How The Animal Lost Its Sensor

Two-Component Systems are one of the major sensory systems used by bacteria to detect and respond to changes in both their outside environment, and their internal state. I cover them in more detail here, but just in summary they consist of two proteins,a sensor and a responder. The sensor senses the change, and activates the responder, which binds to the bacterial DNA and leads the production of a protein that will enact a suitable response.

Although Two-Component Systems (TCS) are found in all three superkingdoms of life (Archaea, Bacteria and Eukaryotes) they are suspiciously absent from the animal kingdom. Plants have them, as do fungi and several protazoa, but they just aren't present in animals. For this reason they've been looked into as potential antibiotic targets as knocking out the Two-Component Systems of most bacteria is fatal.

Why don't animals use TCSs? To answer this you have to start looking at the evolution of the system itself, because despite being nominally present in eukaryotes such as plants and fungi, TCSs are used very differently in bacteria and archaea. Bacteria use TCSs for sensing a wide variety of signals; stress, metabolism, nutrient regulation, chemotaxis, pathogen-host interactions etc. in eukaryotes on the other hand, they are used sparingly, for ethylene responses and photosensitivity in plants and osmoregulation in fungi and slime moulds.

Bacteria (especially soil bacteria which have a lot of environment to sense) can contain up to 50 TCSs although many internal parasite bacteria (with a lot less to sense) contain far less. The maximum for Archaea is around 20 TCSs. Eukaryote number drop right down, with only one in the yeast Saccharomyces cerevisiae (one sensor kinase and three response regulators). None have yet been found in any animal genomes, or in the few partial protist genomes sequences (although I doubt if anyone's had a complete scan through the protist genomes for them).

Comparing the TCSs of Bacteria, Archaea and Eukaryotes leads to the interesting conclusion that the bacterial and eukaryotic systems are far more closely related than the archaeal, and in fact are thought to be monophyletic (all evolved from a single common ancestor). In contrast, the archaeal TCSs appear to be polyphyletic and some archaea lack TCSs entirely. It's therefore thought that TCSs originated in bacteria and spread by horizontal gene transfer to both archaea and eukaryotes (until the eukaryotes developed a nuclear membrane). In eukaryotes very little further diversification took place, whereas the bacterial TCSs diversified widely, and occasionally passed new systems back to the archaea. I've tried to show this in the diagram below:

Diagram made by Lab Rat. Red arrows show the movement (straight arrows) and duplication (curved arrows) of TCS genes. No horizontal gene transfer can take place in eukaryotes after the nuclear membrane (well....it *can* do but very, very rare) although gene duplication may still have occurred.

The eukaryotic kingdom appears not to have contained very many of these TCS genes to start with, and the animal kingdom may just have lost the very few it possessed. This makes sense from the point of view of cellular control because while TCSs are very useful in the small genomed and non-nuclear membrane containing bacteria, it's less clear how useful they are in eukaryotes as a whole. Introducing a membrane around the nucleus makes it harder for proteins to get in and bind to the DNA, and introducing systems of membranes inside a far bigger cell makes it harder for a simple two-component system to sense what's going on. Added to which, cells inside a multicellular organism don't really need to sense what's going on, they get told what's going on by the surrounding cells and circulating hormones.

Whatever the reason though (and any other ideas would be welcomed, the above paragraph is mostly speculation) it is clear that despite this system being vital for bacteria it isn't used widely, or most likely at all, in animals. Research into this would be particularly useful against opportunistic pathogens which tend to have a large selection of two-component systems to allow them to adapt to different lifestyles depending on the conditions of their immediate environment.

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Kristin K. Koretke , Andrei N. Lupas , Patrick V. Warren , Martin Rosenberg , and James R. Brown (2000). Evolution of Two-Component Signal Transduction Mol Biol Evol, 17, 1956-1970

Wolanin PM, Thomason PA, & Stock JB (2002). Histidine protein kinases: key signal transducers outside the animal kingdom. Genome biology, 3 (10) PMID: 12372152

The Impact of Impact!

I went down to London for the weekend to see an exhibition by the Royal College of Art entitled "Impact!" which was a colaboration between designers and research teams to explore the potential impacts and implications of future scientific research. I always like watching when the worlds of art and science collide, and it was a good excuse to get away from my dissertation for a while.

