A Spear-chucking Bacterium

Vibrio Cholerae impales its victims with a metal-tipped, poisoned, rifled, spear.

Life is tough all over, but particularly bad for microbes. Without a glimmer of consciousness, and with hardly any tools at hand, (or hands), they still struggle, suffer, and die in astronomical numbers. One of the more fascinating and classic areas of discovery in the field is the beauty and complexity of the T4 phage, which is a virus that infects bacteria like E. coli. It has a lunar-lander like structure that docks to its victim and injects the DNA (from a highly pressurized head chamber) which then kills it while producing hundreds of new viruses.

But it is only in recent years that a connection has been drawn between this phage injection mechanism and what bacteria do to each other. What has been sedately called the type VI bacterial secretion system, used by Vibrio cholerae and Pseudomonas aeruginosa, among many other species of bacteria, turns out to actually be a violent spearing mechanism they use to kill competing bacteria and also mammalian host cells during an infection.

Model of the spearing system, on right, compared with the T4 viral system on left. The spear is in purple, while the contractile sheath is in green, and the base is yellow. In later experiments (in movie, etc.) the sheath is tagged with a fluorescent protein (GFP), allowing it to be easily visualized in the light microscope.

This was recently described in an NIH talk by John Mekalanos, and also in various publications from his group. One representative film clip is linked below.

Still from movie of the Vibrio spear in action, link here.

One of the more remarkable aspects of this mechanism is its scale. Sausage-shaped bacteria typically are closest to each other at the side, so the apparatus begins assembling at the side of the Vibrio cell. This injector assembles clear across the interior of the cell, forming a thin thread almost 500nm long which has, in addition to the base plate that nucleates the process, an inside spear and an outside sheath.

Electron micrograph of a poised spear assembly in a Vibrio cell. Note the extraordinary length, of about 500 nm. The scale bar is 100nm. T6SS is type six secretion system, IM is inner membrane, and OM is outer membrane. Note the detail of the basal body at the top spanning both membranes.

The outside sheath is spring-loaded, and when triggered (how that happens is unknown) winds down in a matter of milliseconds to half its original length, thrusting out the spear, while also turning it like a drill. In his talk, Mekalanos showed that the spear is tipped by a pointed protein his lab had recently discovered, that contains a zinc-coordinated domain that gives it particular stability. Behind the tip, the spear also is festooned with a variety of toxins, because simply spearing a nearby cell is not enough to kill it. Vibrio injects both eukaryotic-directed toxins such as one that cross-links actin and thus paralyzes the cell and another that modifies the cAMP signalling system causing massive ion and water efflux, as well as several bacterial-directed toxins to clean out the competition, such as inhibitors of cell wall (peptidoglycan) synthesis.

Sample killing, where Vibrio (red) were mixed with Pseudomomas (green) cells. The spearing system sheath is labeled in red and green respectively. In each horizontal set of time lapse images, a spear from a Pseudomonas cell (green) impales a neighboring Vibrio cell and either causes it to swell locally or to lyse entirely, losing its optical contrast (arrows).

An interesting wrinkle in the story is that each bacterium that has this kind of system also has a complement of immunity proteins that neutralize the various toxins that it creates. The bacteria are not terribly bright, and live in close proximity, so they frequently spear each other. One wouldn't want that kind of thing to be fatal. But Vibrio doesn't need immunity from the eukaryotic-specific toxins, which do not affect bacteria, including itself.

Once the spear is thrown, another protein comes along to quickly disassemble the spent apparatus, and another one re-assembles from a new base plate somewhere else inside the bacterium. Quite a bit more is waiting to be learned here, like the triggering mechanisms, and the details of assembly, but not only is this knowledge helpful in addressing a significant pathogen, (though one we hope to not meet in the developed world), but it is an example of the breathtaking complexity, and even beauty, in biology, even in the midst of the most desperate dramas.


Papers:

• Description of the system.
• General review of the system.
• Immunity and warfare issues.
• Investigation of the system in a plant pathogen.

Notes in passing:

• NBA may exit feudal world, go socialist.
• Bird speech and human speech.. not so different, perhaps.
• Am I giving philosophy a bad rap, for being a home for insurgent theists?
• Free market short-term-ism and self-immolation, cont.
• $721 million in mid-term political funding came from fossil fuels. Thanks!
• Not just an increase in the minimum wage, but in overtime coverage as well.
• Religion as psychotherapy, cont. Some people need answers really, really badly.
• Bill Mitchell on Japan: "Let it be noted that the Japanese government 10-year bond yield hit 0.33 per cent overnight. That tells you that all the scaremongering that has been going on over the last twenty years about hyperinflation, the Japanese government running out of money, the bond markets dumping the yen, and the rest of it were self-serving lies designed to advance a particular ideological position at the expense of the broader social well-being."

