Web Conference with Dr. Craig Mello

- 00:00 So welcome to my Cell Physiology class, and this is--

- Hi there.

- Hi!

- 00:09 So I really appreciate your time, and I know you've had a lot of requests to meet and talk. So in the interest of time, we'll just get started with the questions about RNAi. And you should know that my class has read your original 1998 paper in Nature, critically analyzed it, tore it apart, and found absolutely nothing wrong with it, of course. But so, the first question is, just briefly tell us a bit about yourself, your career path over the years, and specifically what led you to the discovery of RNA interference. Thanks a lot for joining us.

- 00:48 Okay, sure. Well, it's hard to ﬁgure out where to start with a question like that, you know. I started out, as a kid, being very interested in all kinds of different sciences. My dad is a paleontologist. My mom was an artist. And they did a good job, I think, of keeping that sort of natural curiosity that you usually, I think, most kids have growing up. They helped, kept that alive for me but you know I always knew I wanted to have a career where I would spend my time trying to learn more about the world, how it works. And I remember as a kid being really captivated by the notion of deep time, 'cause my dad was a paleontologist so the exposure to the museum, the Smithsonian Museum when I was a kid and basically that, seeing the history of life and thinking about the history of life and the origins of us and all that, the human condition, how we got here, those were all fascinating to me and I wanted to be able to spend my career as I went through college I realized that we could essentially see evidence of our evolution in our history, in our relatedness to other living things through the genetic material, the DNA and through molecular biology we could actually learn a great deal about living things. And even make medicines like insulin that's basically a human hormone that's produced in bacterial cells, that that could be that the bacterial cells could read the human Web Conference with Dr. Craig Mello

genetic code. And I read that actually in a newspaper when I was a high school student, and that is what made me want to become a scientist, or not a scientist but a molecular biologist and that's what I pursued ultimately. In college I guess I majored in biochemistry and then in graduate school I went on to essentially do genetics and molecular biology and that sort of led up to my work as a C. elegans researcher. C. elegans is really a simple model organism that had been developed by Sydney Brenner back in around the early '70s as a genetic model system, essentially for its simplicity. It has only 1,000 cells, whereas humans have about 10 trillion, so I guess I started in 1982 at graduate school working on this system at a time when no one had ever injected DNA into it successfully. I remember I told you I got interested in molecular biology having read about the ability of the bacterial cells make and that process of putting a gene into bacterial cells you know was something that hadn't been worked out for other types of organisms really. I mean you could do it for bacteria and yeast but you couldn't really do it for more complex animals. The earliest work on ﬂies and mice delivering DNA into those types of organisms was just beginning. And C. elegans hadn't been transformed as we call it with DNA at that time, so I got interested in that and began working on that. Meanwhile Andrew Fire was also working in that same area, developing techniques for injecting DNA into C. elegans and so the two of us were working in different locations on the same problem. And over the course of the next few years, Andy and I got it working for C. elegans to the extent that you could now inject DNA very easily and reproducibly get the genes that you're injecting into the animal to be expressed. And all that technology, I don't know if you've had that in molecular biology already, but that technology, putting the promoter on the gene, in front of the gene so you can drive it in the animal, in the right tissue, and so on is all sort of worked out now. But it was a bunch of technology that Andy and I were developing during that time, got to know each other very well and develop the techniques for injecting. And these animals are really small, they're only a millimeter long so the injections are done under a microscope and so we were doing that kind of work together and developing technology is, ﬁrst of all, it's extremely important for biology but second it's also Web Conference with Dr. Craig Mello

something that's very difﬁcult because when you fail, usually you don't know why it didn't work. You know, no one's ever done this before and you're trying to get it to work. And so Andy and I talked a lot about solving problems and how to get this really critical tool working for the organism. So we developed trust and a relationship during that time that I think was key to our later collaboration on RNAi. In RNAi, the interference phenomenon was ﬁrst noticed by Su Guo and Ken Kemphues when they did injections, essentially trying to do antisense, which is a technique where you basically, putting lots of complementary RNA to the transcript and try to inhibit it. They noticed that it worked whether they used the antisense or sense and they were using the same injection technology that Andy and I had developed for DNA delivery only they were injecting RNA. And so when Andy and I naturally were already the best at that procedure and we both became interested in essentially trying to understand what was happening. And we began our collaboration then and there. I could go on and give my hour long seminar but I think I probably should let you ask another question, but that's basically how it led up to that.