I've done some work with designers before (during my last summer project, I wrote about it here) and I loved it. Designers bring new ways of looking at a project; they have the ability to take science out of the lab and into the real world, while still addressing social and ethical concerns. What I saw at Impact! was the ultimate in science communication and to be honest I think it showed the reasons most people get into science in the first place. It was fun, slightly geeky (five dimensional cameras!) colourful, thought-provoking and all with a wonderful overtone of sci-fi.

The project I enjoyed most (probably because I've met the designer, and saw little sneak-peaks of of it being constructed) was "Cellularity" by James King. This explored the potential of using cell-like structures to deliver pharmaceutical products into a patient, structures that over time, and years of research became so cell-like that they begin to blur the devide between life and non-life, bringing up fundamental questions abut what life even is.

Cellularity from James King on Vimeo.

Start by considering an empty cell filled with drugs and swallowed, like a tablet. Inside the body the membrane dissolves and and drug is released, similar to chemical pills. Clearly the 'cell' (if it can even be called that) is dead. Move on, design a cell which can both produce the drug itself (from a small DNA coil inside it) and replicate itself. Is that alive - or is it merely a biological drug-dispenser?

Next stage...suggested for patients who respond to no current therapy, allow the little drug-making cells to breed within the pateint, replicating in a semi-asexual manner, so that each offspring is producing a different drug. While James indroduces 'death' as a later stage in the line denoting life from non-life I think that for pure health and safety reasons it should probably slot in here. Cells that produce drugs that could potentially harm the patient must be able to die, either by self-destruction or (as James suggests) signalling to the bodys immune system to come and take them away.

If you start giving these cells the power to sense their surroundings as well (maybe to predict the best drug to produce) you get very close to something that can be called life. It's artificial life, life designed exclusively to serve the humans that use it, but life non-the-less. At this stage, it becomes almost meaningless to talk about 'life' and 'non-life' as separate boxes, and instead they become a gradiant, a sliding scale between the living and the dead. This is something that is starting to be appreciated even now when considering things like virus's, or prions. A prion is an infectious protein element, with no DNA or cell wall yet it is capible of replicating and evolving (and consequently sticking two fingers up to Dawkins a bit). If a small piece of twisted protein has a passing claim to 'life' the definition of what life actually is starts to become somewhat hazy. And scientists have made virus's in the lab, creating what could potentially be classified as living organisms from 'dead' pieces of DNA and protein.

One thing that worries me though, how many scientists went to Impact!? I'm sure plenty of designers did, and I'm sure they got a lot out of it, I certainly did. But this is really something I think more scientists should get engaged with. Designers are fun to work with, and they're good at communication especially to a general audience. They make colourful posters, and five-dimentional photography machines, and wierd spiky machines that hang from the ceiling. They bring the excitment back into biology, they remind you why you went there in the first place.

Also I really, really want a five-dimensional camera...

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Where my bio began...

Sigma is doing a promotion/competition thing at the moment called "Where bio begins" which seems quite interesting. The aim is to get scientists to show, through a variety of media, what sparked their interest in biology, why they decided to study it and why they find it so fascinating. I'm not sure whether I'll enter for the competition (they need my real name unfortunately) but it's a great idea and it sort of got stuck in my head. Why am I doing biology, and where did bio begin for me?

There are a lot of answers to that. Something about my childhood probably, about how I found everything exciting and was encouraged to explore a lot. Something about my schooling, which was quite science oriented. Something about my personality and the traits which allow me to love lab-work, even when it hates me in return, and probably something about science itself, which is so amazing and beautiful.

I think the biggest moment for me though was when I first saw a picture of the inside of a cell, containing all the details of the organelles. I was sixteen at the time, and cells up until then had just been drawn as blobs, with maybe a little blob inside labelled 'nucleus'. To suddenly see the whole crowded, busy and breathtakingly complex cellular interior was a bit of a revelation because basically what my mind saw was something like this:
It was another world in there. And I've been hooked on that world ever since.

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Map is (c) Me. It was fun to draw and I am very proud of the trees and the boat. My favourite bit is the dragon sitting on top of the cell, because it's a dragon sitting on top of a cell.

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Reflections on Lab Work

I've just come to the end of my project for this year. Last week was the usual flurry of tidying, organising the freezer, and making sure all my stuff is on the Lab Computer so that anyone else who wants it can use it. I tried very hard to feel some sort of nostalgia about leaving, but to be honest all I managed was a sense of supreme exhaustion and slight relief. Besides, I'll probably be nipping in an out of there to collect results pictures, protocols, etc. for my write-up.

