 Welcome everyone to the second Aging Zero Science and Longevity Symposium organized by Elive. I am Dario Valenzano and I will be co-chairing this symposium together with Sarah. I'm a group leader at the Max Planck Institute for Biology of Aging in Cologne in Germany. And I am an editor, like a reviewing editor at Elive. And I also was a speaker in the previous symposium, in the first symposium. Yes, so welcome everyone. My name is Sarah Hagen. I'm co-chairing this session today, Symposium with Dario. I'm an associate professor at Keralinska Institutet and I work with the Molecular Epidemiology of Aging. So a lot of human data. And it's a pleasure to doing this second symposium on Aging Zero Science and Longevity, organized by Elive today. So Sarah and I today will alternate in moderating the various talks, but we will be both present at the whole time. And well, I'd like to say that it's very exciting that Elive had this initiative and to bring together a community, not just via like an issue, a special issue on Aging and Zero Science, but also by giving the chance to all the authors to meet virtually with the broader community. So this is really like a great platform. And it's a very interesting experiment. And I look forward to being part of it and to see how it unfolds. So I think that just like the previous symposium that was held in December, also this one really reflects how the field of biology of aging has evolved over the past 20 years from very molecular, very mechanistic, to much broad and encompassing everything from theory, from evolution and epidemiology. And with a very, very, you know, a lot of attention towards applications and medical consequences of the research that we conduct every day. So yeah, so there will be nine talks. And there will be now, I guess, Sarah will explain a little bit the various rules today. Yeah, we have some rules today. Yes, as you said, Dario, we will have nine talks today and they will all be 15 minutes approximately in the presentation. And then we have a few minutes for questions for each speaker. So please, if you have questions, you can write them in the chat and we will post them to the speaker. And if you want, you can also write your name and affiliation and your type of research position. If you want to share that as well. We will also have a 20 minutes break about half time through this symposium. And what else? Yes, we have all the presentations will be recorded today. And this will then be available through the Elife website after the symposium has ended. So you can even watch it afterwards if you miss some parts of it. Yes, did I miss something, Dario? Oh, I think that's pretty much it. So we hope that you will be participating and we encourage also students to be active and ask questions, we'll read your questions. And I think we are ready to start. Right. So then I will move on and introduce the first speaker. And this first presentation is a recording and the speaker is Broke Sankl from Monash University in Melbourne, Australia. The talk of the title talk is dietary sterile trade-off determinants lifespan responses to dietary restriction in Drosophila melanogaster females. So as this talk is a recording it's not possible unfortunately to ask questions like questions to the speaker. So this will be the exception for today but for all the other talks you can pose questions in the chat function. So with that I think we can start with the first talk. Hi everybody, my name is Broke Sankl and I'm a PhD student in the PIPA lab. Thank you for coming and listening to my talk today. I'm sorry that I couldn't be there live at the symposium. I just don't know how great my talk would be at 1 or 2 a.m. here in Australia. But I hope you enjoy it and what I'm going to be talking about is actually my first paper. And we looked at the role of micronutrients in both lifespan determination and reproduction in Drosophila melanogaster and specifically we focused in on sterils. So first I want to quickly just talk to you about diet and health. So we know that what an animal eats throughout its life can have significant effects for its lifelong health. And specifically one way of extending the lifespan of animals has been, which has been shown across a broad range of organisms is dietary restrictions. So this involves restricting the amount of calories that an animal was able to eat throughout its life. And this produces lifespan extension effects. It's been observed across a broad range of organisms including yeast, Drosophila, which we're studying in our lab, monkeys and many different other animals. I'm just going to quickly show you what exactly this effect I'm talking about looks like. And this is in Drosophila. So going forward all plots or anything of that sort will be Drosophila based because that's what we study in our lab. So on the x-axis you can see we have food concentration and this is going from low to high. And on the right y-axis we have lifespan and on the left y-axis we have the quantity. So this is our lifespan here. We see on the high food diet too we've got lifespan coming in at about just under 60 days. Then as we dilute the diet we see the lifespan increases and increases further as we dilute it more. And then it drops off dramatically as once we dilute it too far because then the animals are malnourished. Then here we have the quantity and we use the quantity as essentially