Governance in Venice

Good governance gave Venice a thousand years of prosperity and renown.

The idea that the US is the oldest democracy in the world is far from the truth. Even if you qualify it as  "existing" or "continuously existing", the Swiss have one far older, (making allowances for a brief Napoleonic regime), as do the Icelanders. But more important is that forms of government are very plastic. What we call a democracy in the US is a far cry from actual self-rule, given the vastly greater influence of the moneyed classes and the advertising arts over who gets elected, than anyone in the demos. Oligarchy would be more like it. Republic it may be, but democracy it is not.

But oligarchies aren't all bad. Just think of the Catholic church, which has functioned continuously (give or take a few anti-popes) for about a thousand years, and with less historical certainty for another thousand back to the time of the first bishops of Rome. Its organizational stability has been impressive, even as it has gone through vast changes in theology, morals, and power.

Venice in its heyday was a somewhat similar republic / oligarchy, and an extremely well-ruled one. A wonderful history of Venice tells a story that I had never learned in school, of the long and proud reign of Venice over a commercial empire that grew across the Eastern Mediterranean and beyond. From its extremely humble origins in the 568 in a malarial lagoon, Venice developed a communal government as well as an adavanced (and state-run) ship manufacturing system which, combined with skillful seamanship, diplomacy, a dedication to business, and occasional military prowess, earned her a durable mercantile empire. This was centered on the India trade, which ran through several routes, such as the silk road, the Byzantine ports, and the Arab caravans. Venice was the home of the Polo family, which famously followed these routes to the ends of the earth.

Symbol of Venice, the winged lion of St. Mark.

In 1204, its power was such that, as a particularly horrific and misguided part of the fourth Crusade, Venice led her allies to attack and sack Constantinople, a theretofore unimaginable feat, given the reputation of its walls and military. This was, for the Eastern Empire, a disaster it never recovered from, after which it limped along till its final defeat under the scimitars of the Turks in 1453. Venice, too, was eventually boxed in by the Ottomans, who in their own prime ran a highly capable fleet and threatened Venice in the Adriatic and even its own lagoon, while relieving it of its Greek and other far-flung possessions.

What finally sent Venice into decline (well before being crushed by Napoleon and then assimilated into the modern state of Italy) was the Northern European revolution in navigation and trade, as the Portugese and then Dutch overtook the India trade directly by way of the horn of Africa. Thereafter, Venice became poorer, and more embroiled on its landward side with the complexities of Italian feudal feuds, including with various Popes.

Through it all, Venice had a remarkable system of government. "Byzantine" doesn't do it justice, as there were ten layers of elections to go through among various bodies before the supreme leader, the Doge (a variant of "Duke"), was elected. Each Doge was constitutionally restricted in his scope of independent action, and also given a specific document of restrictions, usually the fruit of past excesses or corruptions that the community had learned from. The civil service was very efficient, and many times over her history, when Venice found herself in a tight spot, she put out the call for various special taxes, donations, and forced loans, which were typically met with great generosity. Each Doge set an example by distributing vast amounts of his own wealth when elected, and many followed that up with other gifts to the city during their rule and in their bequests. The degree of civic committment at all levels is striking, especially in this day when some parties cry about the most infinitesimal increment of taxation.

At the base of the state was the Grand Council, whose membership of about 1500 was originally elected among the population at large, but by 1296 became hereditary to the Venetian nobility, i.e. the rich. Thereafter, new families were allowed in very sporadically, when the state was under stress, and for large payments, but generally, membership was tightly closed and formed the core of the oligarchy, and the voting base insofar as it was a republic. Various more select bodies such as the ministers to the Doge, the Senate, (sixty members), Zonta (sixty more members), and Venice's own Inquisition / Star chamber / NSC- the Council of Ten- were chosen from this Great Council.

One might note that the early Roman republican system was hardly less complex, and also led to hundreds of years of good, if, again relatively oligarchical, government. Universal sufferage was extremely uncommon in large bodies before modern times, partly for technical and ideological reasons, but also because universal education was equally uncommon. But given an oligarchy, the complexity of these great examples such as Venice seem to reflect relatively little cost, and provide an extensive filter of checks and balances that so frequently succeeded in putting the best people in charge.

The effect of this good governance was to provide durable prosperity and promote human values, even in the midst of terrible times, such as several severe bouts of the plague. Its commitment to great art and architecture was legendary. And while Venice was not an intellectual leader in the Renaissance, its enthusiastic and free printing establishments were the largest in Italy and played a central role in transmitting knowledge from the Byzantine archives to the scholars of Europe. In time of our own when political ideologues dream of drowing their own governments in bathtubs, and refuse to govern countries they themselves have conquered, it is important to remember what a blessing (and a lesson) good government is.

