Elife Episode 1: Multicellular life, potato blight and Hepatitis B
How multicellular life began, museum specimens surrender the identity of the bug behind the Irish potato famine, the Hepatitis B and D virus receptor discovered, why fog clouds driver judgement and where nucleosomes came from.
In this episode
01:02 - Origins of Multicellular Life
Origins of Multicellular Life
with Rosie Alegado, UC Berkeley.
How did multicellular life evolve? Ancient ancestors of ours, the choanoflagellates, might give us a clue. Chris Smith spoke with UC Berkeley scientist Rosie Alegado...
Rosie - We study choanoflagellates which are the closest unicellular relatives to animals. They look like little sperms with skirts. They have an ovoid or spherical body with a single flagellum surrounded by a little collar filled with actin. What they do with their little collar is create water currents that cause bacteria to brush up against their collar so that then they could eat them.
There are several different cell types that we can observe in culture when we grow these choanoflagellates. One of the most striking are these little bulbs of cells that we call rosette colonies. The reason why they're very striking is because they look a lot like the morula stage which is one of the very first stages of an animal embryo. Because choanoflagellates are the closest unicellular relatives to animals, we wondered what might regulate their formation and their development.
Chris - So, when you say the morula stage, this is them getting together to form a multicellular or at least an organism with more than one cell linked to it.
Rosie - Correct and I think it's really important to distinguish how they form and they're not formed by individual cells coming together. So, it's not by aggregating together. They form from a single founder cell that after cell division, remains together. That's also very important because that's the same mechanism by which animal development occurs.
Chris - So, do you think then that what this organism is doing lies upstream of how when an egg begins to grow and the cells come from one founder cell to form the trillion or so cells that make a human for example. Is it a similar sort of mechanism here? So, if we understand how it works, it sort of informs how multicellular organisms like us might be doing what they do.
Rosie - So, we think that that's a very provocative possibility and certainly, that may be the case. At this point in time, we don't know. The reason we don't know is because there are about 125 different known species of choanoflagellates, but we don't know if they form colonies in the exact same way. That's important because if they're all the same then that would indicate that the ancestor of all the choanoflagellates also form colonies in the same way. If they did, that would provide stronger evidence that this form of multicellular development might inform us about animal development such as embryonic development.
Chris - So, what triggers them to go into this alternative state where they form cellular derivatives that all remain connected together?
Rosie - It was actually a complete surprise. It was really hard to control the cells in culture. They were very difficult to culture and that was due to the bacteria that was co-isolated with them. And so, there was an undergrad in the lab and his project was to treat the choanoflagellate cell cultures with antibiotics with the hopes that by taming this culture, making it more easy to propagate, it would be easier for genome sequencing. The surprising thing that happened was when we treated them with antibiotics. All of the rosette colonies have disappeared. And so, you can imagine two possibilities. One is that the antibiotics directly affect the choanoflagellates in some way, but the other more tempting hypothesis was that we killed off a bacteria that was important for this developmental transition. And the second possibility turned out to be the case.
Chris - And suppose you could test that hypothesis if you could get the organism to return to that rosette state, having had it lost or taken away if you were to put those bacteria back in.
Rosie - That's right and I have to say, the way that this unfolded was very lucky because most of the bacteria in the world are not easily culturable on plates by lab methods. And so, even if it were a bacteria that caused it, it's not even sure that we would've been able to grow that bacteria. So, we did two things. One was, we took just whole cell environmental bacteria and added it back and that seem to induce a return of the rosette colonies. And so, we kind of had some idea, "Okay, there's something in here. Maybe it's something that's secreted by the bacteria. Maybe it's a metabolite." But it turns out that we were able to culture 64 different environmental isolates from this culture and then add them back individually and only a single environmental isolate was responsible for inducing this transition.
Chris - I'm sorry to interrupt. It wasn't a contact phenomenon then. Could you do it with conditioning media if you grew the bacteria in some media and collect just the media, no bacteria and put that in? Was that sufficient to make them form this rosette?
Rosie - Indeed. It was sufficient for them to make the rosettes. And so, that also gave us the idea that perhaps what this molecule was, might be something that was intrinsic to the bacteria, something that they were making. Not even in response to the choanoflagellates. That led us to think that maybe it was something that was very key to the bacteria's biology such as something that was released unintentionally and that's what turned out to be the key. So, it turned out to be a component of the cell envelope.