- Great. Thank you for the history there. We'll start student questions now. And each student will come up to the computer here. Read the question to you, and then we'll move on. So our ﬁrst student is Jesse.

- Hi Dr. Mello.

- Hi Jessie.

- 08:14 Can you please describe your current model of RNA interference, and what types of approaches your lab has taken in the past few years to achieve this model?

- 08:26 Okay. Models are yeah, they're really important. That's a good question. Basically we're always trying to come up with new models. We have probably eight or nine models at any given time. And quite often they are contradictory and they couldn't Web Conference with Dr. Craig Mello

both be right. So you basically have a model that's a working model, you know, all the parts of it fit, the pieces fit together, you draw little circles and arrows and everything and it all makes sense. But you realize it's a work in progress. So that's where we are right now with RNAi. And in C. elegans RNAi works a little bit differently from other organisms, other animals, in that there's an amplification step involved in the silencing which probably helps explain why RNAi was discovered in C. elegans and also related phenomena were discovered in plants and fungi because those organisms all have a very potent amplification step where they respond to foreign double-stranded RNA and they have an enzyme called RNA-dependent RNA polymerase that amplifies silencing signals. So we had basically in our model, we have these enzymes called Argonautes. I don't know if you've read about those. Certainly in the 1998 paper there's no mention of them because we hadn't discovered them yet. 1999 we identified the first gene involved in RNAi in C. elegans and it turned out to be this highly conserved gene which had been identified as a developmentally important gene in plants. And it had been named by the plant people as Argonaute as the plant gene name. We named it RNAi-deficient gene number one. Kind of a boring name, Rdg1. And our name didn't catch, their name caught. The Argonaute name is what people usually refer to this group of enzymes as, and these are enzymes that hold on to the little RNA, they're called the siRNA and they use it to hunt for and search out targets in the cell by base pairing. Base pairing is a very efficient way of identifying a target because as you know there's a lot of information in each of those interactions between the base bair interaction so by a high degree of sequence specificity it allows even a short RNA to uniquely identify almost any gene in the genome. So the mechanism basically involves this protein called an Argonaute that holds on to the small RNA and uses the sequence information in the small RNA to identify other sequences that can base bair with it, forming those hydrogen bonds that make up the Watson-Crick base pairing in the DNA, only it's an RNA-RNA base pairing interaction in this case which is really very similar, even a little bit stronger than the DNA base pairing interaction. Then you get this very high affinity, very specific interaction that allows this enzyme, and it's really cool what Web Conference with Dr. Craig Mello

this enzyme does, it essentially, probably, this is a model, again the Argonaute holds on very tightly at one end of the siRNA, the ﬁve prime end, and it lets go at the other, and lets the little siRNA wrap around the messenger RNA, sort of like give it a big hug, and when it does that, the helical, a structure hold point so you have a linear messenger RNA that's the target and it's unstructured really, and then it base pairs up with this siRNA and by doing that it forms a helix that pushes the backbone of messenger RNA up against the catalytic center in the enzyme, and that leads to cleavage of the messenger RNAs backbone, and no cleavage to the small RNA. The small RNA can then get peeled off, probably by helicases that help to unwind this after the cut that they made, and then they can go on and cleave additional targets. So it's a catalytic mechanism. So it's catalysis at that early step and then after the cleavage, the RNA-dependent RNA polymerase, which is our model, gets on to the cleaved product, the cleaved messenger RNA and copies it to make more double-stranded RNA. So the double-stranded RNA is formed at that second step then gets incorporated after further processing by this enzyme, Dicer, into an additional complex. Now we just learned that our model which has involved Dicer at two steps, Dicer functioning at the initial delivery of the RNA itself, Dicer's this enzyme that makes the short RNAs from the longer RNAs. So you got the double-stranded RNAs, let's say 100 nucleotides long, Dicer will chop it into 20 nucleotide pieces, 22 nucleotides or so, and those get loaded onto Argonaute and then the Argonaute does what I just told you. So backing up a little bit, Dicer functions at the ﬁrst step, Argonaute, then Rdrp, then we pull out Dicer again because the Rdrp is gonna take the single-stranded cleaved message, make double strand again, Dicer functions again, and Argonaute gets loaded again and so on. Now we've realized that that model's probably partly right but that there may be another way that the RNA-dependent RNA polymerase can make siRNAs that get loaded onto Argonautes without Dicer processing them, in other words, the RNA-dependent RNA polymerase, we call it an RdRP just for short, is able to make short RNAs directly without Dicer processing. And that's something that's brand new, it's very exciting and that you basically get a catalytic step when the initial Argonaute is cleaving multiple targets, then you get a Web Conference with Dr. Craig Mello