"The more one studies the domestic history of Venice, the more inescapable does the conclusion become: by whatever political standards she is judged, she compares favorably with any nation in Christendom- except, arguably, in the days of her final dotage. Nowhere did men live more happily, nowhere did they enjoy more freedom from fear. The Venetians were fortunate indeed. Disenfranchised they might be; they were never downtrodden. Although, being human, they might occasionally complain of their government, not once in all their history did they ever rise up against it; such few attempts as there were at rebellion were inspired by discontented nobles, never by the populace." 

"Alone of all the states of Catholic Europe, it had never burnt a heretic."

• We need a war on cars.
• "Christians more supportive of torture than non-religious Americans."
• Still some problems in the intersection of race and genetics.
• Investing vs disinvesting, in our environment, in ourselves.
• Just how hosed is the middle class in the US? Part 1.
• Does Obama gain anything by caving to cave dwellers?
• Bill Black #2: Appeals court says insider trading is OK.
• Sony lets the terrorists win.
• Death and youth.

The American dream comes true.

With Shifafa on the Side

Poetry in America is OK.

The state of formal poetry is rather grim these days. Poet laureates are named, nationally and at other levels, and are then mostly ignored. Books of poetry are indigestable, and progressive forms are militantly unreadable. The New Yorker and other magazines gamely continue to publish poems, but to me, it seems a vain pursuit. It is a sad tale of a vibrant form in the humanities being academi-sized, perhaps like philosophy and economics, to irrelevance. But perhaps there is more going on.

Some fields do extremely well in academia, particularly the hard sciences. But the softer the field, the less it can be transmitted by formal methods, and perhaps the more it is killed rather than nurtured by the conformity, the drive to explicit formulations, the competitiveness, the prosody of the logos.

Poetry, however, is doing fine outside of formal institutions. It lives as always most happily with its musical muse. And that great American art form, jazz, is one of its finer incarnations, with word play aplenty. An example is "The Frim Fram Sauce", a standard from the Nat King Cole era.

I don't want french fried potatoes,
Red ripe tomatoes,
I'm never satisfied.
I want the frim fram sauce with the ausen fay
With shafafa on the side. 

I don't want pork chops and bacon,
That won't awaken
My appetite inside.
I want the frim fram sauce with the ausen fay
With chafafa on the side. 

A fella really got to eat
And a fella should eat right.
Five will get you ten
I'm gonna feed myself right tonight. 

I don't want fish cakes and rye bread,
You heard what I said.
Waiter, please serve mine fried
I want the frim fram sauce with the ausen fay
With shafafa on the side. 

~interlude~ 

A fella really got to eat
And a fella should eat right.
Five will get you ten
I'm gonna feed myself right tonight. 

I don't want fish cakes and rye bread,
You heard what I said.
Waiter, please serve mine fried
I want the frim fram sauce with the ausen fay
With shafafa on the side. 

(now if you don't have it, just bring me a check for the water!)

- Redd Evans

Some singers (Diana Krall, I'm looking at you!) leave out the concluding line, i.e. the punchline, leaving the audience mystified. And then she has the audacity to intimate that it is all sexual inuendo! Anyhow, the song is a great example of poetry that looks pretty dry on the page coming alive with music, since as sung, it is smart, melifluous wordplay.

Perhaps poetry is not meant to be dry, to be subject to "readings", with apologies to Shakespeare. Country artists are another class of homegrown poets who know how to make a line sing.

No, we're not the jet set
We're the old Chevrolet set
Our steak and martinis
Is draft beer with wieners. 

-George Jones

• Ten commandments, modernized version.
• "However, Pakistan has a history of releasing jihadists who seek to destroy the Pakistani state if the government feels it will further its goals of destabilizing Afghanistan or India."
• Obamacare is doing just fine.
• Minimum wage increases are paid by customers, not through unemployment.

Measures of Consciousness

Can EEG be used to tell whether someone is conscious, and to what degree?

The remarkable discovery that some people with no outward sign of consciousness are still, within, quite conscious indeed, has been both sobering and a spur to better ways to detect consciousness directly. The orginal studies used fMRI, which is extremely laborious and expensive. Far cheaper would be to be able to tell a patient's state from an EEG, i.e. reading their brain waves from electrodes on the scalp. A recent study attempted to do that, using a high density array of 96 electrodes.

