Kuro5hin.org: technology and culture, from the trenches
create account | help/FAQ | contact | links | search | IRC | site news
[ Everything | Diaries | Technology | Science | Culture | Politics | Media | News | Internet | Op-Ed | Fiction | Meta | MLP ]
We need your support: buy an ad | premium membership

[P]
Biofilms

By Sgt York in Science
Fri Oct 09, 2009 at 11:19:10 PM EST
Tags: science, bacteria, evolution (all tags)
Science

It is one of the most ancient cooperative systems. It is the most widely accepted candidate for the ancestor to all multicellular life. It has been observed ever since men have looked through microscopes; Leeuwenhoek himself was the first to describe it. It's on your teeth. You've seen pretty pictures of them in Yellowstone. It's clogging your drain. You showered with one this morning. You slipped on it the last time you crossed a stream.


OK, given the audience, maybe those last two are a bit of a stretch, but you've probably never heard of them, and if you have, you've rarely thought of them.

Take one (1) happy bacterium floating in a pond, or your sink, or a drop of water on your shower curtain, or the saliva in your mouth. It's free swimming. It may have a handy little flagellum that is uses for tooling around, avoiding bad environments, seeking food, doing its little buggy thing. It is currently in what is called its planktonic stage. As a planktonic bug, it can accomplish the dream of all life everywhere, to become what every little bacterium the world over wants to become : Plural. Preferably measured in powers of ten.

Like I said, it can do this perfectly well planktonically. It can swim around and eat and divide and evade and eat and divide and evade.... until eventually it fails at one of those things, and some instance of itself dies. But usually, it doesn't just keep swimming happily around. For some reason, given the chance, this bug will abandon its free swimming existence, shed its flagellum, and find some nice little surface to adhere to. Why?

Because it would be nice to do away with one of those three things that bugs have to do: Evade. I know, at first glance that make no sense at all. If you can't move you can't evade, and the the thing you are evading will do whatever it is that it would do that made it worth evading, right? Bear with me. Planktonic bacteria spend a lot of time evading. They move away from toxic environments like pH or osmolarity changes. They move away from hunting phagocytes like amoeba and neutrophils. They often have trouble finding a nice, stable and safe environment to settle down in. They solve this problem by creating a nice stable and safe environment to settle down in. They make biofilms.

That lone planktonic bacterium makes a class of seemingly useless compounds, the quorum sensing molecules (QS). For the most part, these are n-acyl homoserine lactones (AHSL), small nonpeptide molecules. Simple guys, a 5-member ring on one end, a short chain of carbons with a carbonyl or two hanging off it. Simple bugs, simple signals.

QS are part of an autoinducer loop; the bacterium talking to itself. Normally, this doesn't do anything, because the molecules simply diffuse away before they can reach a level high enough to trigger any kind of response. Selection dictates that these things have a purpose, though. A bacterial population can experience hundreds of generations in a week, and that's a hell of a driving force for selection. Therefore, they are usually quite streamlined. They make nothing they don't need....so why these?

Let's go back to our lone bug. Call him Phil. Phil eats stuff and makes stuff. One of the things he makes is AHSL. But it doesn't do much good. AHSL has to get up to a certain concentration before it can do anything, and the rate at which Phil makes AHSL is not anywhere near the rate at which AHSL diffuses away from him. Eventually, Phil will set down and attach himself to some kind of surface. At some point after that, Phil strives for the dream and makes the ultimate thing any bacterium can make, Phil Jr.

Phil Jr also eats stuff and makes stuff (like AHSL), and he cranks them out next to Dad. Mom. Whatever. Doesn't really matter, it's a fucking bacterium. Bacteria. Whatever.

Anyway, Phil and Phil Jr both make AHSL. For you math whizzes out there, that's doubling the rate at which AHSL is made. Unfortunately, this just means they diffuse away even faster, reducing the increase. But the increase is there. Phil Jr strives for the dream, just as Phil does the same, for a second time. Meet Phil III & IV. Eat, divide, repeat. Meet Phil V-CCLVI. Hey...that QS level has gone up a bit. Phil has become PHIL. PHIL is talking to himself, and for the first time, diffusion isn't silencing him. So he silences diffusion.

OK, enough with the anthropomorphic prokaryotes. We now have the makings of a biofilm. QS molecules like AHSL have reached a respectable concentration, and signaling has begun. New genes are turned on, and new behaviors kick in. Flagella go away. New types of pilli form. Each cell begins to crank out proteins, thick, gooey carbohydrates, and DNA, which come together to form a sticky coat around each bacterium. Due to their proximity, these thick, gooey coats merge, and grow. Other proteins are made. Beta-lactamases to protect against incursions of fungus, catalase to fend off superoxides in the environment, proteases to chew up anything near the biofilm to make more room to grow. New properties arise. After a time, the bacteria have made a viscous liquid surrounding them, a glob of slime in your drain, the black goo in your trap, or the yellowish slick on your teeth. It's not really a solid and it's not really a liquid. Think snot or the watery phlegm that you cough up when you have the flu. It's actually very similar stuff.

After a while, you have millions of bacteria, all living in and as a single biofilm. At first, the film is a fairly homogenous hydrogel, a water trapping 3-dimensional mesh. This mesh impedes normal diffusion, greatly reducing the rate at which molecules can move in or out of the biofilm. Inside, QS concentration accumulates, and the rising rate causes further changes (the mechanisms of which are not fully understood). The biofilm now no longer has to evade. It can control the pH inside itself by regulating H+ export. It can regulate ion and osmotic gradients by sequestering and pumping ions around. It avoid being eaten by roving phagocytes by sheer size; biofilms can be large. They can grow from a few bacteria that barely affect the turbidity of a test tube full of media to a visible, tangible thing you can actually pick up with your fingers after only a night of growth.

It's easy to see why these are thought of as the precursors to multicellular life, they are multicellular life. It's practically an organism. As the biofilm develops, it even develops organs of a sort; fruiting bodies. Small pockets will form inside the biofilm, pockets devoid of the carbohydrate/protein/DNA scaffolding that makes the structure of the biofilm. In these pockets, planktonic bacteria start to grow, and the pocket starts to move to the periphery of the biofilm. After a time, the fruiting body buds off and spills the planktonic bacteria out into the environment, to seed new biofilms and exploit new territories.