Chris - So, why do they form these rosettes in response to this secreted molecule from the bacteria?
Rosie - That is the million dollar question. We don't know why the rosettes form. We do have some choanoflagellates seem to be feeding better when they are organised in this multicellular state as opposed to being a single cell swimming around. And so, that might be one possibility that there's a feeding advantage. Certainly, we don't know why a bacteria would put out a signal that says, "Eat me!" So, what we think might be happening is that the choanoflagellates might just be eavesdropping in on bacteria that are happily growing in. this might actually be a signal to say, "Hey, this is a good patch of food. Stay around here. Divide and make a rosette colony."
07:40 - Potato Blight
with Sophien Kamoun, Sainsbury Laboratory; Hernano Burbano, Max Planck, Tubingen
An international team of researchers from Germany and the UK have made headlines around the world by identifying the bug that caused the Irish potato famine which killed more than a million people in the mid-19th century. Sophien Kamoun heads the Sainsbury Laboratory in Norwich and spoke to Chris Smith...
Sophien - We knew that the pathogen called Phytophthora infestans, a fungus-like organism was the agent of the potato blight that caused so much havoc in the 19th century and essentially triggered the Irish potato famine. What we didn't know is which strain caused the disease at that time. So, what we did is we went back to herbarium specimens from museums, extracted DNA from the specimens and we were able - using the latest DNA technology - to sequence the genome of the pathogen and identify the strain that caused the disease in the 19th century.
Chris - I'm intrigued to think that people kept leaf specimens from affected plants from more than 150 years ago.
Sophien - There's a lot of interesting hidden treasures in all these museums. There's millions of herbarium samples and were stored in museums that are studied usually to identify the species by looking for instance at flowers and the morphology of the leaves and so on. But in this case, we were able to do something quite cool with it, but actually look like the genetic makeup of the organism that were in those leaves.
Chris - So, you ground up some of the samples of leaves and extracted genetic material which would've included both the genetic material of the potato and the genetic material of the blight that killed the plant.
Sophien - Yes, exactly. So, we cut small pieces of the leaf and we're able to analyse both the plant and the pathogen. In this case, in this study, we focused on the pathogen, that was the interesting bit.
Chris - But people have - as you say - known that this was a fungus that was knocking around that did this. What was the big question that needed to be answered here that your research has enabled us to fill in a missing gap with?
Sophien - Well first of all, it's not a fungus. It's a fungus-like organism. So, I'm corrected you. Sorry about that, but it's a different type of microbe. But it does look like a fungus. So often, people refer to it as a fungus. There are many strains of this pathogen. What we discovered was that it's a new strain. We called it HERB-1 that caused the blight in 19th century and this strain apparently is gone. It's not around anymore.
Chris - Why do you think that is? Is it that it was so good at devastating potato plants that as a result, people just stopped growing susceptible species and it ran out of plants to infect?
Sophien - No, we don't think so. What probably happened is that as potato bidding started and took off in the 20th century, scientists starting bidding better potatoes by crossing them to wild relatives of the potato. Probably, HERB-1 was at a disadvantage compared to other strains. We know that in the 20th century, HERB-1 was replaced by another strain we know as US-1. And then later on in the 20th century, US-1 was replaced by additional strains.
Chris - So, is this sort of model then that you have plants that are susceptible to one of these organisms? The organism becomes more successful at working its way through those plants and then the plants change or new types of plant come along which are more resistant and so, the pathogen changes, and we're just seeing a sort of arms race playing out.
Sophien - That's certainly part of the equation, but in fact, what's amazing about this pathogen Phytophthora infestans - the potato blight pathogen - is, how adaptable it is. It's very good at adapting to new resistant varieties that breeders are releasing.
Chris - How does it do that? What makes it so successful?
Sophien - Well, this is actually work we've been describing in the last few years and we discovered that this pathogen has an amazing genome. In fact, we described this genome as a 2-speed genome. It's composed of 2 different types of compartments if you like. One compartment contains the housekeeping genes, the key gene the pathogen needs to be a microbe. The second set of compartments contains all the villains genes that are important for the pathogen to infect plants. That second compartment is evolving and changing much more rapidly than the slow evolving housekeeping compartment if you like.