Patients with disorders of consciousness come in many kinds. From the EEG perspective, one is tempted to say that all happy brains are alike; every unhappy brain is unhappy in its own way. But the very unhappy ones are put in two classes these days, those in a vegetative state, and those in a minimally conscious state, which is significantly better, particularly with the ability to follow action with the eyes, or some similar ability to react to the surroundings, at least sporadically.

The researchers used high density EEG recordings, ten minutes long, from 26 normal people, 13 in a vegetative state, and 19 in a minimally conscious state. This status was evaluated according to a standard checklist of tests, called CRS-R. The hunt was to develop some kind of algorithm out of the large amount of EEG data that would reliably classify their patients, using the CRS-R tool as a standard of comparison. One would assume that if these researchers are successful, they could go on to run blinded trials to validate their  EEG analysis for clinical use, but it didn't sound like they got that far.

Brain wave power vs frequency, for human controls, those with minimal consciousness (MCS), and those in a vegetative state (VS).

There are dramatic differences, though, between the subjects. A summary of their brain wave power vs frequency (above) shows that the alpha band has a strong peak in normal people, which is utterly missing in those who are impaired. Conversely, patients with disorders of consciousness (DoC) had much higher delta band power. As discussed in a recent post, alpha waves are a sign of consciousness, but not really of active thought, rather they characterize an alert, relaxed state, especially in the visual cortex, ready to process data, but not actively processing. Theta and delta bands are more common in sleep and coma. One might surmise that they have something to do with healing and regeneration, which fits with this current data. The graphs also show raised gamma wave power in those with impaired consciousness, which the authors interpret as an incidental phenomenon due to involuntary muscle movement that causes electromyographic noise, i.e. electrical activity from muscles, not from brain waves.

But is there a quantifiable test to be made out of this? While there are significant differences in the wave pattern, it is merely statistically detectable, not an absolute, every-time kind of distinction.

Connectivity metrics among EEG electrodes, clustered into color-sets. Note long-range and strong connectivity in delta band of VS patients, theta band of MCS patients, and alpha band of controls. 

The researchers also provide more detailed maps of connecting / correlating activity among their electrodes, which makes interesting viewing. Note here again that the most active and longest-range connections are in the alpha waves in the control group, the theta waves in the MCS group, and the delta waves in the VS group. They point out that not only strength, but also the distance of interaction is a significant metric. The VS group shows mostly localized connections in the alpha waves, for instance.

Comparison of EEG node connectivity computed for three patients in a vegetative state,  after being asked to imagine themselves playing tennis. Patient P3 is clearly following along. Colors come from an arbitrary clustering algorithm that groups more-connected nodes.

They even go on to reproduce the "locked-in" patient test, asking patients to imagine playing tennis. As shown, two of the three patients show quite disorganized patterns in the alpha waves, while the third had dramatically long-distance and strong wave action. The third patient was classed as being in a vegetative state, but scored highly on all the EEG metrics that this group used, and thus appears to be a case that is more accurately diagnosed by newer methods of EEG or fMRI than by the standard CRS-R diagnostic checklist. Indeed, this patient appears to be a locked-in candidate, but the researchers note that in other brain wave frequencies, this patient looked more impaired, and say little more about her or him.

"In particular, though these prominent differences between the brain networks of P1 and P3 could perhaps be attributed to aetiology, it could not explain away the differences between P2 and P3, as both had suffered traumatic brain injury. It is also interesting to note that though P3's alpha network properties were clearly very prominent outliers as compared to P1 and P2, delta and alpha power in P3 were much less exceptional. Hence characterising network signatures of spectral connectivity could considerably improve our understanding of residual brain function in behaviourally uncommunicative patients who nevertheless demonstrate covert awareness."

The researchers go on to analyze their data in various ways, but while very interesting, none looks like a slam-dunk for telling the classes of patients apart in definitive fashion, quite yet. Such methods are sure to arrive at some point, however, as better tools are developed, and now that we know how much can be hidden behind the facade of immobility after traumatic brain injury.

• Finance is pretty much all bad.
• The Ferguson prosecutor played defense attorney, not prosecutor. And the grand jury played regular court jury, not grand jury. Police deserve some leeway in this respect, but then there should be an alternate form of review, like a civilian oversight board that would fire a policeman and could ban them from future police work after a culpable or unnecessary killing.
• An industry of fear and hate.
• Oh, those nasty atheists!
• A small hate problem at the fringes.
• The American dream is now dead. And would be dead-er if people knew what is going on.
• You will never guess who is the mastermind behind the global warming hoax.
• Antarctica is melting.
• Cruise ships? You don't want to go there.
• Krugman- just who is stupid?
• TNR in throes of death.
• Destruction of the state as normal policy.
• History of capitalism, another iteration of feudalism, colonialism, etc.