a measure of health because the more offspring that an animal is able to have the more evolutionary success it's going to have in the wild. And you can see that here where the lifespan is reduced is actually where we have the highest number of offspring and then this goes down as we dilute the food further and further. And this what you're seeing here is being formalized into a mechanistic theory. It was done by Tom Kirkwood in 1977 and it essentially postulates that animals that the soma and reproductive organs competing for limiting resources. And so when resources are abundant then the animal will reproduce as much as they can. And this comes at the cost of the soma. However, when the animal is resource limited what it will do is it will basically switch off reproduction. And as a result, the soma will receive priority supply of nutrients. And this allows the animal to put off reproduction until a later date when nutrients are more available So moving on to some more recent findings we've actually been able to mimic the effects of dietary restriction by modifying the macronutrients in the diet. And so what you can see here in the details of this unimportant but what I want to show you is that we have two different traits. So these are pairs of data and on the left we have lifespan and on the right we have lifetime aids for different animals and protein here and carbohydrate here. And what you can see is that these traits are actually maximised at different protein to carbohydrate ratios. So we see that it's not necessarily the amount of calories that an animal is eating. It's just that these two traits are maximised have their different optima. So they're maximised at different points on these food maps, which is where I don't know if I mentioned, but where it's red. This is where that the trade is maximised. And you can see that in general, a high protein diet encourages high levels of reproduction, while low protein diet is where we see lifespan be maximised. Now, moving slightly different, I want to actually talk about what goes into making an egg and the decision for a Drosophila to lay an egg. So when the fly is eating these really high protein diets, they're laying a lot of eggs and there are nutrient-sensing mechanisms in Drosophila that tells them when they have the amount of protein or carbohydrate that is in their diet and it tells them how many eggs that they should be laying because these macro nutrients are, of course, very important for both egg production and survival. And they don't want to overcommit to reproduction and run out of these resources. So when they're eating the high protein diet, they're laying lots of eggs. But the thing is that eggs are not simply comprised of protein and carbohydrates. They're made up of an abundance of different nutrients, including micro nutrients. And the thing about these is that micro nutrients are not as tightly regulated or monitored in the body of Drosophila as the macro nutrients are. So when a certain micro nutrient is running low, the fly might not actually realise because it's a shame that it should be receiving these micro nutrients from its diet. However, this is not always the case. So in the case in which the fly is eating really high protein diet, so it's laying a lot of eggs, but its diet is lacking in a specific micro nutrient that is really important for egg production and somatic maintenance. The fly may therefore overcommit this micro nutrient to reproduction. And as a result, deplete itself of this resource completely resulting in no death. So we wanted to look at this in Drosophila to see if maybe this is actually what's going on. And this is what explains the effects of dietary restriction. And so we chose to look at sterols. And the reason for this is because it doesn't appear that Drosophila actually has a sensory mechanism for sterols. So you can't actually tell when it has run out of sterols almost completely. And this micro nutrient is also very important for both egg formation and in production, as well as somatic maintenance. So to look at this, we designed a series of diets. You can see another heatmark here and the details a bit aren't important. It's just some preliminary findings that we had that helped to inform our decision for creating each of these diets. And so we have three PTC ratios and three caloric levels here. So we have a prepping deletion series and a carbohydrate deletion series. And the open circles are our diets. So we have five different diets. And this is all done using athletic medium, by the way. So this allows us to manipulate each individual nutrient independently of the others. So this is perfect for looking at variants in cholesterol levels at different PTC ratios. So what we did was we had our five diets and we had four different cholesterol levels for each diet. So that was zero, low, medium and high and medium is called medium because it's our standard amount of cholesterol that we put into our flat diet. So quickly, I'm just going to show you what these phenotypes look like when we vary the PTC ratio. And here you can see we have our survival plots on the left. Protein is in red at the top and green is carbohydrate. And here we have lifespan as circles. And it's the same as this diet here, basically, just to conduct and the same for carbohydrates. Now, we see, as we would expect, that the lifespan is maximised on