It's an elegant and useful system. The problem is, it shouldn't exist. According to all we know about evolution, it should be a highly unstable system due to a phenomenon well known in the geek world: The tragedy of the commons.

Making this stuff takes work. It is work to make the biofilm scaffold, to pump ions and make protective proteins. The bacteria do this because working together this way is of benefit to them all. It's not really altruism, but mutualism. Prokaryotic quid pro quo. It's worth the effort because everyone is pitching in. Some of you may see where this is going.

You can break down mutations into 4 broad categories: Gain of function, change of function, loss of function, and invisible. In prokaryotes, the most common of these, by far, is loss of function. Think about computer code. If I were to randomly change a few characters in your code, it is possible that it could be an improvement. But it's not very likely. Most likely the program will break. Same with bacteria. Change something at random, and you break it. Most of the time. I hope you see where this is going.

Take one bug in that biofilm and hit it with a stray bit of radiation. That stray bit of energy happens to strike the DNA strand in the alg promoter region just as it is being replicated for mitosis. The radiation lends its charge to a guanine residue in the strand, rendering one of its three hydrogen bonding participants temporarily uncharged. As a result, instead of the cytosine that should settle in opposite it, a thymine settles in instead. The charge on the guanine dissipates, and the error becomes obvious. Repair structures move in and assemble to fix the damage, but by now, mitosis is complete. What is correct, the T or the G? Coin toss....the G loses. alg no longer works. Not that that is a bad thing.

alg, among other things, happens to control the production of some of the carbohydrate scaffold in biofilms. This bug just lost the ability to help make the scaffold. But like I said, that's not a bad thing. Its neighbors still make the scaffold. It still gets food, protection, and stability from the biofilm, but its load in maintaining the biofilm is less. So it has an advantage. It can grow slightly faster than its neighbors, and it does. And its progeny also don't make the scaffold. In the context of a biofilm, this bug has an advantage and it uses it.

After a short period of time, selection runs its course and the biofilm+ bacteria are outcompeted. The scaffold dissolves, AHSL diffuses away. The biofilm capable bugs lose their signal and stop making all the things that made them a biofilm. Phil is back, and he goes on his merry way.

But biofilms DO exist. They are stable, that is the inescapable observation. There are even multispecies biofilms; the crap on your teeth is a perfect example. So why doesn't one bug get mutated and wipe out the biofilm with its own selfishness? It's not clear.

There are explanations for parts of this, but none are satisfactory, and I've gone on long enough. I'll get you started by saying this: There are two primary ways by which cooperation can arise in a population. One is mutualism, each individual derives a benefit from the others behavior. That's how biofilms get started. But when one bug stops helping, it's all qid and no quo. Mutualism is gone.

The second way is kin selection, and from 1964 (Hamilton) to the early 1990s (Kelly, Queller, Wilson, and Taylor to name a few), it was thought that this is what accounted for the stability of biofilms. But that last set of authors voided Hamilton's theoretical work with their own theoretical work and showed that if you invoke limited dispersal to get kin selection (as Hamilton did), then you get your ass handed to you by kin competition after a short period of time. Back to square one, just by a different mechanism.

But that highlights the problem right there: It's all theoretical. Theoretically, biofilms shouldn't exist. Theoretically they will form in a certain way that dooms them to dissolving in a certain way after a period of time. That wouldn't be a problem; we could deal with biofilms that exist for a while and then disperse. But that's not what we see. We see biofilms grow and stay for very long periods of time. They are very hard to get rid of. Don't believe me? Go clean out the trap under your sink. Wait a week and look again. That black shit that smells like death died and rotted for a while? Biofilm.

Go brush your teeth.

Sponsors

Voxel dot net
o Managed Hosting
o VoxCAST Content Delivery
o Raw Infrastructure

Login

Related Links
o Beta-lacta mases
o catalase
o Also by Sgt York


Display: Sort:
Biofilms | 49 comments (39 topical, 10 editorial, 0 hidden)
perhaps there's some way cells punish freeloaders? (none / 1) (#3)
by Blarney on Thu Oct 08, 2009 at 12:43:25 PM EST



Perhaps (none / 0) (#4)
by Sgt York on Thu Oct 08, 2009 at 12:54:14 PM EST

but it's not even clear how they'd detect freeloaders, much less punish them.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Yes! It's like objectivisim for bacteria! /nt (none / 0) (#22)
by ksandstr on Fri Oct 09, 2009 at 03:36:00 PM EST



Fin.
[ Parent ]
That ones easy (3.00 / 2) (#5)
by GhostOfTiber on Thu Oct 08, 2009 at 01:36:35 PM EST

You're assuming the biofilm is static, but in a running river (or in your mouth with all the cock pushing on the biofilm in your case) its constantly being eroded. Since the freeloader isn't pushing against the flow of biofilm because he has none to make, he's eventually pushed out of the group.

Anyway, +1 FP even though your a homo.

[Nimey's] wife's ass is my cocksheath. - undermyne

Most (none / 0) (#9)
by Sgt York on Thu Oct 08, 2009 at 04:05:47 PM EST

biofilms are in stagnant or low flow conditions, so erosion doesn't have much of an effect here.

Also, what does

Since the freeloader isn't pushing against the flow of biofilm because he has none to make
mean? What flow? What push? Has none of what to make?

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

biofilm (none / 0) (#10)
by GhostOfTiber on Thu Oct 08, 2009 at 04:08:27 PM EST

everyone else is making biocraps and he's not making biocrap so he rides the delicious wave of biocrap right back into the water. Then he'll grow a flagellum and FUCK YOUR MOTHER.

[Nimey's] wife's ass is my cocksheath. - undermyne
[ Parent ]

Ah (none / 1) (#11)
by Sgt York on Thu Oct 08, 2009 at 04:38:00 PM EST

I made the mistake of assuming you intended to make sense.