Chris - Do you know why those bits of the genome change so fast whereas it's elsewhere in the genome don't? How does the organism do that?
Sophien - I wish I knew. That's a very interesting topic we're studying.
15:01 - eLife, a revolution in publishing
eLife, a revolution in publishing
with Fiona Watt, Deputy Editor, eLife
Chris speaks to scientist and eLife Deputy Editor, Fiona Watt, about the free journal and its funding.
Fiona - eLife is all about a publishing revolution. It is a journal which is fully funded by three organisations, the Wellcome Trust, the Howard Hughes Medical Institute, and the Max Planck Society. And by 'fully funded', I mean that at present, none of the authors pay and none of the readers pay for the content. ELife was launched with three main objectives. The first was a feeling that it was important to push the concept of open access further, that the open access movement in a sense had started well but had stalled. Second was that if we published a journal which is purely digital with no paper copy, we could completely revolutionise the way content is explored by the authors and by the referees. And then thirdly, very importantly, we felt there was a need to take science publishing back into the hands of scientists. As I like to say it, we wanted to end the tyranny of that bastard referee 3. So in other words, we wanted to avoid months and months of papers being revised and resubmitted. Young scientists career's are really on the line because of a single referee wanting more and more data.
Chris - Well, let's look at each of these things in term but since you've brought the review process first, I was sitting in a meeting at my institution the other day, and one of the professors of one of the research departments at Cambridge University said it's not uncommon when you send your papers off somewhere that a referee will come back with some suggested additional experiments that basically, they're a year's work.
Fiona - I don't know really at what stage this really began to spin out of control, but certainly, my experience in my lab is that when we submit the paper, we are probably looking at at least 6 months work even if we have favourable reviews.
Chris - So, how will eLife address that?
Fiona - The process of handling papers in eLife is distinctive in a number of ways. All of the people involved in the review process are practicing scientists. So, we're all accountable. Secondly, when a paper is submitted, we line up members of our - if you like - college of referees who are actually paid to be part of the review process, and we have an online discussion about the paper. So, we ask our office to submit a simple pdf and we aim to make a quick decision about whether or not the paper is likely to fly at eLife. And if a decision is made that the paper should go out for review, we've already lined up people who are familiar with the content of the paper and are committed to steering it through the full review process.
Chris - But how does this solve the problem of that nasty referee number 3 who then says, "I want 18 months more experiments to be done before I will consider this adequate for publication"?
Fiona - Well, if your paper has gone out for full review, it will typically be three reviewers. When the reviews come in, we consider do we in principle - want to publish this paper and if so, can the authors fix the bits that need to be fixed in a short timeframe. We would say, ideally 3 months. Instead of regurgitating the reviews in verbatim, we draft a high end summary where we highlight the things that we think are important and doable.
Chris - The second point you made was about not being tied to print. So, how does that benefit eLife and the people who want to send their papers to you?
Fiona - "Not being tied to print" has been a very interesting experience. We could say, "Well, do we really need a two column with format?" If most people are going to be reading the paper on an iPad, how would we want to look at the figures? We're experimenting now with something called lens which means that as you pass the cursor over the text, figures will appear exactly where you want them. Movies for example are fully embedded in the paper so you can play the movies as you're reading the text. It's a process which is still evolving, but we want people to be able to essentially play with the data, interact with it in ways that have not been possible up until now.
Chris - And I suppose the benefit of beginning with something which is built for that purpose rather than taking a traditional print process and trying to turn it into something that has that functionality, it must be easier, more streamlined.
Fiona - Yes, I think that's right. One rather amusing, effective of being purely digital is that when we've had stands at scientific conferences to publish eLife, people are so used to picking up hard copies of the journal that we felt we didn't really have enough beyond a few t-shirts and an iPad. So, we really had to think about better ways to display what we're up to.
Chris - I think if you gave away iPads, that would probably attract quite a curious crowd.
Fiona - I'm sure if you'd like to suggest giving away iPads, that would be fine, but we're going to have them anchored to the stand. But it will be a great chance for people to see what we mean about handling the data in different ways.