catalytic step with the RNA-dependent RNA polymerase copying those cleaved RNAs to make more, and then ultimately you get turnover again of more message, and so the whole thing is very catalytic, very much ampliﬁed at multiple steps, and so that explains why it's able to achieve such a high degree of balancing in C. elegans. And that's just one part of what happens. That's the response to foreign double-stranded RNA. There's a whole additional set of mechanisms that relate to naturally occurring forms of this silencing that are involved in gene regulation just naturally in the cell. And I think that another one of your questions will probably address that, so I'll stop there. But that's, models are fun, that's what you spend most of your time, in science you spend a lot of time just brainstorming about models and then trying to ﬁgure out how to test them.

- How often would you say you end up changing your model?

- We probably change it two or three times a day.

- That's what I thought your answer was gonna be. Our next student is Aileen.

- Hello Dr. Mello.

- Hello Aileen.

- 15:50 We were wondering how this non-native double-stranded RNA is not degraded when it's injected into the cell? We were wondering if it's somehow being processed by you know, adding a ﬁve prime cap, or the three prime poly-A tail? Which would help it dodge the degradation that would occur naturally if you didn't have these things and we were wondering if it's actually the size of the double-stranded RNA being injected that is perhaps facilitating this as well?

- 16:23 Well, that's a good question. There are modiﬁcations that happen after Dicer processes the double-stranded RNA. First of all, double-stranded RNA is much more stable than single-stranded RNA. The ends get trimmed off of it but where it's double Web Conference with Dr. Craig Mello

helical it's harder for enzymes to degrade it so it's already much more stable than single-stranded RNA would be, so it doesn't disappear very quickly in cells. It even lasts for a few minutes in human blood without any protection at all. But if you modify the RNA chemically, you know the backbone, if you change it a little bit you can achieve a great deal of stabilization. And there are naturally occurring enzymes in all eukaryotes that basically modify RNA, and for example there's an abundance, so far we don't know of any modifications that occur naturally to the siRNAs, you know the double-stranded RNAs that you were referring to, to answer your question simply, there isn't any capping that we are aware of, and there's no modification to the end, but rather once it's loaded on the Argonaute, the protein is detecting it. If you knock out the Argonaute protein, if you have a mutation in that gene, then the siRNAs are not stable. So we think that once it gets into that protein complex it's stabilized by it's association with the protein, it's protecting it. But we do know of modifications that occur to naturally occurring small RNAs that are present in the animals, and those modifications do exactly what you were suggesting and protect it from nucleases that would otherwise degrade it. So as far as we know there are no such modifications to the double- stranded RNAs that are entering the RNAi pathway.

- Our next student is Valerie.

- Hi Dr. Mello.

- Hi Valerie.

- 18:41 Okay, I was wondering if you could clarify how double-stranded RNA or other forms of RNA interference can be passed onto the progeny of C. elegans and also how many generations can that interference be detected for?

- 18:56 Okay. Yeah that's an exciting area right now. Not just because it, well ﬁrst of all it was exciting when we ﬁrst discovered that. That was one of the reasons why I got Web Conference with Dr. Craig Mello

interested in pursuing this, you know, as a mechanism, or trying to understand the mechanism because inheritance indicated to us that there had to be an amplification step in the process and that it had to be an active response in the organism to the double-stranded RNA. What we believe is happening with the long-term inheritance is that the RNA that we're injecting is silencing the gene at the chromatin level. So one of the really exciting things about RNAi is it connects with other gene regulatory mechanisms in the cell, and one of those mechanisms that is fascinating right now, and we're just learning about this too from work in other labs, is all of these really cool modifications that can happen to these proteins called histones that make these core, almost like the spools, that around which the DNA thread is wrapped. These histone cores have little tails that stick out and those tails are little peptides that stick out near or past the DNA and those can be modified by these enzymes that control how compacted the DNA is, whether it's in an open conformation or really all coiled up and wrapped up in the silent conformation, because of its packaging and that regulation is achieved by these enzymes that have been now recently identified as interacting with the Argonautes in yeast mainly, and plants. But we believe it also occurs in C. elegans and human cells as well, and these interactions have been defined genetically in worms from an early time actually but not biochemically yet. We're looking for biochemical evidence for interactions between the Argonaute proteins in the chromatin remodeling protein. So here we have RNA regulating the accessibility of the DNA to the transcription factors and so on that would allow the DNA to be expressed. Now the interesting thing about that in terms of the inheritance is it implies a mechanism by which modifications to the chromatin can be passed from one generation to the next. And that that is really amazing, and also it's really cool in the sense that you could imagine that organisms use that kind of variation adaptively in an evolutionary sense. So you could imagine that an organism can change the packaging of a gene without changing the nucleotide sequence of the gene. So an adaptive, heritable, change without an underlying change in the nucleotide sequence. And that's something that people have not been thinking about at all, as far as I can tell. I mean epigenetics is something people talk about Web Conference with Dr. Craig Mello