the intermediate PTC ratio on the high protein diet. The flash lifespan drops off and on the really low protein diet. It does the same, it drops off. And we see that egg output increases as we increase our protein. Carbohydrate is almost the same, except it's just reversed. Except we see that the lifespan is actually maximised across all of these PTC ratios, which is also too big to be expected. And we see here that on the low carbohydrate is when they are laying the most amount of eggs and this reduces the more carbohydrate that we add to the diet because this is decreasing the PTC ratio. So now what happens when we modify cholesterol? And I'm going to show you first, this is our protein series. And then after I show you our carbohydrate series. So we have cholesterol going from low to high here, you can see. And this is our in the square, we have our medium level of cholesterol. And this is what I showed you before. So we see that on the high protein diet, lifespan is reduced and it increases as we reduce the protein and then drops back down as we reduce the protein too much. When we add more cholesterol to the diet for our high cholesterol diet, we don't really see any changes in these effects. We see that the high protein diet still is where lifespan is reduced. However, when we begin to reduce the amount of cholesterol in our food, we see that there is a significant decline in lifespan on the high protein diet and that egg laying doesn't really change. And then when we dilute it even further, we see that lifespan decreases even more for the high protein diet and also drops down for the intermediate PTC ratio. Interestingly, our low protein diet, there's very little variation in lifespan. And this is also the diet which plays the least eggs. And the diet which is most affected by the reduction in cholesterol levels is the diet in which the highest level of egg production occurs. So now for carbohydrate. So again, we have our medium cholesterol level in the square and this is the same as I showed you before. Where lifespan remains high across all of these conditions and egg production is highest at the low carbohydrate level. And then for the high cholesterol level, we see that there is no real change in these phenotypes. They remain the same. But then we begin to reduce the cholesterol. We see the same as before. The diet which encourages the highest level of egg laying begins to drop off dramatically in its lifespan here. And as we reduce the cholesterol even more, it drops off further and the intermediate level of protein and carbohydrate so drops off. So what we're seeing essentially is that when the fly is staying more eggs, they're more susceptible to a cholesterol deficiency. So if this is true and there is an indirect trade-off occurring here, then we would expect that by stopping the flies from laying eggs, by making the move fertile, we would be able to alleviate them from this sterile deficiency in their, for instance, the lifespan. So we thought a great way to do this is, of course, to add rapamycin to the diet because that stops egg laying. And that's exactly what we did. So here you can see we have our low cholesterol diet. And when we add rapamycin to this diet, we see that lifespan is extended here in the open red circle. Lifespan is significantly increased when rapamycin is added to the diet. However, oh, sorry. Interestingly, when we add sufficient levels of cholesterol to the diet in the black circles, we see that lifespan is also increased to the same extent as which rapamycin increases lifespan. And we see the same when both cholesterol and rapamycin are added to the diet. So this essentially shows us that the way in which rapamycin appears to actually be extended in lifespan is that it stops egg laying, which stops flies from depleting themselves from steriles. Now, finally, we wanted to check that these effects aren't just limited to the holistic diet, that we can replicate these results in a yeast based media, which is what is used by many different labs to study dietary restriction and the mechanisms behind dietary restriction. So what we did was we had a low use diet and a high use diet. And to each of these diets, we either left them as they were or we added 0.3 grams per litre of cholesterol. Now, what you can see in this open dark orange circle is our high use diet, and it lives a significantly shorter life than our flies fed a low use diet in the open yellow circles, which is exactly what we would expect. Interestingly, here, though, we see that when we add cholesterol to the diet, so to both the low yeast and the high use diet, we are able to extend lifespan significantly to the point at which these two diets are almost indistinguishable. And interestingly, it actually seems that the low use diet is sterile limited. So summarise all of kind of what I just showed you. We have a fly, it's about its life, it's got a full level of steriles. And then we put it on to a high protein diet. One diet is sterile sufficient. There's a high level of steriles in this food. The other one is sterile limited. Both of these flies and these different diets, they go on to lay lots of eggs and have lots of baby flies. This fly, however, who has a sterile sufficient diet, goes on to live a long, happy life with a full amount of steriles because they have enough steriles to allow them to support both reproduction and maintain