GENETIC DETECTION OF SPUTA FROM THE rRNA OF LOCAL ALIGNMENT SEARCH TOOLS COULD PROVIDE AN ASSESMENT OF RESPIRATORY PICOPLANKTON IN ENDOBRONCHIAL MICROVASCULATURE.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Hi welcome to K5. (3.00 / 5) (#12)
by GhostOfTiber on Thu Oct 08, 2009 at 04:43:03 PM EST


[Nimey's] wife's ass is my cocksheath. - undermyne
[ Parent ]

Weak spots and holes (none / 0) (#13)
by Blarney on Thu Oct 08, 2009 at 11:04:23 PM EST

I think GhostOfTiber has the right idea. Suppose that we get a mutant cell that freeloads on the biofilm. Well, it multiplies, right? You end up with, I'd guess, a weak spot in the biofilm. Right where all these cells are not contributing. Eventually you have a hole in the biofilm. And all these lazy cells drift away, carried by currents and Brownian motion and their own urges, never to enjoy the benefits of being in a biofilm. Meanwhile the neighboring cells that have continued to participate will fill in this hole, secrete biofilm components, and the biofilm will heal as if the mutation never happened. Isn't this more likely than the whole biofilm just breaking up at once?

Not really (none / 0) (#14)
by Sgt York on Fri Oct 09, 2009 at 01:44:36 AM EST

I may have given the impression that biofilm scaffold is rigid; it's not. It's actually a viscous gel. Think slime. I should probably address that in the story.

When a freeloader stops making matrix, it's not like there's a gap in the biofilm around that bug; scaffold from other areas will move in to fill the gap. You wind up with a net movement of scaffold materials towards the freeloaders due to the diffusion gradient.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Experimental test then (none / 0) (#24)
by Blarney on Fri Oct 09, 2009 at 05:38:25 PM EST

Suppose you somehow selectively kill a group of cells in one region of the biofilm, then, so they're visibly there but not actually contributing to biofilm synthesis. Perhaps they've been treated with some light-sensitive pro-toxin and you laser them. Something like that? You're actually saying the biofilm would remain intact in that area, forever and ever? Because I'd doubt that. I'd guess that not only is there a concentration gradient towards the cells, but towards the solution environment at large.

[ Parent ]
Not for ever and ever of course (none / 0) (#26)
by Sgt York on Sat Oct 10, 2009 at 12:20:30 AM EST

Everything decays eventually. But it would recover.

If they're dead, they will break apart and the hole will be filled in by other bacteria. If you just remove the ability to make biofilm, what we expect is

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Fixed, I hope. (none / 0) (#19)
by Sgt York on Fri Oct 09, 2009 at 09:55:14 AM EST

I added a bit to the end of 10th paragraph.
...fend off superoxides in the environment, proteases to chew up anything near the biofilm to make more room to grow. New properties arise. After a time, the bacteria have made a viscous liquid surrounding them, a glob of slime in your drain, the black goo in your trap, or the yellowish slick on your teeth. It's not really a solid and it's not really a liquid. Think snot or the watery phlegm that you cough up when you have the flu. It's actually very similar stuff.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Biofilm Shmiofilm (none / 0) (#20)
by mirleid on Fri Oct 09, 2009 at 10:59:13 AM EST

+1FP

Chickens don't give milk
''planktonically''? (none / 1) (#23)
by LilDebbie on Fri Oct 09, 2009 at 04:34:09 PM EST

you lost the FP for that one

My name is LilDebbie and I have a garden.
- hugin -

fuck you, man (none / 0) (#38)
by Sgt York on Tue Oct 20, 2009 at 12:41:02 PM EST

that was my only non FP upvote.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Why are human societies still around? (3.00 / 2) (#25)
by Pentashagon on Fri Oct 09, 2009 at 07:10:32 PM EST

There are people who keep order, and other feedback mechanisms that harm freeloaders or at least negate their advantages.  What keeps the human body together?  Lymph cells that explode on anything that doesn't look right.  I would almost bet that biofilms have a functional immune system of some kind.

How often do individual bacteria die off in a biofilm?  All the biofilm has to do is provide a little extra pressure on bacteria that aren't spewing more biofilm components, and the attrition will more than make up for any reproductive advantage a freeloader would have.  A simple molecule/protein that inhibits reproduction would probably be sufficient; it doesn't even have to be smart.  It just has to be pushed toward the bacteria that don't produce as much biofilm as the rest by the very action of all the other bacteria producing biofilm.

We are highly ordered systems (none / 0) (#27)
by Sgt York on Sat Oct 10, 2009 at 12:33:16 AM EST

with dedicated tissue types. We arose from something like this, but we are far, far removed from it. You don't even find T-cells (the cells you're talking about here) in invertebrates. You'd have a hard time convincing anyone that something like T-cell function exists in a prokaryote.
I would almost bet that biofilms have a functional immune system of some kind.
Hells, yeah! Let's team up and get a Nobel. Great idea, but how would that work?

It all boils down to "how do they know?" The system you propose does have to be smart; it has to know what bacteria is cheating, and what bacteria have just lost scaffold due to mechanical processes or some grazing protozoan, or is a fruiting body, happens to be dividing, or whatever.

I'm not saying it's not a good idea, it's a great idea. But how would it work? Where's the evidence that something like that exists? What would the mechanism be? How would it discriminate?

In vertebrates, we've got highly complex systems that are geared to find the cheaters and kill them (cancer surveillance). But that requires dedicated cell types and tissues. And even then, it fails from time to time. How would a bug know? There is something there, there has to be. It always makes sense in the end.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

The feedback has to be a property of the biofilm. (none / 0) (#29)
by Pentashagon on Sat Oct 10, 2009 at 05:17:49 AM EST

I'm not enough of a biologist to explain it at a detailed level, but here's the basic idea of how it could develop:

A plain old bacteria starts producing both a molecule that inhibits cell division and a molecule that negates the first (a toxin and an antidote, basically).  Maybe the toxin knocks the flagella off of other bacteria or something.  In any case, a few of these bacteria are able to survive a little better through chemical warfare against their neighbors who don't have the antidote.

Once a lot of these bacteria gather in one place, the toxins and antidotes end up producing a natural barrier in the form of a simple biofilm that has advantages for the colony as a whole.  Because each bacterium is forced to produce the antidote to make it immune to the toxins in the biofilm, whatever it produces will just end up contributing more material to the biofilm.  Anything that degrades the biofilm won't help the bacterium producing it, because it loses the benefit of the biofilm at the additional cost of pumping out biofilm-destroyers instead of dividing.  A bacterium that produces only the antidote could have an advantage, so it may be that the toxin and antidote are the same product at different times.  For instance, a toxin that is harmless until it degrades into a second form would be its own antidote if produced at a rate that keeps the harmless form near the cell wall and pushes the toxic form toward the under-producing bacteria.  This would not require that the cells know anything about each other; the toxin would do all the work.  If a stable antidote to the toxin is much more expensive than just producing half-activated toxins, it's pretty clear what the best choice for each cell is.  It's Mutually Assured Destruction for bacteria, I suppose.  Produce too few nuclear weapons, and your neighbors will kill you and absorb your nutrients.