a lot but they talk about it like a minor phenomenon, something that happens on one or two genes, and oh isn't that cute, you know there's a couple good examples of this type of epigenetics that have been described but this is something that's potentially, could be applied to any gene in the genome and it's very likely I think happening in humans as well, such that there's an additional source of variation, we've thought of the DNA nucleotide sequence as the thing that was varying when we've had mutation. And Mendelian genetics is all based on this very simple idea that the DNA is the source of inheritance. The DNA is the source of variation in the sense of mutation effecting the DNA. The theory in how the source of inheritance is not the DNA primary sequence, it's a three-dimensional interaction between the RNA, the protein, and the DNA, that results in a heritable on or off state for a gene that can be transmitted from one generation to the next. And I like to make the analogy that you have the DNA as part of the hardware of your computer, you know, and the RNA and the proteins are the programming and also keep in mind that all the differentiated cells in our bodies have the same DNA but represent different programs that are turned on in those cells. So what I'm suggesting is that there are, changes in programming can occur that effect the germline as well as the differentiated cells of our bodies, so that we have evolutionary change happening at the level of RNA-protein-DNA interaction that are heritable. That's I think very exciting and could help explain how such a small genome with only about 30,000 genes can make organisms that are so complex like ourselves and in C. elegans. So that's a long-winded answer but I think the RNA that we're putting in is interfacing with an existing mechanism in our cells, whereby RNA can control the DNA heritably, in a heritable way, and that's you know, really exciting. Something we're studying right now in the lab and I think beginning to get some further insight into.

- Thank you very much. That is truly exciting. Thanks for sharing your interpretations of that. Our last student is Catherine.

- Hi Dr. Mello. Web Conference with Dr. Craig Mello

- Hi, Cat.

- 25:14 We were wondering what evolutionary roles has RNAi played and what would be advantageous about having RNA that functions to break down mRNA after the initial energy investment of transcription, and how did RNAi evolve?

- 25:29 Okay. That's another great question kind of related to the last one but the evolution of complex organisms is thought to have begun with molecular evolution that began soon after the oceans formed on the planet, three and a half, four billion years ago. And it was a mystery really how life began. But it's hard to conceive of life beginning with a whole cell already organized, but rather with maybe a self- replicating molecule of some kind and RNA would be a good candidate for that kind of molecule because as you probably have heard there are things called ribozymes which are basically RNA molecules that are capable of carrying out catalyzing reaction in a way that you can imagine might allow an RNA molecule to essentially copy itself. So once you have a self-replicating molecule and you know if it's a nucleotide sequence like an RNA, then it can copy itself, it can make more. You've got sort of the proto-living thing. It's almost like a virus that just started leaping off. One notion that I think is interesting to think about is that the RNA molecules that represented those primordial living things never really gave up control, what they did is they ﬁgured out that DNA was a stable and useful type of information. It was sort of like, the DNA can't catalyze reactions but boy it sure can store information very efﬁciently, very stable-y, so, and the other thing that I would like to say about this is that living things, we've continually and consistently underestimated living things. The Nobel Prizes are given out almost as a rule to discoveries that demonstrate how we were wrong before about how things really work. So I think that one thing we have to do and that's why I've been speculating about the importance of RNA interference and evolution for example, I actually had a lecture recently where I chose the title "Return to the RNA world: "rethinking gene expression and evolution" because I think basically we really have to now begin to think about this idea that the RNA Web Conference with Dr. Craig Mello

is a source of inherited information in the cell. That the interactions between the RNA and the chromatin are very important. I think the ancestral function of RNAi might go so far back in time that it really evolved right along with other mechanisms of information storage and transfer in cells. And it's now becoming clear that even bacteria have small RNA mechanisms that are quite intriguing. So we may learn more as time goes about how ancient these mechanisms really are. But I think they're gonna turn out to be very important. They already have turned out to be very important. But I think they're gonna turn out to be even more important as we go and learn more about them. And you know, it's an exciting time in biology because we have genome sequence as you know, and we have lots of great technology that allow us to move very quickly in studying genes, and yet there's still so many fundamental things that we don't understand, and I would really encourage all of you to think about going into biology as a career as you know, something you could enjoy.