somatic maintenance. The difference is this fly here that's on a low sterile diet will still have a lot of offspring, but it will die young. Because that it is depleting itself of its steriles to maintain that high reproductive output and the result is early death. So to summarise all of this, essentially, the effects of diet on lifespan depend on balancing several nutrients simultaneously. And what happens is we're seeing one thing at a time, right? But there may actually be multiple causes as an indirect trade off or protein toxicity, for instance. So it's important to consider that just because we're seeing same phenotype occurring, there might actually be different mechanisms behind this same effect. And finally, and importantly, all those steriles aren't likely to be an issue in mammals because mammals can synthesise their own cholesterol. The effects of diet restriction in these animals may actually be explained by the micro-neutering deficiencies. So we know that we can extend the lifespan of mammals by diet restriction. And it's very possible that there may be indirect micro-neutering trade-offs occurring here as well. So thank you so much for listening. And I'd also like to thank the Piper Lab, as well as the Murph and Skrowlabs. So I hope you all enjoy this symposium and have a great day. Thank you. All right. So thank you so much, Brooke, even though you're not here and we don't have the chance to ask questions. I think we can already move to the next speaker. Sara, is there anything you wanted to add? I could just add that all the papers, the published papers are available on the Elife website from the talk. So if you're interested to read more about some of the topics, you can find it online and also for Broke. That was not available live today. Exactly. Thank you, Sara. So before we start with the next talk, I also would like to say that at the end of each talk, I will read the questions that will be asked on the chat. So please, as soon as you have a question for the speaker, write it down, you write it in the chat and maybe at the end of the question, you can tell your name, say your name, your affiliation and whether you're a student, postdoc, PI or if you work in the industry. And I will read your question. So and then we can move to the next speaker. So Fernando Monje Casas, I hope I'm pronouncing correctly the name from Cabimea and the Spanish National Research Council in Spain. And the title of his talk is Aging at the Poles, Asymmetric Inheritance of Spindle Microtubular Organizing Centres. And Fernando, it's a pleasure to have you here. Thank you for for giving the talk. I will turn off my video and I will turn it on after 12 minutes to signal that the time is arriving to an end. OK. OK, great. All yours. Oh, I cannot share my screen. You have to allow me to to share my screen. So all here we go. OK, great. Thank you. Great. So thank you. I would like to first, obviously, thank you by thinking alive for organizing this symposium and inviting me to present the research that we have been doing in the lab for the last few years has to do with a regulation of asymmetric subdivisions. And obviously also all of you for for attending in in a virtual manner. You think about an asymmetric subdivision basically two things must happen. And this is first depolarization of the cell and on predetermined axis. OK. And then to ensure that the genomic material is distributed according to this polarization, the cells must ensure that the mitotic spindle is oriented parallel to this polarity axis. The mitotic spindle is a bipolar array of micro tubal that allows for the segregation of the chromosomes. And interestingly, this structure is itself an asymmetric structure in some nature. And this is particularly true at the level of the micro tubal organizing centers, the M-tox, which are these structures located at the poles of the bipolar array and from which the M and the micro tubals that form the spindle M and A. The M-tox are known at the center of something higher eukaryotes or the spindle pulvates in mudding gifts. Now, when the cells start duplicating, they only have one of these M-tox. But soon after they start cell division the M-tox are going to duplicate. And interestingly, it was found originally in the budding gifts that the micro tubal organizing centers can be asymmetrically inherited. As specifically in Cervicea, what was found is that the all spindle pulvary is always inherited by the daughter while the new spindle pulvary is retained by the mother cell. This was later shown to be an evolutionary conserved phenomenon that can be observed in the division of many stem cells in different organisms, including humans. We have recently shown that the asymmetric inheritance of the spindle pulvates is necessary to maintain the replicative lifespan of the budding gifts. And this is due to the fact that this process is essential to maintain normal levels of expression of the C2C2in and also to maintain also the transport of functional mitochondria into the daughter cells. Two processes that are required to preserve the protein aggregates and malfunction proteins within the context of the mother cell during cell division. Therefore, when this spindle pulvary inheritance is somehow preserved, we have an accelerated aging. Thus, we are interested in understanding mechanistically how asymmetric distribution of the micro tubal