I imagine that biofilms are not very socialist.  If you get chomped by something, you die and perhaps take a couple of your neighbors with you while the biofilm is repaired.  Budding off would have to derive from what is effectively time compressed version of two bacteria swimming close to each other, then swimming away.  Differentiated layers in the biofilm, perhaps even mediated by a series of toxins, counter-toxins, etc. from the evolutionary history of the particular biofilm that allows progressively more typical bacteria to divide off from their ancestors.  Eventually they're in a series of bubbles with layers of slightly different modes of bacterial behavior keeping the whole system in balance, then the bubble migrates to the edge and pops open, spewing the "normal" descendants at the center into the drain trap.  If the intermediate layers die, so what?

Further along the line, this could develop into a more symbiotic relationship.  Inside the biofilm, cell metabolism may be changed enough that survival requires cooperation.  It's possible to paint one's self into a corner, and perhaps the bacteria do this on purpose, making it impossible to leave the biofilm (what are you going to do outside a biofilm without a flagellum?  Growing a new one is a lot of work).  Their only hope is to work towards producing buds with brand new bacteria, probably in a milder soup of chemicals than the biofilm proper, and then let them go off to start new biofilms.  As long as the biofilm itself is sufficiently deadly to normal bacteria, it would take a huge evolutionary leap for a biofilm bacteria to do anything other than what it's expected to do.

There's also a similarity between the biofilm and the cell wall; both protect the vulnerable innards at the expense of the DNA in those innards which have to spend energy building cell walls or biofilm instead of just replicating all the time.  This is possibly due to the surface area/volume trade-off of maintaining an entire cell wall versus contributing energy to maintaining the biofilm in exchange for a cheaper (but weaker) cell wall.  Why don't organelles go crazy and start making copies of themselves?  The cell is full of self destruct switches that cause it to lyse if things get out of balance.  My guess is that biofilms are basically run the same way; in some ways it's a more (potentially) dangerous environment than swimming in a pond, but it's a much more stable environment.

[ Parent ]

Allow me to quote my Statics teacher (3.00 / 2) (#32)
by localroger on Sat Oct 10, 2009 at 10:22:27 AM EST

On the first day of class he stacked up a bunch of blocks and rods and casually asked us what the force was at one of the intersections in the stack. Of course none of us knew how to even figure that out; that's what we were there to learn. "Well, these are just dumb sticks and they figured it out," he taunted us. "These dumb sticks know how not to fall down. We have to become as smart as they are."

The biofilm bacteria obviously have an anticancer mechanism; the fact that the biofilm exists is proof of that. The question is not whether it exists but how it works. There is certainly signalling involved; lots of signalling is necessary to maintain the integrity of the biofilm. And the most likely punishment mechanism is apoptosis. Long before our fabulously complicated immune system gets involved, that's what stops most would-be cancer cells in our bodies; they aren't hunted down and eaten by lymphocyte samurai, they commit seppuku.

And screw the Nobel, whoever figures out how to trigger biofilm cancer (at least by some mechanism that doesn't poison eukaryotic life) is going to make a fortune in the toothpaste and drain cleaner business.

And that is what is so great about the internet. It enables pompous blowhards to connect with other pompous blowhards in a vast circle jerk of pomposity. -- Bill Maher
[ Parent ]

Dynamic equilibrium (none / 1) (#28)
by cym on Sat Oct 10, 2009 at 05:05:18 AM EST

Like mentioned before, your objections applies not only to biofilms, but to any multicellular organism, group of organisms, colonies, societies or even the different components within the cell. None of them should likewise exist.

The answer is dynamic equilibrium: The appearance of freeloaders will reduce efficiency of biofilm function, not immediately destroy it. The fraction of cooperative bacteria will continue to decay until a minimum is reached. This minimum correspond to the point where the biofilm ceases to function. If the fraction of cooperative bacteria decreases further, then it will be advantageous to be a cooperative bacteria, just like it was advantageous when there was no biofilm at all.

Don't just assume binary responses, like in chemistry the reality is dynamic, so anything persistent is the result of a dynamic equilibrium between opposing forces.

The rate of cooperative bacteria could even be higher than the minimum required for the biofilm structural integrity, for instance in the case where several types of biofilms are competing for resources. This is basic selection theory at work, there is no logic gap whatsoever.

Furthermore, the advantage of cooperating must be present at all stages of biofilm formation. Because of the high rate of mitosis, if there was a period of biofilm formation where to cooperate is costlier than non cooperating, then biofilm development would not proceed any further than that point.

So instead of wondering if the selection theory is wrong without any concrete reason for doing so, one should wonder why is it advantageous to participate in biofilm production at all the stages where biofilm production and maintenance is observed.

As for the circumstances where producing biofilm is disadvantageous, you should investigate instances in which biofilm does not develop, or can't be maintained.

BTW, your essay was lively and entertaining. And kudos for the controversy at the end, I could not avoid biting it.

it's funny (none / 0) (#30)
by donnalee on Sat Oct 10, 2009 at 05:31:46 AM EST

how you go from:

"It still gets food, protection, and stability from the biofilm, but its load in maintaining the biofilm is less."

to:

"But when one bug stops helping, it's all qid and no quo. Mutualism is gone."

less help != no help. Could they be doing something else? Maybe they contribute to the formation of the fruiting bodies ("pockets devoid of the carbohydrate/protein/DNA scaffolding that makes the structure of the biofilm").

Also, how does the biofilm absorb food? If there is enough food surplus, the biofilm+ bacteria can coexist with, not be outcompeted by, the mutated ones.

---
Guess I'll be adding this to tomorrow's comment dump!

The point (none / 0) (#37)
by Sgt York on Tue Oct 20, 2009 at 12:37:56 PM EST

which I guess I underemphesized, is that there is something else going on that we don't understand. "Could they be doing something else?" is a valid idea; but what?