So I thought I'd just do a quick run-through, for anyone who's interested, about my lab experiences. I've done four lab projects to date, and enjoyed every one of them. I can't even say which one I enjoyed the most, they're all special in different ways.

1) My First Project Ever was working on bacteriophages (viruses that infect bacteria). It was fairly heavily supervised, I was working with a PhD student and helping her on her project. It was probably the most fun I've ever had in a lab; there was no pressure for a big write-up (just a small report for the funding body) or excessive pressure for me to get results, and I felt very useful being able to help. It was also the first time I'd been paid for lab work, and got that amazing rush of "woah....people will give me actual money for this!"

I started this blog during that project. The entries from back then make me laugh now. :)

2) Second project was for my course, where I switched to working on Streptomyces. Again, I was working quite closely with a PhD student, but she had kids and used to have to leave the lab about midday to take care of them. By the time the first week was up we'd worked out a system; in the morning we'd go over all the work I needed to get done, and in the afternoon she'd leave and I'd get on with it. It was quite a nice step up, as I was still being supervised, but I was doing the work all by myself.

The blog kind of died in that period, because it was insanely busy and I hadn't found out about research blogging yet.

3) The third project was the synthetic biology one, which wins the award for Most Stressful Project so far. As it was meant to be student-based we (there were seven of us, only four of whom were biological scientists) were pretty much dumped in the lab and left to get on with it. We had meetings with the supervisors once a week, and we had a PhD student to help us out (for which we were incredibly grateful) but other than that it was all down to us.

Considering I was the only one in the lab who'd ever done a project before it's amazing we managed anything really! I've got a lot of blog posts from that time, mostly because I spent about four weeks in the middle of it failing to make two point mutations. This involved lots of waiting around for gels to run, PCRs to happen, and ligations to fail, during which I would happily type away at my computer. Also at some point around then I discovered researchblogging.org and completely took off with the research side of things.

4) The project I'm currently writing up. This one has been the first project I've really thought of as my own (which makes the lack of conclusive results a bit harder to take). The lab is quite small, and as the PI and post-doc were quite busy last term, I was organising experiments and procedures pretty much on my own. I'm looking forward to the write-up, especially now I've drafted it and discovered that I actually do enough to write up. I feel more personally attached to this project than any one I've ever done before. The knowledge that my little samples are going to be sitting in the freezer for a long while before anyone bothers to dig them out actually does hurt a little. The next three weeks of write-up are going to be fun though :)

I'm quite surprised and proud that I've managed to keep up regular blogging throughout the course of the project.

5) I've already organised a summer project! As usual, there will probably be only the vaguest details of what I'm actually doing on the blog, especially as this project really should lead to a paper. But stay tuned for more bacteria-related posts. I'm sort of hooked on blogging now, and it'll probably take a lot to get me to stop.

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Norovirus and Clinical Research

I've written a lot about bacteria and plants over the last few weeks, so in celebration of the fact that my project is finally on it's way out (with a whimper rather than a bang, unfortunately, but that's how it goes sometimes) I've decided to descend into the world of viruses. I've also decided to have a go at deconstructing some clinical papers, to make a change from academia. The difference between clinical and academic research can probably be described as follows (and I'm pretty sure I've stolen this quote from somewhere else, but can't remember where):

Clinical Researcher: "That's interesting, but it is useful?"

Academic Researcher: "That's useful, but is it interesting?"

[Industrial worker: "That's useful and interesting....can we sell it?"]

Clinical papers therefore have a different slant from academic ones. They tend to be a lot more precise, a lot clearer. Most will include data from actual people rather than just biochemical data, and the real-world applications come across as the main focus of the study, rather than just tacked onto the end for funding purposes. It comes across in presentations as well, I went to the Society for General Microbiology conference last year and you could usually tell within about ten minutes whether you were listening to an academic, industrial or clinical researcher.

But anyway...on to the papers, which deal with Norovirus, a viral infection usually thought of as a mild (if unpleasant) winter vomiting disease. Norovirus is an RNA virus, which means that it consists of a coil of RNA wrapped up inside a spherical protein coat, as shown to the right

(image taken from the Naked Scientists):

The virus enters the body through the mouth, and invades the small intestine, resulting in lesions in the small intestine tissue and an increase in mucus production. This affects the absorption of nutrients, and leads to build-up of food in the stomach (which can't move down into the intestine) which in turn leads to vomiting and diarrhoea and generally feeling unwell.