- 29:37 One quick wrap-up sort of discussion. How do you just, see your discovery of RNAi potentially influencing medicine? Specifically, currently in your opinion what's the most pressing questions in the field of studying RNA interference, and what steps is your lab specifically taking to address those questions? Thanks a lot.

- 29:58 Okay, sure. In terms of its impact on medicine, RNAi is having impacts in two ways. The primary way right now is in the laboratory where it's used as a tool for studying gene function. And it speeds everything up because you can actually do genetics now in human cells in culture using RNAi to knock down genes in human cells and looking for how the cells behave when you knock these genes down. I'll give you an example. A colleague of mine, Michael Green here at UMass, has been doing these high-throughput screens in vertebrate cells looking for genes important in cancer, and so he's, we have a library of short hairpin RNAs which essentially are RNAs that are expressed by viruses. We built, not we, but actually a company called Open Biosystems, provides free of charge, RNAi targeting every gene in the human or the mouse. And so you can take the vertebrate cells in Web Conference with Dr. Craig Mello

culture, and you can transform them or transect these viruses into those cells, and the viruses will enter the cell and bring with them a small RNA that will silence the speciﬁc gene in the genome of that cell, or turn over the RNA more precisely. So the knock down gene, and then you can ask how the cell behaves. So then you have an assay for example, a simple assay could be cell growth. Does the cell still grow or does it not grow? Or if you have, let's say you have a cell that's not a cancer cell yet but it's on the verge, it's got two genes or something, mutated, that sort of predisposes it to becoming cancerous, you could ask well what other genes could be inactivated to push that cell over the edge so it will begin to glide out of control? You can do screens like that and you can basically screen by transecting the whole library of short hairpin RNAs into these cells and then looking for cells that now exhibit a change of behavior. And when we recover those, you can then recover those cells from the Petri dish, let's say the cells now grew where all the other cells didn't, and recover that colony of cells and then can ask what hairpin RNA is in there? Which gene is it targeting? And then you do that over and over again and you get let's say you've done this 1,000 times and 50 times you hit the same gene, I mean you've identiﬁed these genes involved in promoting or changing the behavior of these vertebrate cells in culture and then you can ask in the animal you can do further experimentation later by making the knockouts in the animal which takes a lot longer. So you can use cell culture to rapidly go through all the genes searching for genes that are important in that particular assay that you There's just so many applications like that. And people are using it in research to quickly identify new genes that are in pathways that might be relevant to other diseases as well. So that's one of the primary ways it's impacting medical research. That route will take several years before they'll give you drugs because it's a long process of identifying their standing genetic pathways and then developing the drugs later on. The other way that's sort of quicker, is if you know that there's a gene related to the disease, and you know that it's just a bad gene, you have to down-regulate it, then you can essentially on your typewriter, design a drug based on an RNAi that would shut that gene down or lower the levels of that gene, and thereby achieve a therapeutic effect. Those types of drugs are now Web Conference with Dr. Craig Mello

in development or in testing in humans for a couple of indications where delivery is straightforward, for example, delivery by injection into the eye to treat macular degeneration. That's already shown efﬁcacy in phase two trials and I believe is now in phase three. There is a trial going on for respiratory syncytial virus where it's an inhalation drug. The RNA is inhaled and absorbed into the lung epithelium directly. Again that is in trials in humans now. But there are many others that are sort of getting close to being tested in humans. So those are sort of the two ways that RNAi is impacting medical research and probably the biggest impact is through the effect on the laboratory because it's already a multi-billion dollar reagents business for the laboratory and that's having a huge impact in developing really understanding of mechanisms. I'm trying to remember the other questions that you had.

- 35:22 What is the most pressing question in the ﬁeld of RNAi and what's your lab, just give us some insights as to what your lab's speciﬁcally doing to address that question.