Chris - Also, you mentioned that you felt that Open Access, which is obviously a major driver for eLife, you felt that process had stalled. Why do you think that?
Fiona - I was really referring to data from the Wellcome Trust. So, they have been pioneers in the UK of the Open Access movement and they monitor compliance. People who are funded by the Wellcome Trust must publish their work, make it openly available to others. And compliance was rising steadily until a couple of years ago when it seem to sort of level out at about 55%. So, they actually now have introduced the stick as well the carrot to make sure that their grant holders make their work freely available. So, I think eLife gives scientists a platform to publish their very best work, Open Access and completely, at no cost to them. So, I do think that is going to be a boost for Open Access.
22:03 - The receptor for Hepatitis B and D
The receptor for Hepatitis B and D
The liver virus hepatitis B is thought to have infected up to a third of the world's population - 2 billion people, half a billion of them, chronically. Hep B also has a partner in crime called hepatitis D which is the subviral particle that borrows the coat proteins of hepatitis B and quite literally rides on its coat tails. And although there's an excellent vaccine that can prevent infection with both of these agents, scientists still didn't know how the virus was recognising and entering liver cells. And this is important because if we know how it gets in, we can potentially design drugs to block the process, helping people who are already infected. Wenhui Li from the National Institute of Biological Sciences in Beijing has made just that discovery.
Wenhui - We want to find the functional receptor for hepatitis B and hepatitis D virus, which infected a third of the world population, and how it enters our hepatocytes, which is the target cells of that, has been unknown for several decades. We just want to identify the receptor for this virus.
Chris - So, although we know that hepatitis B and its relative hepatitis D get in to liver cells, until now, no one knew exactly how they were doing that.
Wenhui - Yes. So, we've identified very specific bile acid transporter which could function as a functional receptor for this virus.
Chris - So, this is just a chemical which is present on the cells in the liver which the virus is docking onto and using to get in.
Wenhui - Yes. It's a protein mainly expressed in the liver, the protein name is sodium taurocholate cotransporting polypeptide. In short, NTCP and is a multiple transmembrane protein and predominantly expressed in liver. It's in the sinusoidal membrane that is that blood side of the hepatocyte and then it functions as a bile acid transporter and is critical for the enterohepatic circulation of bile acid.
Chris - Can you explain to me how the technique worked? How did you home in on that protein in the membrane in order to prove that this was the receptor that hepatitis B and hepatitis D were using?
Wenhui - Yeah. So, we combined biochemistry and biological approaches. We developed a unique and very highly efficient approach for tandem purification of complex membrane proteins. We utilised a viral protein ligand in which we incorporate three tags of photoreactive or nonnatural amino acids in the receptor binding sites epitope and adjacent to the receptor binding site for antibody recognition and a biotin tail. So, the photo-leucine allowed zero distance cross-linking of the peptide ligand and then expand the receptor upon UV irradiation.
Chris - So, what you've done is to insert some fake - for want of a better word - amino acids into the virus protein which when you shine ultraviolet light on them will form bonds to anything nearby them. You've also got a tag on there so that you can grab it with an antibody or biotin. And you incubate this modified virus protein with cell surface membrane and then zap them with a light to make the proteins stick on irreversibly to anything it's bonded to at the moment, at that moment in time and then you purify it. Is that right?
Wenhui - Right, yeah. That's exactly what we did and that we then use the MS that is mass spectrometry to analyse the target protein.
Chris - So, by basically asking, what is this molecule, you come up with a chemical formula for the protein. But how do you work out what that is in the cell because there must be lots of proteins in the cells that are going to have a similar chemical formula?
Wenhui - Yes. We took several approaches to prove that is the molecule that we want. First of all, we used small interfering RNAs to knockdown this molecule in the cells. We checked whether the infection of HBV and HDV is affected by this gene silencing. And then we express this NTCP molecules into the Hu stem cells, which normally does not express this protein, and we evaluate whether the viral infection could be enhanced by transfecting this molecule.
Chris - That's quite neat. So effectively, you turn a cell which is uninfectable into a cell that becomes infectable as soon as you express this molecule in it. Thus, proving that this is necessary and sufficient for hepatitis B and hepatitis D viruses to get into these cells.