Norovirus usually peaks during the winter months, probably because during the winter people tend to spend a lot of time stuck in the enclosed areas with other people. However in 2002, this decline during the summer months halted, and incidents of the disease began to increase, particularly in the over-65 age range. This is due to the emergence of a new variant of the virus which has a higher human-to-human transmission and is therefore more likely to spread in areas where people are living in very close proximity, such as nursing homes and hospitals.

As these places tend to be full of people who are not at their most healthy, this can lead to serious complications in the disease. The damage to the intestines can lead to dehydration which in some cases has been severe enough to lead to kidney failure. Diarrhoea and vomiting cause potassium loss, which can lead to problems in the heart. Deaths are thankfully uncommon, but have been reported.

What this means is that norovirus shouldn't really just be thought of a mild disease any more particularly (which one of the main issues with for clinical researchers) when it happens in hospitals. There's no vaccine against it, as multiple strains exists and having norovirus doesn't really give you much of a defence against getting it again. There has been some work done with the capsid proteins (which surround with RNA of the virus) but as yet this is still happening in mice, and I'm not sure whether it's even entered clinical trials. The best current defence against norovirus is still just to keep things as clean as possible, especially in places where many people are in close proximity, to keep very ill patients away from as many other patients as possible, and to tell doctors to stay at home if they wake up vomiting.

One of the great things about Medical science of course, is that it's an ongoing process and we are the data-sets. This summer rolling around (soon, hopefully!) will bring more information about the state of norovirus, whether the trend in increasing virulence is still around, or whether it's starting to die down. Either way it's a good reminder that sometimes all it takes is a particularly busy hospital and a blip in a piece of floating DNA to turn a relatively harmless disease into a much more problematic one.

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Hane Htut Maung (2008). Norovirus Infection: An Underestimated Danger Cambridge Medicine, 22, 22-24

Lopman BA, Reacher M, Gallimore C, Adak GK, Gray JJ, & Brown DW (2003). A summertime peak of "winter vomiting disease": surveillance of noroviruses in England and Wales, 1995 to 2002. BMC public health, 3 PMID: 12659651

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International Women's Day

"What does “equal rights for all” mean to you?"

It means being able to look at any other person in the world and being able to think: Yes...I would be not be significantly more unhappy if I had been born you. I would not be any more ashamed, I would not be any more afraid, I would not be any more unable to be happy. I might have a different lifestyle, different thoughts, different feelings, maybe even a different set of values than I do now, but I would be no less able to enjoy my life.

It means watching what I say, examining what I think, trying to see inside my head from other peoples point of view. If I were you, would I laugh at that joke? If you were me, would you say it? It means making myself listen to other people, listening for clues as to how they see their world, why they act how they do, making myself imagine thinking like that, acting like that.

It means accepting that just because two views are different, doesn't mean that one of them is 'Right' and the other 'Wrong'. Finding more dimensions to see the world in, more colours to paint thoughts with, more ways of being. Not to laugh when someone does something I find odd, but to watch, to understand, and to accept. To be able to feel differently to you, in the knowledge that neither of us considers our point of view superior, just different.

It means being able to look at You, and seing someone that could be Me.

Other participating blogs can be found here.

Redecorating

I'm trying to personalise my blog a little more, so that it doesn't look the same as all the other blogs written by people who like olde-style parchment. I made a bit of a change a few months ago, when I expanded the size of the actual writing area, but unfortunately it left me with a weird dark line down the right hand side of the page that I would love to get rid of.

I got the new background from squidfingers which has a lot of really great tilling patterns in case anyone else was thinking of doing some blog redecorating. It's pretty similar to the old one, but let me know if the repeating little white lines make anyone's eyes go funny!

So there might be some changes over the rest of the week. I'm really looking for a new parchment-style background that I can fit under my words (without the dark line - I got that by just repeating my current template sideways), preferably with some kind of ornamental line under the "Lab Rat" title. I'm also considering a wholesale move to black and white. It might be cleaner, but I think I would miss the gentleness of the browns. Besides, my partner once told me that if I was a colour I would be brown. (I'm still trying to work out if that's a complement - I've always seen myself more as yellow, which I guess isn't that different...).

What I think would be great is a little picture in the top right hand corner (opposite the "Lab Rat" title) but I have no idea how to do that, and I don't quite have a suitable picture. I'm tempted to push a little more of the money I don't have towards Alice, to get an awesome spiky-haired rat in a lab coat. Maybe once I get a job.

Anyway. There probably won't be another science post this week, because I've only technically got one week of my project left *FLAILPANIC*. Times like this you really wish the ligations had worked.