- 35:38 Sure. The pressing questions involve mechanism. The better you understand mechanism, the better you are able I think to utilize the tool. So it's clearly a very important tool. It's fundamentally just really interesting because it's fascinating basic biology but even more importantly it's a tool and therefore if we understand how the mechanism works we can design better reagents for utilizing that mechanism and inducing the silencing in the cells. So understanding mechanism is a key area of focus in my lab. I'm particularly intrigued by how the RNAi mechanisms play a role in regulating gene expression naturally. We didn't talk about it but maybe you've read about the microRNA genes? Our humans and worms and all animals and even plants have little genes that incur these natural hairpin-like RNAs that are very important developmentally, they control the expression of other genes and they're also these mechanisms that are referred to whereby the RNAi can inﬂuence the chromatin structure of a gene itself and those mechanisms are again, very poorly understood, and yet, if we knew how to manipulate those mechanisms, we might be able to achieve some really powerful therapeutic effect, Web Conference with Dr. Craig Mello

because you're not only regulating the mRNA, but you're regulating the gene itself. We have no idea really how to look at those pathways, how to utilize this to develop better tools. Those are the sort of things that we'd like to understand. So we're still working very much in C. elegans because the worms still have a lot to teach us. It's a really amazing little system and we're taking some of the knowledge that we've developed working in the worm into work now in the vertebrate cells because the genes we've identified in the worm, like Dicer and the Argonaute are conserved, along with several other novel genes that are conserved in the human, and we're trying to understand how the same genes function in the human cells. So those are the kind of things we're doing in the lab. There's probably, worldwide there's probably on the order of two or 3,000 people in about three to 400 different labs that are basically working on RNAi full time. There are hundreds of labs around the world working on RNAi full time, trying to understand how it works. It's a great field. We have really nice meetings. Last one was in Keystone, Colorado and you know it was a very beautiful setting, but also great science. One of the fun things about being a scientist is the meetings that you get to go to. They're really awesome. And the other thing that's really fun about it and it's something I didn't stress in this discussion, but if there's anything at all that I would recommend to you in terms of becoming not only a better scientist but just a better person is talking to people. The importance of dialogue and discussion and argument is so important to achieving anything worthwhile. Andy and I, we're collaborators who shared ideas with each other and through that work we accomplished much more together than we would ever have been able to do by ourselves. I mean, there are some people who are, you know, absolute geniuses like Einstein, and they can just sit in a room probably and rattle off great new ideas, but most of us the most effective way of developing new ideas is by sharing your own ideas with someone else and they will share their ideas with you and together, that dialogue will produce a new idea and it's not your idea and it's not their idea, it's just a new idea. And it comes from that dialogue. If you start doing that, you'll find that you can come up with really great ideas that you weren't even thinking about before, and that's always a great way to start. You Web Conference with Dr. Craig Mello

have a project or something and you have to start out, talk about it with your classmates and your teachers especially, and you'll be amazed how productive those kinds of conversations are. And that's what I love about my job. I spend most of my day talking to my students about our ideas and how do we address, how do we design experiments. So that's the one thing I would recommend that you start doing no matter what profession you're in, if it involves a problem that's complex, getting every brain cell in the room focused on that problem and thinking about it from eight different directions is the ﬁrst step in creativity. And it's gonna be something that will always work for you. So that's what I would encourage you all to do, is make sure you take advantage of your friends and colleagues and even your enemies. Try to get them involved in a discussion. And that's what science is all about too and that's why it brings together people from all over the world, from any religion or philosophical or historical background, because science is all about sharing ideas and that's all about talking to people and it's a great thing to be involved in. So I encourage you all to get involved in that kind of discussion. Join the fun.

- 41:42 Well thank you very much for being a perfect example of sharing your dialogue with us and taking the time today. You could have easily said no but you didn't, and we appreciate beginning that conversation. All of your insights and discussions are just really greatly appreciated. Can we give him a round of applause?

- Is it true that you guys will be participating in that thing out of Amherst?

- Yep, we're looking forward to seeing you then too.

- Great. Look forward to give you my whole lecture. You get to see all these little fun movies I have lined up.

- We're looking forward to it.

- Good. Web Conference with Dr. Craig Mello

- I don't want to take any more of your time. I know we went over and I appreciate that. So have a fantastic day and discover more. We need you.

- Good luck to all you too.

- Take care.

- Thank you.

- Bye.