Food absorption: I left out a LOT. There are book sets written on biofilms. The bioflim is actually a very complex structure; it has something akin to a circulatory system, channels cut through the biofilm.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

could they be contributing to the formation (none / 0) (#42)
by donnalee on Sun Nov 08, 2009 at 02:12:51 PM EST

of the fruiting bodies (which don't contain the structure that the 'normal' bacteria produce) or of the channels

---
Guess I'll be adding this to tomorrow's comment dump!
[ Parent ]
Unlikely (none / 0) (#44)
by Sgt York on Mon Nov 09, 2009 at 10:47:18 AM EST

If fruiting bodies are a way of exiling the cheaters, then you would expect the bugs that come out of those fruiting bodies to be unable to make biofilms; this is not the case. Genetically, the bugs that come out of the fruiting bodies are identical to the major components of the rest of the biofilm.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Seems like what you are asking... (3.00 / 3) (#31)
by localroger on Sat Oct 10, 2009 at 10:06:27 AM EST

...is why biofilms don't get cancer.

You could ask the exact same question of us, and of course the answer turns out to be incredibly complicated, and so deeply interwoven with the rest of our functionality that a surprising number of seemingly innocuous things break the anti-cancer mechanism, and when that mechanism breaks and lets a cancer cell through all hell breaks loose.

You have beautifully described some of the intricate mechanisms that make the biofilm possible. Then, right after you say:

Inside, QS concentration accumulates, and the rising rate causes further changes (the mechanisms of which are not fully understood)
...you loudly wonder, after a simple first-order analysis, why this fabulously complicated and well-adapted system doesn't self destruct from cancer. I'd say that while humans have been spending few years working that out, the bacteria have been working on it for three billion years.

We know the biofilms have intercellular signalling because without such signalling you can't regulate the pH or the scaffold-to-cell-mass ratio or a lot of other things necessary to make a stable macroscopic growth. So obviously there is anticancer signalling; it's almost certainly one of those things that kick in as the QS concentration goes up and behaviors continue to alter.

As to the mechanism of punishment for deadbeat cells, I'd guess the most likely thing is apoptosis -- voluntary suicide. The same thing is true to some extent for us -- most would-be cancer cells simply kill themselves before they come to the attention of the immune system, like an embedded device that refuses to boot when its ROM fails the checksum.

So you can't use normal mutation rates, which are very high, to predict the occurrence of would-be biofilm cancer cells; you need at least two mutations, one to make them deadbeats and another to disable apoptosis.

I think this is the basic error being made in wondering how biofilms are possible. If the rate of biofilm cancer is low enough it doesn't matter that it happens once in awhile, just as the occasional incidence of cancer in humans doesn't make us go extinct; the cancerous films will simply dissolve, the free-market supply-side bacteria will never be able to create another one, and the surviving non-cancerous cells will be free to start the process all over again.

And that is what is so great about the internet. It enables pompous blowhards to connect with other pompous blowhards in a vast circle jerk of pomposity. -- Bill Maher

Wondering why they persist (none / 1) (#35)
by Sgt York on Tue Oct 20, 2009 at 12:24:52 PM EST

doesn't make biofilms evaporate in a puff of logic. There are a lot of things we simply do not yet understand, it's why I still have a job.
We know the biofilms have intercellular signalling because without such signalling you can't regulate the pH or the scaffold-to-cell-mass ratio or a lot of other things necessary to make a stable macroscopic growth. So obviously there is anticancer signalling; it's almost certainly one of those things that kick in as the QS concentration goes up and behaviors continue to alter.
They have one form of signaling, but you can't just say "so they obviously have this other kind of highly complex signaling." You have to actually show it, or at least propose how it's possible. How does a bug know that another bug is a cheater? How does it order the other bug to die without killing itself? Why do we never see that system go haywire?

Yes, of course there's something that make biofilms persist, that much is obvious to anyone with a few neurons to rub together. We just haven't found out what it is yet.

Just saying "It's there, because it has to be, QED we're done" shows a profound lack of curiosity. Saying "it has to be signaling" is closed minded; we don't know it's signaling; it could be anything. It's unexpected and remains unexplained by current theory. That makes it infinitely cool.

The most exciting phrase to hear in science, the one that heralds new discoveries, is not "Eureka!" but "That's funny ..."
Isaac Asimov

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

This is what drives me nuts about science (none / 0) (#41)
by localroger on Sat Nov 07, 2009 at 08:13:55 PM EST

Look, I was trained in this. Here is your error:
They have one form of signaling, but you can't just say "so they obviously have this other kind of highly complex signaling."
YES I CAN. I can say it because the behavior they demonstrate is impossible without such complex signalling. So while educated people like you go around wondering how oh how do these organisms do it, I am saying THEY HAVE COMPLEX SIGNALLING LOOK FOR IT. It is demonstrably not a question of whether such signalling exists, it is only a question of HOW IT IS DONE. SO GO LOOK FOR IT OK?

And that is what is so great about the internet. It enables pompous blowhards to connect with other pompous blowhards in a vast circle jerk of pomposity. -- Bill Maher
[ Parent ]
You're making unwarranted assumptions (none / 0) (#43)
by Sgt York on Mon Nov 09, 2009 at 10:43:44 AM EST

And no, you have not been trained to look for this sort of thing. You are not a scientist, and you know as much about biology as I do about programming. That you you are incapable of thinking like a scientist has been made very apparent to me in our past discussions. I have seen you get duped by the most transparent of charlatans, and you are a sucker for confirmation bias. You latch onto the first idea you have and then pursue it. If you can't disprove it, you take it as truth. You don't even bother looking for alternatives, because damn it, you found something that works. You don't want to be confused by alternate theories. And the really sad thing is, you think that's doing science. I think you honestly believe that.

You assume that this behavior is impossible without complex signaling; you do this out of hand without any thought to the matter. If you knew a damn thing about what you are talking about, you would know that diffusion effects are a very likely culprit in the matter. And that is far from being any kind of complex signaling pathway.

Do you even know what the requirements of such a signaling system would be? Have you given a moment's thought to that? What other things would you expect to see if such a system existed? Complex signaling systems + high mutation rate = fucked up signaling. So where are the side effects? Or do you propose that somehow prokaryotes have evolved the world's first mutation proof complex signaling system?

