that has been around the block, and this is used in some forms of post-replication repair. But if the mutation slipped through that process, or happened at a different time, again we would not know where to turn for the right way to correct such an error.

But there is one place to turn ... another copy of that DNA, if the organism has one. Diploid organisms always have another copy of every gene lying around, and other organisms, even bacteria, do as well, if they have already replicated their DNA but not divided the cell, quite yet. This is called homologous DNA repair, because the other copy is called a homolog of the first. Organisms have developed ornate methods of homology detection, strand invasion, copying, and repair to use the information from a good version to repair a defective one. And one of the key actors is RAD52, or its colleague, BRCA2.

Structure of RAD52. The ring is composed of multiple subunits of the same protein, each colored differently here. DNA, in single stranded form, can wind around the outside.

RAD52 looks like a big donut. Well, in relation to DNA, at least. The DNA doesn't thread though the middle, as it does in some other important machinery, but rather winds around the outside, in single-stranded form. That way, RAD52 can expose the single stranded DNA to other donuts with their respective single stranded DNA, and figure out which ones match. It is evident that the energetics of DNA-DNA binding and DNA-RAD52 binding are engineered to be very similar, so that DNA engaged in mismatches does not get tangled up in unproductive knots, but prefers to wrap around these massive protein donuts. But then on the other hand, when a true DNA-DNA duplex match is found, it smoothly zippers up, out of the Rad52-wrapped state.

Model of the energetics of SS DNA binding to RAD52, with subtle transitions from SS and protein-bound, to double-stranded if a proper match is found (pink).

This is the mechanistic secret of homology search between DNA stands, at least in principle. How this happens at the scale of full super-coiled duplex genomes, in the crowded milieu of the cell, with the DNA studded with countless other proteins and complexes, plus a few chemical alterations, remains a bit hard to understand, however. And the rest of the repair story, of directing a large invasion of homologous DNA into the defective duplex, editing out the defective strand, re-duplicating the donor DNA, flushing the ends, and all the other odds and ends, are of course another story entirely.

In the current paper, the researchers looked for inhibitors of RAD52, intended for breast cancer treatment. On of the traditional treatments is to break the patient's DNA with toxic chemicals, because the cancer cells typically are missing the function of some DNA repair proteins like BRCA2, or BRCA1 which acts in the same pathway. Missing this repair function has generated the cancer in the first place by creating new mutations, but there can be too much of a good thing. That is, an excess of DNA damage causes cells- even cancer cells- to kill themselves or to die outright. So it would be helpful in this mode of therapy to be able to turn off what turns out to be, for humans, a backup DNA repair pathway- RAD52.
"RAD52 forms an oligomeric ring, where the primary ssDNA binding site is located in the narrow groove spanning the ring circumference. We designated this ssDNA-binding groove as the feature to be targeted by small molecule inhibitors. While disrupting the protein-ssDNA interaction with small molecules presents a formidable challenge that has only been overcome in a handful of cases, the ssDNA binding groove of RAD52 is a promising target and is distinct from the ssDNA binding sites of other ssDNA binding proteins."

The technique to screen for RAD52 inhibitors used fluorescence energy transfer. Donor and acceptor fluorophores were put on opposite ends of 30-nucleotide DNA strands. This happens to be exactly the length that fits around one RAD52 donut, so when the DNA binds to RAD52, the ends meet and the acceptor efficiently absorbs fluorescent photons from the donor, and re-emits at its own characteristic wavelength. Wow!

Compound found by screening for impaired fluorescence energy transfer between ends of DNA wrapped around RAD52 protein complexes.
Example of screening data, showing that binding of double-stranded DNA (gray) is unaffected by the compound "1", while the binding of single-stranded DNA is strongly impaired, at very low concentrations. The assay (Y axis) is fluorescence by the red fluorophore, which is excited by the green fluorophore only when the green fluorophore is nearby and also excited by the researchers shining appropriate (green) wavelength light onto the experiment.