Wenhui - Correct.
Chris - So, now that you know this target, is it the only target on cells? In other words, if you take cells in a dish and as you have done, put this receptor molecule into them, do you get the same sort turnover or growth of the cells or efficiency of infection of the cells that you would in a human who is challenged with hepatitis B?
Wenhui - That's a very important question. We think that its molecule is a predominant one. If not, the only of the receptor. However, if you transfected this receptor into cells, it can support viral infection but not as efficient as in vivo. There's many reasons for this and detailed mechanism is still unknown.
Chris - Now that you know what the receptor is though, what do you think the implications of your discovery are?
Wenhui - This discovery advances our understanding of HBV and HDV infection and I think it also may raise a possibility to develop new therapeutic approaches for these viral infections. There are some drugs available against this NTCP, but we don't know yet if they can block the viral infection.
29:03 - Why does fog cloud driver judgement?
Why does fog cloud driver judgement?
with Paolo Pretto, Max Planck, Tubingen
What happens when you drive in fog? Paolo Pretto from the Max Planck Institute for Biological Cybernetics in Tubingen, Germany, clarified a few issues related to driver's perceptions of their speed in foggy conditions. He explains to Martha Henriques.
Paolo - So, there are a lot of accidents in fog, right? We blame low visibility condition and there were a couple of papers in the past that just said we can't explain why this happens and they provided an explanation which was quite comfortable when you drive in fog, because fog is simply a natural way to reduce visual contrast. And we know that a low visual contrast leads to lower perceived speed then what happens to the driver is they perceive their own speed being lower and therefore, you accelerate to match what the actual speed will look like in clear visibility conditions. So, what this paper did was they brought this to a situation of self-motion. So, when there is no object moving, but the observer itself is moving in the environment. So, they considered this for the first time. And, probably, the way they reduce their contrast was the same way that used in the laboratory conditions. So, they reduced the contrast of the visual seeing of the driver in a driving simulation uniformly, independently of depth, independently of the distance from the observer. And so, that's what was missing in the previous study and that's what we did in our new study.
Martha - And in those previous studies, their simulations were more like driving with a misted up or dirty windscreen whereas you adjusted contrast so that it seemed more like actually looking at fog outside through a clear windscreen. Why is that an important difference and how does that affect human perception of speed when driving?
Paolo - The difference is quite simple. So, no one ever tried before to simulate a gradient of contrast reduction. So, a contrast which is not reduced uniformly all over the visual field. But different regions have different contrast reductions and that's basically the new part.
Martha - So, you created a more realistic simulation of driving in foggy conditions and the initial models used were essentially just too simple and you've added a few more factors to make it...
Paolo - So, we're not appropriate. We're simulating a realistic fog.
Martha - Once you have this better idea of the way fogs should be modelled, what happened when you got some drivers into these simulators, the testing?
Paolo - Well, we used different methods. We did some study on the perceptual side. So, studies which are basically passive for our subjects so they don't have to do a driving path. But with this kind of methodologies, we can somehow quantify their perception. We can relate their internal representation of their speed to the actual current physical speed that they're driving at. So, we measured what is technically called the point of subjective equality. So, when two different speeds are met to different scenes, moving scenes. With that, we derive the perceived velocity, the perceived speed of the driver. And now, the method that we use was more like a behavioural neuroscience method. So, we asked our participants, our subjects to drive, to do some driving tasks and by trying to match a given target speed. They were trained to reproduce before the real experiment. With that long, we could measure the other side. So, we could measure the production of speed. By that, we could relay the effect of perceived speed that we measured in the psychophysical experiments with the observable changes in the behaviour of the drivers that we measured in the second experiment.
Martha - So, you found that the perceived speed was significantly higher than the speed produced by the drivers in the simulator. Meaning, they would actually tend to drive more slowly in foggier conditions. So in fact, our instincts seem to be telling us to do the sensible thing and slow down. So in fact, speed perception doesn't seem to be a factor that would make us more prone to traffic accidents in fog after all.