Yes, it is possible that something like this exists. But it is not a necessity. It can be something simple, like the recently proposed precursor diffusion limitation theory. Or it could be something intermediate, like the (unproven) idea of "uncommon common goods," in which bugs make new scaffolding and common-good type products, but hang on to them in a unique way. Or, it could be a combination of things, each common good being regulated by a different mechanism, with some being unregulated. If you had any experience with biological systems at all, you would immediately recognize that last possibility as the most likely scenario.

But you don't give a shit about that, because you found something that you think should work, and damn it, all those edumacated people with their fancy degrees and experience don't know what they're talking about.

Shit, man, do you actually think no one has looked for complex signaling systems? Do you really have that low of an opinion of the rest of the world?

Moron.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Yes (none / 0) (#45)
by localroger on Mon Nov 09, 2009 at 09:58:04 PM EST

Do you really have that low of an opinion of the rest of the world?

Two words: Plate tectonics. You are a very smart person and I know I don't have to explain that to you.

In this post you throw out a bunch of mechanisms as alternatives to the idea of complex signalling. OK, maybe I got the language wrong but to me if any of those ideas had panned out, I would call that "complex signalling." I don't know what your criterion is for that, or your field's. Mine is that if you have a mechanism involving more than a few bits of information that get transmitted and a result gets processed, well signalling happened and if it's more than "high PH, die" that's considered complex when you're talking about bacteria.

Maybe this is the problem (as I in my personally infinite wisdom [/snark] see) in your field. More likely it is the general hacked fuckup that is the genetic code in all organisms. I have spent enough time reverse engineering the hack jobs engineered by actual humans that speak my language that I don't envy you at all for trying to hack the Great Randomizer that is God.

I will admit I have only a bare understanding of your field -- but more than you realize, because I really was trained as a scientist, though it was many years ago and as a physicist. And in those days there was an emerging idea, then considered radical, of a fusion between the fields of biology and physics. Before my personal life imploded I was aiming for that point. So I have been exposed to more than you might realize, though it is also probably more obsolete than I like to think.

The thing is, this behavior can be figured out. If it's as simple as you think it is then it's a matter of reverse engineering the fractal algorithm that creates it. By definition there are a very limited number of those that are possible and if nothing else we can use computers to iterate them. It's a doable thing. Someone should be thinking of ways to work on it.

And if it's not so simple, as I have assumed, then people should be looking for other information transfer mechanisms. Have you considered proteins? There are some who think that before DNA there was basically protein genetics where the disadvantage is that the gene is always "on" because it's "there." Maybe that's a nonstarter for reasons you can dismiss in three sentences. But I'm just tossing 'em out here. Maybe you know that such protein transfers aren't possible in a biofilm. Go back to the last paragraph.

I do not think you are a moron, nor that anything you are doing is unworthwhile. But I think that the cry for help you posted here in the form of the biofilm article -- oh, it wasn't a cry for help, what was it then? -- reflects the fact that you are on the wrong side of a plate tectonics like thing. Maybe you should look at the history of the plate tectonics thing before replying to this. Because it's not a slam in any way, it's meant as a helpful suggestion. And if you run with it you might really get the Nobel Prize.

There is no chance that I will, because I don't have the skills for it.

And that is what is so great about the internet. It enables pompous blowhards to connect with other pompous blowhards in a vast circle jerk of pomposity. -- Bill Maher
[ Parent ]

Biofilms (none / 0) (#46)
by Sgt York on Mon Nov 09, 2009 at 11:33:30 PM EST

The mechanisms I listed would not be considered complex signaling in molecular biology, not by a long shot. Both of the processes I mentioned involve states; the way the biofilm is formed stacks the deck against cheaters.

The genetic code is not a hacked fuckup, especially in something as streamlined as a prokaryote. It's a common misconception, even in biology classrooms. But the genome itself is elegant, and the way the parts interact is a thing of beauty. It's incredibly complex, headache-inducing when you try to understand even a part of it, but in no way is it a hacked together fuckup. The way it comes about is pretty fucked up, but the result, oddly enough, is downright elegant.

Of course the behavior can be figured out. But not if we insist on saying things like "It MUST be this, because." You quickly learn in biology that you should never limit yourself without cause; biology uses random chance as its raw material. That allows for a lot of innovation.

Your attitude of algorithmic deconstruction of biologic phenomena is a common one among physicists. I work with a bunch of physics types as part of a collaborative effort to generate a computer model of lung function. They love to reverse engineer everything in biologic systems, and it always seems like a great idea...unfortunately, I have never seen it work very well. And this is by top-notch physicists and mathematicians. For example, according to the best data and math models we have, it is impossible for bronchial epithelium to maintain a fixed level of fluid on its surface....but whenever I grow culture it hits 7 microns and regulates. Even after I put an extra centimeter or two of depth on top of it. It's back to 7 microns within 24h. How? We have some ideas, but the modeling guys don't like them. We can block certain signaling pathways and block or even hijack the regulation in cell culture. But when you take it back to the math models and do the same thing to the formulas, it doesn't work.

What it boils down to is that although the combinations may be finite, we have no idea what the limits, or even the players are. Just bashing away at it doesn't generally work because we don't know all the variables, and we don't know how they relate to each other.

Lastly, biofilms are not my field of study, I work in inflammation and chronic remodeling. It's merely something I come across from time to time as a mechanism. How they form, how they maintain, etc is currently outside my field of professional interest. It's an interesting side issue, and I follow the literature on it like other people follow baseball or fashion. This article not a request for assistance, I was just sharing one of the interesting things I have come across.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Thanks (none / 0) (#47)
by localroger on Tue Nov 10, 2009 at 10:18:06 PM EST

I know I come off as a blowhard at times, and to someone like you probably an ignorant one. That's a hard thing to adjust to becuase IRL I'm usually the genius in the room. The perspective is useful.

Your attitude of algorithmic deconstruction of biologic phenomena is a common one among physicists.

Well I doubt the way I would do it is common among them -- I'm the son of a physicist, not a physicist myself, and after 20 years of field programming and service work I don't see the world quite the same way my Dad did.

They love to reverse engineer everything in biologic systems, and it always seems like a great idea...unfortunately, I have never seen it work very well.

This surprises me not at all. That's almost something you need an actual mathematician for, as opposed to a physicist or biologist. Funny aside, in the original printing of Jurassic Park Crichton headed each chapter with a fractal diagram intended to show how complex things emerge in ways you can't reverse engineer. I reverse engineered the fractal algorithm he used to make the images.