After screening a bunch of chemicals, they came up with one that really works well, at low concentration. It clearly inhibits single strand DNA winding around RAD52. It also, in other work, interacts directly with RAD52, indicating that, as it looks quite a bit like a couple of DNA bases, it probably just binds better than DNA itself. By computational modelling they find that this compound binds into the single-stand DNA binding groove of RAD52, very snugly positioned in a way that is tightly bound and will keep anything else at bay.

 Model, using the known structure of both RAD52 and compound "1", of how they fit together. The chemical fits tightly in a groove that would otherwise bind single stranded DNA.

Finally, they ask whether this candidate drug works in cells. In tissue culture cells, the very low concentration of 500nM makes the cells behave as though they had no RAD52 at all, which is very promising indeed. Indeed, it kills cells lacking BRCA2, if treated with a chemotherapy regime, validating the idea of the work. How all this will work in whole animals and in humans is, of course another story entirely, in a long road to drug development. But this is an example of beautiful work with its origins in studies in model organisms (yeast) and structures in basic molecular biology.
"We also show that [compound] ‘1’ selectively kills cells depleted of BRCA2, further supporting the importance of the RAD52-ssDNA interaction in BRCA deficient cells and the potential therapeutic value of RAD52 inhibition."

  • It is OK to be just OK?
  • Religion as fully imaginary... and for some, that is OK. "Secret knowledge that everyone else was too blind to see" "God is the ultimate transitional object".
  • The decades of lies and meanness that led to this.
  • Who are you gonna believe?
  • Of ~400 districts in Afghanistan, the Taliban controls 40 and is actively fighting to control 44 more.
  • Costa Rica- a beautiful, just, peaceful, and low-carbon country... "Traffic is offensive."
  • Is there a problem with the sources of Islam, and our understanding of its history?
  • Our friends the Saudis, cont... balkanizing the Balkans.
  • Krugman detects evil and greed on the GOP side.
  • In return, one of the steps of grief ... denial and lashing out.
  • Is America breaking bad?
  • Obama leaves a great deal undone.

Saturday, August 13, 2016

I Like Looking at Westerns, Not Being In One

Where are the gun nuts taking us?

I admit it.. I love Westerns. The classics channels have my number. When you carry the law on your hip, drama is liable to break out at any moment. For a family show, Bonanza sure stacked 'em up. For dramatic purposes and moral edification, the Western is one of the finest genres, right up there with medieval-ish science fiction-ish power-drama. If our love of drama (like our experience of dreams, in part) is about social play and modelling, focused on training to face tough choices that come up rarely in real life, but have extraordinary consequences when they do, the payoff from Westerns is obvious.

But watching is one thing. Being in the middle of it is something else. The point of our communal "real" life, politics, law, etc., as opposed to our dramatic alter-lives, is to make horrible choices, compelling a they are dramatically, as rare as possible, not more common. I get it that you as a gun-toting open-carry-ist are the last hero standing between my precious bodily fluids and that cold-blooded robber. Or worse yet, a Hillar-ized jack-booted federal agent. But the reality is quite different.

The reality is that guns make us less safe, not more. The police are going into conniptions about facing an ever increasing tide of guns. They don't want the "help" from all those open-carry-ists. The Republican convention is a no-gun zone. Why? Not because crime won't happen. It is because guns are dangerous to everyone around them, even to the very well-trained. Western towns set up gun controls as soon as they could- as soon as the law became stronger than the outlaws. Why? Because guns are unsafe, and if everyone has one, someone with less training and dedication to the constitution than the dedicated, well-trained, and articulate gun nut is going to come stumbling out of a saloon and shoot up the town for whatever purpose, or none. Look at Afghanistan, a country awash with guns, and a sort of contemporary wild west. Is that a safer place to live? A better place to live?