Paolo - What we did in our study was to concentrate on the perceptual effects, on the visual motion that was processed by the drivers. But we know that when we drive that it's not only perception going on. We have also our own motivations. For example, if we are in a hurry, if we have an appointment, we will speed up. We will drive faster. So, we simply will ignore what our perception, what our brain will suggest that. there are a lot of higher order cognitive factors that overcome the perceptual effects. If we try to simplify and we try to analyse separately the perception, that's what we found. So, the brain would kind of suggest that to drive safely. We don't do it obviously.
Martha - And so, how do you think these findings might influence say, road safety campaigns or policy? What do you think might be the practical applications that your work could contribute to?
Paolo - At the time we conceived the study, we were not really concerned about practical applications. But after seeing the results, we actually realised that this could be kind of useful for many different fields. For example, we know that in the driving simulation community, there is always a problem, and the problem is that in the simulation, all the drivers over estimate their own speed. With our findings, we could think of some application in the future where playing around with the contrast of the visual scene, we could somehow adjust more or less efficiently towards higher or lower levels of perceived speed in the simulation.
35:26 - How did nucleosomes evolve?
How did nucleosomes evolve?
with Corey Nislow, University of British Columbia
How did our cells come by their nucleosomes? These are short stretches of DNA wrapped around a sort of protein ball. The nucleosomes help us to pack DNA tightly inside our cell nuclei. They also control when genes get turned on and off.
Corey Nislow at the University of British Columbia has discovered that these structures were probably doing something else before they were turned by our sorts of cells into spools for DNA. He explains this to Kat Arney.
Corey - We noticed that nucleosomes occupied in very characteristic positions in the genome. As any good biologist would do, we set about trying to perturb that, and we spent a lot of time looking at mutants in yeast and some of these mutants in yeast were extremely informative, but most weren't. And so, we went to look at an extreme environment - to push the system to failure to the point where we knew we were going to see some kind of - if we didn't see a dramatic change in the location of nucleosomes, we would really start to wonder what's going on. So, we started to look for extremophilic organisms - organisms that live in near boiling water or near saturated solutions of salt. As we were looking for eukaryotes in those environments, we kept finding archae-bacterial organisms. They were all over the place - in boiling water environments, in environments where the salt concentration is so high that the salt were actually precipitating out of solution, like in the Dead Sea. Rather than keep looking for eukaryotes in those environments, we started to say, "Well, maybe we should look at these Archaea." Archaea have nucleosomes that are remarkably similar to eukaryotic nucleosomes except they're half-size. Using new mapping technology, specifically next-generation sequencing, we prepared nucleosomes from archaea and we also prepared RNA at the same time. So, we can ask - one, is the organisation the same in archaea as it is in eukaryotes? Two, is this correlation that we see between gene expression and nucleosome positioning maintained? I wouldn't be talking to you if the answer to both questions wasn't yes!
Kat - So, what did you find?
Corey - So, we found that this parallel universe is exactly what's going on in that the entirety of the archaea bacterial genome is wrapped up in nucleosomes. It's the frequency of the wraps, they are twice that of what you see in eukaryotes because they're half the size and they only wrap half as much DNA. The regions that have fewer nucleosomes are regions at the beginnings and ends of genes where other proteins need to fight for access to the DNA to turn things on and off.
Kat - And that's exactly what you find in eukaryotes as well.
Corey - That's why I was saying it's really like a parallel universe for us.
Kat - When you found this, what did you think because traditionally, there's been views that archaea and eukaryotes are very separate in terms of their evolution and they're separate branches of an evolutionary tree? What do you think now in the light of your experiments?
Corey - I really, really feast on the thought of as a wonderful device for packing DNA into a tiny space. Each of us has over 4 meters of DNA, but we have to squeeze down into a nucleus that's only 5 millionth of a meter in diameter. And so, one of the things they must be doing is serving as - we often use, 'packing material'. But archaea don't have nuclei and therefore, don't have the same constraints of really shoving all of their DNA into such a tight space. And so, we let ourselves wonder if given the fact that the nucleosomes in archaea are intimately linked with the gene expression patterns that maybe evolution first used nucleosomes to control gene expression. And then shortly thereafter, when came a time to get everything into the nucleus, it co-opted that mechanism. This wouldn't be the first case we're approaching this moonlight and served multiple functions.