Of course while it was very hard it was a lot simpler than the genome, but the principle stands; it can theoretically be done.

For example, according to the best data and math models we have, it is impossible for bronchial epithelium to maintain a fixed level of fluid on its surface....but whenever I grow culture it hits 7 microns and regulates.

Look, I hate to get you PO'ed again but this is kind of the same thing. You're looking at a structure that has had four billion years to organize itself, and people are are saying "We can't make the math work right to describe how it works" because they're applying some single-parameter schema out of Dynamics 101. These systems have multiple, interlocking communication mechanisms. How old are the estrogen and NO pathways? How many other pathways are in there that didn't make it to the eukaryocytes? There could be LOTS of them. I'm sure the modeling guys don't like this idea because it means they are trying to model an actual national railroad with software more geared to a model layout.

There is lots of DNA out there, and when I described it as a hack I wasn't really being derogotory; it's more like the spaghetti code written by a brilliant but untutored person like me who does not care at all whether anyone ever understands it. It overlays totally unrelated stuff that happens to be similar enough for reuse to such an extent that the human genome project people have been astonished. Certain things have to be exactly right and others are subject to drift. An the fact that any of it works at all seems like a miracle when you're trying to figure out the object code.

But it has happened over and over again that the appearance of such complexity has indicated a simplifying influence that was not understood. There is clearly one, and likely many more than one, waiting to be discovered in your field.

And that is what is so great about the internet. It enables pompous blowhards to connect with other pompous blowhards in a vast circle jerk of pomposity. -- Bill Maher
[ Parent ]

Your arrogance is showing again (none / 0) (#48)
by Sgt York on Wed Nov 11, 2009 at 10:32:03 AM EST

And it is not very flattering.

These are not guys fresh out of Dynamics 101. These are the guys that turned down the offers to write the texts used for Dynamics 101. Bear in mind that I work at a major university, known historically for its math & physics programs. I'm the lowest of the low among these meetings; I consider it an honor to even sit at the table.

They are bona fide geniuses, top of the field. There are at least two guys in there that will in all likelihood have either a Nobel or a Fields at some point in the next 10-20 years. All of them are top notch; physicists, mathematicians, chemists, computer scientists, physiologists, molecular biologists, microbiologists. The idea that you have some Lamarckian math and physics ability that is superior to them is not even laughable in its absurdity. I have a doctorate and a few decades of experience in conducting original molecular biology research. I am considered an expert in certain aspects of molecular biology and biochemistry. And these guys would still be my intellectual betters if they were lobotomized.

You seriously compare reverse engineering a fractal used as a prop in a novel to reverse engineering physiology? You don't have any idea what you are talking about. We don't even know all the players yet, much less know all the rules of how they interact. We're still discovering whole new classes of regulation. Not new regulators, but whole new regulation systems, classes of regulation that we had no clue existed just 10 years ago. And this is the stuff several layers below the effects in the article.

You could reverse engineer that fractal because you knew the basic rules of mathematics. The specifics of that fractal were not known to you, but you knew the rules that govern fractals in general. When it comes to biology, we don't know all the rules. We don't even know all the players.

That's what the math models are good for, though. Finding the holes. The problem is that the math & physics types think they can do it all in silico, and when the wetlab numbers don't match their predictions, they get frustrated. Bio types have the opposite problem; we think we can find all the holes in the wetlab, but we can't. The systems are son complex that we need the math models to point out the holes.

The problem is twofold, but neither aspect has anything to do with the math types we work with being blind fools. One is that the math types and the bio types don't speak the same language. We need to improve our math knowledge, and they need to improve their bio knowledge. The other is that we both still have this mindset that the "other side" exists to support us. But that's only half true. We are each other's flashlight, and the sooner we figure out how to do that effectively, the sooner we'll get results.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Let me try this again (none / 0) (#49)
by localroger on Thu Nov 12, 2009 at 08:11:12 PM EST

Look, I was not in any way trying to suggest that my little hack of Michael Crichton's line replacement fractal was comparable to what you and your colleagues are doing. It was meant to suggest a similarity of methodology, much the way one might compare the matchbox tic-tac-toe playing machine to an actual computer, to illustrate a principle.

You yourself have said in this response some of the very things I've been trying to say; you've just used language you like better.

The problem is that the math & physics types think they can do it all in silico, and when the wetlab numbers don't match their predictions, they get frustrated.
I'd call that a major problem, and you said it, not me. The problem is that what physicists do is they attempt to simplify things. This is written into the DNA of physicists the way rhythm is written into musicians, and if you don't think that way physics makes no sense at all. What physicists do is make models, apply them, and look for the model to match reality, with the idea that as the model gets better it will come closer and closer to reality.

But that doesn't work for biology because biology is a fractal system and the math of fractals doesn't work that way. You can only get so far trying to describe living things (even very simple living things) in terms of physics, just as you can only get so far describing a computer system; ultimately you run up against the fact that both systems contain a lot of information and that information becomes more important than the physical system. Physicists really don't have a mechanism for dealing with that. So their basic methods are failing, and instead of taking the lesson a relatively untutored person like me would take -- that maybe their methods are not appropriate to all the parts of this problem -- they get frustrated, which is a totally useless response.

I am going to arrogantly tell you what your problem is right here: Living things contain a lot of information. Trying to understand what a living thing is doing on the basis of physics is like trying to understand what a computer is doing based on physics, and for exactly the same reason. This information content is what confounds you on the biological side because it's the cause of those "holes" you can't explain, and it's what confounds the physicists because it prevents them from simplifying the system because a system can't be simplified if it contains a complex information state.

Going back to the toplevel article, what got me started on this is this fundamental error: Your brilliant math guys are assuming that the bacteria do not possess a complex internal state. (I may have been mistaken about missed communication channels, but I doubt that; I am definitely not mistaken about this.) In all the angst over why biofilms don't get cancer it has apparently never occurred to anybody that after a few billion years, evolution has created a stateful mechanism that the bacterium itself might use to figure out it is supposed to be a contributing member of a biofilm, it's not contributing, and so it's time to commit seppuku for the greater good. Just as eukaryotic cells do, unless they manage to get broken in one of the ways the that cause us cancer.