If you miss the science, head over to researchblogging.org. We've had a sudden rush of new people joining due to the Awards getting publicised!

Evolving Molecular Machines: The Plant Edition

Over at Thoughtomics, Lucas has a post up about the evolution of mitochondrial import systems. He starts by going back in time two billion years:

"Life was well underway at the time, with proto-bacteria already populating the oceans for over hundreds of millions of years. One of the cells alive at the time, swallowed an alpha-proteobacterium. Something remarkable happened: the alpha-proteobacterium did not die but survived in the host cell. Over time, the host and symbiont became to be dependent on each other." That symbiont became a mitochondria.

He gets massive brownie point for writing 'proto-bacteria' rather than bacteria, and it is a very remarkable event to have happened. However from the point of view of a plant, it's only half the story, because plants carry two endosymbionts within them: the mitochondria and the chloroplast.

Their stories are remarkably similar. After becoming engulfed by the surrounding cell, two major things happened to them: First (and it had to be first otherwise major problems would have arisen!) a protein import mechanism arose, creating more communication between the symbiont and the host and allowing things to pass between them. Second, the symbiont lost bits of its genome, transferring them into the nucleus of the surrounding cell to create the cooperative arrangement seen today:

Picture above from the amazing science illustration gallery by California state University. Nucleus is purple, chloroplasts are green, and the mitochondria are orange.

Lucas's post covered the evolution of the import mechanism for the mitochondria. I'm going to write about the same thing, but for chloroplasts. After all, the plants already have mitochondria so they can't use exactly the same import process, they have to be able to differentiate between the two.

Like mitochondria, the chloroplasts are surrounded by two membranes, and outer membrane and an inner membrane. Two transporters are therefore required to get proteins across. In the mitochondria these are called TOM and TIM (Transport of Outer, and Inner Membrane respectively) and in the chloroplasts they are called TOC and TIC, just to keep things simple (Transport of Outer and Inner Chloroplast membrane). They look fairly similar to TIM and TOM, but recognise different sequences attached to the proteins. While the mitochondrial transport machines recognise sequences that contain a lot of the amino acid arginine and form a specific helical shape, the chloroplast machines (TOC and TIC) recognise sequences rich in serine and proline:

TOC and TIC. The proteins of the TOC machine are coloured green, and the TIC machine proteins are all the rest. Diagram from here.

One of the questions that Lucas asks in his post is: where did all of these proteins come from? After all, before you have an endosymbiont, you don't need any kind of apparatus to transport proteins into them. Once you start looking closer at the transport machinery it starts looking suspiciously like a rather rushed and last minute job. Different proteins with different functions have been cobbled together, and while there's still a bit of a debate as to whether these proteins came from the surrounding cell or the endosymbiont I suspect that it may be a bit of both. The cell needs to communicate with the little alien inside it, and once the endosymbiont started loosing genes, it needed a way to keep resources coming in.

So how do you make a protein importer? What do you assemble it from? Plants had a slightly easier task with the chloroplasts as they already had a perfectly serviceable TIM/TOM transporter present. Looking at the TIC complex, the first two components to come in contact with the imported protein (TIC22 and 20, shown in the diagram above in dark purple and orange) show homology to components of the TIM machinery; TIM23 and 17 for anyone interested in the detail. However TIC22 also has far stronger homologues in cyanobacteria, which means it is likely to be a protein owned by the chloroplast, similar to the proteins owned by the mitochondria that got roped in to help with protein import.

The TOC proteins (all green in the diagram above) all appear to have no other function in modern plants other than protein transport. Toc 34 is the GTPase, and as there are many GTPases in cells (used to provide energy) it could have arisen from any one of them. The other TOC proteins are involved in membrane and may have arisen from ancestral membrane receptor proteins, while some components, (including TOC64, not shown above) appear to be rather redundant, as the machinery works perfectly well without them.

The research on this is a little sketchy, there are no good solid biochemical means as yet to discover what might of happened somewhere around two billion years ago in order to create a transport mechanism between the cell and it's organelles. There are plenty of different views out there as well, about where the different subunits might have come from. The only thing that seems clear is that like the mitochondrial import system, this was clearly pulled together from bits of old machinery lying around. It had two billion years to get better after all, and reach the efficiency of the modern-day protein import machines.

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Gross J, & Bhattacharya D (2009). Revaluating the evolution of the Toc and Tic protein translocons. Trends in plant science, 14 (1), 13-20 PMID: 19042148