organizing centers is established and maintained by the cells. And we are in the lab not only looking at the consequences, but also trying to understand the mechanisms leading to this phenomenon. We know already some of the players and not surprisingly, maybe some of them has to do with the process of the spindle positioning. Maybe the most important protein in this sense is the carnaine protein, which is the homolog of the adenomatose polyposis coli, the APC protein in humans. And this protein is interesting because it loads the old spindle pulvary in metaphase and from there is going to travel through the micro tubal towards the tip, what it's going to buy in a myosin motor that traveling through acting cables is going to push the old spindle pulvary into the daughter cell. We know also other players such as the Sui-1 kinase components of the men pathway, which is the equivalent of the hippo pathway in mammals, or the most upstream component that we know so far, which is the SPC-72 protein, which is a gamma tubulin complex receptor, all of them working to the red carnaine towards the old spindle pulvary. However, the picture is still not complete. And as I mentioned before, we're still looking for new factors that intervene in regulating asymmetric spindle pulvary inheritance. We knew that the polo, which is a protein kinase of an important family of cell cycle regulators, was necessary to establish the mother daughter sensual asymmetry in the neutrons of the soft blood. And CDC five, which is the cheese polo homolog, was shown to localize to the spindle pulvary since it's a kind of service here. So we decided in the lab to analyze whether polo kinase could have a role in the asymmetric inheritance of the spindle pulvary symbiotic use. To analyze the spindle pulvary inheritance in bandages, we use a methodology that you can see here that basically is based on using an inner component of the spindle pulvary tagged with a slow folding red fluorescent protein. And due to the conservative measure of the spindle pulvary duplication, this allows us to distinguish the old from the newest spindle pulvary based on differences in fluorescence. Because the newly generated spindle pulvary mostly incorporate no fluorescent protein. Now, if we do this and we compare wild-type cells with cells that express a CDC five allele that we can conditionally inhibit with an ATP analog, you can see how in exponentially growing cells after inhibition of CDC five activity, we get a randomization of the spindle pulvary inheritance. This indicates that polo kinase does indeed plays a role in asymmetric spindle pulvary play determination. We then start looking for possible targets of the kinase of the spindle pulvaries. And obvious first candidate was SPC 72. As I mentioned before, it's a gamma tool in complex receptor from the CDK five rep to family because it was shown to be phosphorylated both in vivo and in vitro by CDC five. SPC 72 referentially localizes to the pre-existing spindle pulvary in saccharomyces cerevisiae. It's not a full asymmetric localization such as that of car 9, but it is obviously enriched. So the first thing that we did is to check whether the distribution of SPC 72 changed after inhibition of polo like kinase activity. Indeed, similarly to what happened with the spindle pulvary fate, as you can see here, distribution of SPC 72 was randomized after inhibition of CDC five. However, when we look at the association of SPC 72 with the old spindle pulvary, you can see how it was still maintained after CDC five inhibition, which indicates that the CS5 role in SPV fate determination was either downstream or impalavent to that of SPC 72. In any case, since CC five, as I told you before, phosphorylates SPC 72, we check whether there were other aspects of SPC 72 function that could be regulated by the kinase. To this end, we synchronize cells in G1 and allow them to progress synchronously into the cell cycle. And we analyze more carefully the localization of SPC 72. SPC 72 localizes to the oldest spindle pulvary that is there in the cell end of the cell cycle. But soon as the spindle pulvary duplicates, as I mentioned before, it's going to asymmetrically localize to the old spindle pulvary. However, as cells reach another phase, the protein becomes more symmetric. However, in the absence of the CDC five activity, you can see how the protein remains mostly asymmetrically localized. Not only this, what we observe if by looking at the total fluorescent levels is that you get a reduction in these levels, indicating that CDC five regulate both the timing and loading of new molecules of SPC 72 to the spindle pulvary. We then continue by looking at other possible targets. And then we focus on nine again. Carnite was a randomized in an asynchronous of culture. But in contrast to what happened with SPC 72, in this case, the lack of CC five activity did perturb the preferential association of Carnite with the preexisting spindle pulvary. Carnite, in fact, has other localization in the cell, not only to the spindle pulvaries, and we also observe that the protein was nuclearly retained in these conditions and localized to the inner microzooms of the spindle. So these are other observations led us to propose that what CC five doing is to promote post-translational modifications in Carnite that promotes this nuclear export and its association to the old spindle