This thing about information is the fundamental lesson of fractals, which remember weren't even known until the 1970's and were only barely understood until the early 1990's and continue to hold big question marks for the math types even today. Fractals allow simple systems to create complexity because they contain information -- it requires memory and processing to create one. And when you apply that memory and processing even a relatively simple algorithm can create complexity that blows up on you and becomes all but impossible to predict. Physics can't deal with that, at least by its normal methods. When you make a small change to a fractal system you do not get something a little closer or a little further from your target; you get something totally and completely and often unexpectedly different. The usual hill-climbing algorithms simply do not work.

This does not mean your colleagues are blind fools. It does mean they are missing something, something important, and they stand with many great men of science in missing important things for a long time. I would say this in their defence, but I would also say of anybody of any skill level in any field that if they are getting frustrated at their methods not giving expected results, then they are missing something, and getting frustrated does not help. You have to step out of the box and look for the thing you are missing.

Look at all the years Kepler spent trying to stuff the Solar System into Pythagorean solid spherepoints. Fortunately, he kept seeing he was wrong until he got it right. Look at all the time Einstein wasted pursuing a non-quantum grand unified theory. Unfortunately, he never did accept quantum theory and so it never went anywhere. Look at all the energy poured into string theory today, which most of the top people in the field will admit if you get a couple of beers in them is totally unverifiable and useless and likely wrong besides.

So to take a stab at your final paragraph, let me say your problem is not communications between the math and bio types; it is that neither the math nor the bio types are approaching the problem in a way that can solve it. Attempting to reduce the behavior of organisms that contain large amounts of information to equations, even very fancy equations, is doomed to fail. You truly need a new and third approach. But given the complexity of the problem you are facing, you will probably need a lot of computational help to get there, possibly at a scale that doesn't exist yet.

And that is what is so great about the internet. It enables pompous blowhards to connect with other pompous blowhards in a vast circle jerk of pomposity. -- Bill Maher
[ Parent ]

Weeping sore. (none / 0) (#33)
by Ward57 on Mon Oct 12, 2009 at 09:28:11 AM EST

Any successfull more efficient self replication would likely dominate a small local area of the biofilm, which would degrade faster than other areas. Which wouldn't work if biofilms were too stable.

future generations (none / 0) (#34)
by dark ally on Tue Oct 13, 2009 at 03:04:46 PM EST

IANAB

Let me see if I got this:

  1. A bacterium mutates so it no longer produces part of the biofilm.
  2. From the colony's perspective, the loss of one worker is insignificant - the remaining members of the colony produce enough to make up the loss.
  3. However, the mutant now has a slight advantage and may out-reproduce a normal bacterium.
  4. So why don't the mutants eventually outnumber the normals - why does biofilm exist?

I think the answer is at the other end.  Imagine enough of these mutant bacteria exits to spawn off a fruiting body and release the mobile (mutant) bacterium into the environment.  However, when these mobile mutants settle down and try to form a colony, they are at a significant disadvantage because they don't form the biofilm, or form a lesser one.  Then the environment does its best to wipe out the mutants.

I suspect biofilms are actually a result of earlier mutations.  Say you have a colony of bacteria which doesn't form a biofilm.  It's probably in a less hostile environment as well.  A mutation happens which causes the mutant to emit a proto-QS molecule.  Nothing happens because it diffuses.  But that mutant reproduces - creating a mutant sub-colony.  The proto-QS no longer diffuses faster than it is produced, and causes the colony to produce a proto-biofilm.  The proto-biofilm makes it easier for the mutant sub-colony to survive even though it's spending energy & atoms making the proto-QS and proto-biofilm.

Meanwhile, other organisms in the environment are also adapting - consuming the colonies which don't have the biofilm and adapting to those which do.  Meanwhile, the colonies are continuing to mutate - refining the proto-QS and the proto-biofilm through an endless game of random chance and survival of the fittest.


The problem is that (none / 0) (#36)
by Sgt York on Tue Oct 20, 2009 at 12:33:58 PM EST

the mutants won't leave the biofilm. They'll stick to it and stay in it; they wouldn't, they'll stay with the biofilm. Why would they leave?

Think of it this oversimplified way: Each bug has a little clamp that lets it grab onto a big scaffold. Each bug also makes scaffold. Each bug grows.

One bug decides to not make scaffold anymore, but instead focus on just growing. Because of its reduced workload, it now can grow faster; and each of its offspring also make no scaffold.

But they never stopped making clamps. they can still grab onto the scaffold made by everyone else, but that population is now growing faster. Eventually, there won't be enough bugs that make scaffold to support the deadbeats, and whenever a new bug pops up, it won't have any scaffold to clamp on to.

But this system won't stay for long; life is just a limited reprieve from entropy. The scaffold will start to break down, and the film will fall apart.

This is what is predicted, but it is not what is observed. Therefore, something in our course of events there is off. The facts are correct, the outcome is not. Therefore, there is something we dn't know about or haven't fully accounted for.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Nobel Prize, here we come! (none / 0) (#39)
by meijer on Wed Oct 21, 2009 at 08:32:07 PM EST

Do you believe that the mysterious mechanism that makes biofilms work, also contributes to us not getting cancer most of the time?

(As far as I know, even people with very weak immune systems often DON'T get cancer...)

No (none / 1) (#40)
by Sgt York on Thu Oct 22, 2009 at 10:20:23 AM EST

But is may give us hints as to how our immune systems evolved. This may in turn give us insights into how the finer points of how our immune systems work, and how to better model its behavior.

This may eventually give us insights to cancer, but that's really just a very small part of it all.

We're talking about first appearance here. Is it possible that the predecessor to the immune system existed before there was even true multicellular life on Earth? How freakin' cool is that?

And I wouldn't call it mysterious....it's just not understood.

There is a reason for everything. Sometimes, that reason just sucks.
[ Parent ]

Biofilms | 49 comments (39 topical, 10 editorial, 0 hidden)
Display: Sort:

kuro5hin.org

[XML]
All trademarks and copyrights on this page are owned by their respective companies. The Rest 2000 - Present Kuro5hin.org Inc.
See our legalese page for copyright policies. Please also read our Privacy Policy.
Kuro5hin.org is powered by Free Software, including Apache, Perl, and Linux, The Scoop Engine that runs this site is freely available, under the terms of the GPL.
Need some help? Email help@kuro5hin.org.
My heart's the long stairs.

Powered by Scoop create account | help/FAQ | mission | links | search | IRC | YOU choose the stories!