Unfortunately, addressing the fantasy life of gun nuts is easier said than done. Apparently statistics and reason doesn't make you stop wanting to buy, clean, stroke, and carry your gun around. And this fantasy extends to insurrection and terrorism. A blog I follow regularly issues threats, both veiled and explicit, against the "gun-grabbers". Unique among grievances in our country, the gun nuts think the very power they are guarding so zealously is the means of its and their defense, leading naturally to revolutionary rhetoric- the kind of thing that would be unconscionable in any other community or venue:
"Yea, Hopkins just went from being a stooge to being an enemy.  Understand, Hopkins, that universal background checks will bring out the guns, and not in a good way, if you know what I mean.  That is a line that cannot be crossed by anyone.  It won’t happen, and your willing adultery with the Bloomberg position is most disappointing."
"Finally, it gives me amusement and pleasure to point out the obvious.  You can never effect this outcome because we have the guns.  Understand?  You can’t take them from us because you eschew them and we don’t.  What?  You didn’t really think we’d give them up, did you?  And you didn’t really think those cops would want to be gunned down as they try to confiscate weapons, did you?"
"God grants me the right (and even duty) to go armed and conduct myself in a manner consistent with self defense.  Not you, and not the constitution, and not black robed tyrants.  That means that whatever the outcome of this “day in court” to which you refer, the right to self defense is still present because God said so."
"But it’s not over with.  Know who your local police are.  Make sure to let them know in no uncertain terms, without them knowing who you are, that it is all just beginning.  Make your points in the shadows, not in the light of day.  Make it clear to them that you will not tolerate infringements on your God-given rights. ... Make your points until the police no longer want to wage war on otherwise peaceable citizens.  Make your points in the shadows. That’s how this should go down.  Do you understand?"

And this is not to mention Donald Trump's recent casual incitement of the assassination of his opponent.

So we are faced with a heady macho-psycho-Freudian-theological brew of perceived potency, with strong ties to American history and heritage, but also to much more deeply seated emotional issues.  Other countries have been able to lower the temperature around guns. Why not us?

Saturday, August 6, 2016

No, I do Not Really Feel Your Pain

Where is pain in the brain, and is empathic pain in the same domain?

Our culture has gone through some interesting evolutions in the experience of pain. From antiquity through medieval times, inflicting pain was entertainment and justice, in forms such as gladiatorial fighting, bull-fighting, burning at the stake, trial by torture, and plain old torture. In modern times we seem to have softened to the point of respecting human rights, one of which is to not inflict gratuitous pain, even on deserving criminals. Perhaps this is due to the subjugation of pain in medical and dental settings, which has raised our expectations for an untroubled existence. Or perhaps it is due to dominance of secularism which continues to guide moral innovation and refuses to countenance twisted theistic rationalizations for our painful existence, let alone explicit theological torture. With the current election, we seem to be taking a step backward in the excruciating-ness department, but still one may hope for the best!

Despite such variation in cultural tolerance of pain, (especially that of others), our intrinsic capacities are naturally long-standing and common to most animals. In particular, pain is perhaps the most immediate and insistant instance of consciousness, and thus a particularly interesting case for investigating the nature of consciousness and its presumptive basis in the brain. One thing we have learned is that pain comes in many forms, from vague itches to excruciating burns. And in addition, we experience various abstract pains, such as bereavement, the sudden horror of devastating news, and the empathy we feel for the pain of others- at least some people, in some cutural settings(!)

So it is not a simple field. A recent paper made some new observations about empathic pain. Prior work has shown substantial overlap of self-pain and empathic pain as represented in the brain, leading to theories that it is involuntary and almost as vivid as one's own. But we know that that is not quite true- that spectators at the colosseum could, though social training and construction, view the torture and dismemberment of unfortunate gladiators with enthusiastic bloodlust rather than squeamish sympathy.

So where is it, and how do we feel pain of both types? These authors discuss various defects with the current model, particularly that brain locations that have been noted as activated during both own-pain and empathic pain are not really pain centers per se, but are activated by many non-pain events, to the point of being some of the most frequently activated areas in the brain across all fMRI studies.
"Only a small minority of dACC neurons are pain-related, and the dACC encodes emotional events, including rejection and general negative emotion, in a way that is distinct from how it encodes pain."

Indeed, the best anatomical correlate of pain perception in the brain is not found at a single anatomical location, but in a network (the Neurologic Pain Signature or NPS) that is something of an abstract, deduced entity, built up out of statistical measures of where the hurt shows up, specifically and reliably, in fMRI. The authors try to refine these anatomical claims with their own fMRI studies.
"The NPS has over 90% sensitivity and specificity in predicting somatic pain relative to several other salient states, including non-painful warmth, anticipated pain, pain recall, social rejection, and general negative emotion."

Active sites from actual, somatic heat-induced pain on the subject's arms or legs.

Their hapless subjects where seared on their forearms or feet with about 10 seconds of high temperature (46, 47, pr 48˚C, which is 115, 117, 118˚F). This doesn't sound too bad, really, perhaps just noticeable. The vicarious / empathic pain was more interesting: being shown pictures of a person putting a shovel through their foot, or cutting a finger with a knife, and asked to imagine this happenening to themselves. Trained actors were used, I assume. What they showed certainly causes a chill to go down your spine, but it does not share specific aspects with the experienced pain. The vicarious pain was not heat- better to show someone taking a blowtorch to their extremeties. Interestingly, however, the researchers claim to be able to differentiate between pain vicariously induced at the arm vs the leg. Even by this very indirect means, there are site/anatomy-specific signatures evident in the brain signals.

For exprienced pain, they validated the NPS network, seeing it reliably show up in their scans. But the vicarious / empathic pain was a different story. In fact, they found that the NPS areas were negatively correlated with empathic pain, and devised a newly calculated network to describe what was activated, which they call the vicarious pain signature, or VPS. Helpfully, however, they compared their VPS pattern with those prompted by negative affect pictures, and by romantic/social rejection and found no correlation. So evidently, fMRI is getting good enough to distinguish between several negative emotions.

Active sites from vicarious pain, i.e. from pictures of other people doing painful things to themselves. Note the quite different patterns of activation vs above.

Global correlations between experimental fMRI patterns and the modeled networks of the NPS or VPS. Note the complete lack of correlation of the somatic pain with the VPS, and conversely the vicarious pain with the NPS.

One question is how high-level the NPS network is. Does it represent the actual physical pain but not the consciousness of it, or does it represent both? In prior work, the same lab showed that they also saw complete distinction between NPS and efforts the subject might make to cognitively control that pain, seen as dampening from the frontal cortex. Insofar as the subjects of these studies could manage their pain, it did not affect the NPS intensity at all, but worked at another, higher level. So no, while the NPS correlates with some aspects of pain and its cognition, it does not seem to encompass the highest level processes of conscious pain management, and perhaps even perception.

But back to the main study. The authors observed the NPS quite consistently, and saw its intensity increasing with increasing amounts of applied pain. And as noted, the VPS was quite different. But it did overlap in very few areas- the anterior insula (aINS) and the dorsal anterior cingulate cortex (dACC). These have been previously cited as the points of overlap, thus as sites showing that we feel the pain of others. The aINS is a profound part of our brain, functioning in awareness, consciousness, sense of self, and sociality and other emotions, like the sense of importance of things- meaning. Thus it is natural that it might be activated in both these experimental regimes, without "meaning" quite the same thing.

Likewise, the dACC also functions in emotion and consciousness, as well as reward evaluation, internal conflict, and impulse control. Again, functions that might come up in both experimental scenarios without meaning quite the same thing. Whether a pain is on your own body or elsewhere, at some very high level it is just another input to evaluate for what we are going to decide to do next. In any case, the researchers give additional evidence from lack of correlation on the finest voxel-level scales within both regions to suggest that what is going on in these locations is quite different in the two pain scenarios.

So, despite our mirror neurons and the best intentions, pain is not readily shared, and counts quite a bit less when it happens in others rather than in ourselves. Which is good, since being incapacitated by someone else's pain would probably help neither person. It also points to the importance and privacy of personal consciousness. While we keep coming up with technologies to share / immerse our mental selves in other worlds (telling stories, books, movies, VR), we do so because our capacity to do so unaided is quite limited. We are naturally wired first and foremost to feel ourselves.

  • If the 1% gain a lot from free trade, and many low-wage workers lose what is to them a lot, but to the 1% a little, is it a good thing?
  • Growth is dead. No wonder the fight to split the pie is getting fiercer.
  • We all needed someone to blow up the GOP.
  • Marxists vs Trump.
  • We are not a healthy country.