pulvary. Finally, we look at the independence in SPC 72 and Carnite localization. And we demonstrate that in a Carnite mutant, SPC 72 localized normally, but SPC 72 is essential for Carnite to localize. Then we look at the possible interdependence between the three proteins that interplay between SPC 72, Carnite and CC five. And then by coin monoprecipitation, we demonstrated that SPC 72 and Carnite as a seed and the association diminished in the in the absence of CC five. And not only that, in the absence, there is an increase in the number of cells that display opposite localization of protein. So finally, just to conclude, this is the model where we propose is the CC five as a molecular timer that establish a spindle pulvary fade by promoting an official temporal and special recruitment of SPC 72 and Carnite to the spindle pulvary. OK, so with this, I just want to end by thanking the funding and the people, acknowledging the people that did the work. It's mostly don't work done by a really talented student in the lab, Laura Matilla, with the help of also extremely talented postdoc, Javier Manfaro. And with that, I just want to thank you for your attention and happy to take any any questions. Right. Thank you very much, Fernando, for a great, great talk. I would like to remind everyone to please post your question on the chat so far. There's just one message from from Anya. So please do post questions for Fernando and for your team. So maybe I can start with a with a question for Nando. So I was actually. So this is definitely not my my field, but I was surprised to learn that the daughter cell receives actually the old M talk. So and so that means that mother cells always get it's a new one, right? Because every every time they form the new one, they will then transmitted to the new daughter cell. Is that right? Is it said again? Sorry. So I was surprised to to to to learn that daughter cells inherit the the the, you know, the the the old M talk, in a way, like the mother. Yeah. And so that means that at every division, the mother cells forms a new a new one, basically, right? Yeah. And so the mother cells is always provided with a new with a new M talk. And so that means that somewhere in a clonal line of of yeast, there will be one daughter cell that has a very, very old, you know, I was wondering whether it's possible to trace the age of this M talk and whether that has any impact on on replicability or any age related phenotype in in this cell. Well, you know, I guess at some point, you know, so it's true what you're saying. So basically, the there's always going to be a cell that is going to maintain the the the older. But at some point, you know, there is even though the the the duplication of the spinable body is conservative, it's not fully conservative, it's semi conservative. There is some, you know, there is some renewal of the protein. So at some point, you know, that that is renewal and that is a reconstitution of the spinable body. You know, it's just that it takes longer. It's but but the the the thing with the difference in the inheritance is that you inherit different post translational modifications in this in the in the proteins that, you know, that made the spinable body. And to me, I think that that this is the most important is the information that you are receiving together with that spinable body, you know, is not that much. So if you consider the history in that sense, then, yes, it is important. Now, one thing is that in not in all organisms is always the the same is is the old spinable body that one of the goes to the daughter cell in some divisions, OK, for example, in Drosophila in the neuroblast is the same pattern, but in the germline stem cells is the opposite. But interestingly, as supporting our idea of the of the relationship of this process with aging, what is true is that it is the cells that that that retains the old spinable body, that one, the ones that is going to live the longer. That's right. On the contrary. Yeah. And so, you know, following up on that, maybe. So I mean, I guess yeast cells, correct me if I'm wrong, they can also undergo sexual reproduction, right? So you can also have meiosis, right? I was I was wondering in the context of meiosis, rather than, you know, fission due to to mitosis, whether you have a reorganization or whether this this symmetry breaks in a different way. Or whether this is little to do with the whole duplications observed. It's a very interesting question, but we haven't haven't checked anything in terms of, you know, meiosis and and and and, you know, since it's it's a, let's say, you know, you have two divisions in which you have, you know, it's actually more symmetric or asymmetric state. I don't I haven't actually, you know, look into into into this, you know, we focus on the mitosis because it's, you know, the stereotype of asymmetric salivation. So, but then can you connect causally the inheritance of the older SPB to the longevity, like as or as an organizer of yeah, so how can you how can you, you know, mechanistically tie this event with with the cytoplasmic reorganization that may have some activations gene expression or epigenetic activation that may be understood in the field of molecular biology of aging as kind of like. Well, that's what we are trying to do right now, because so so basically, we're moving into the elections one make the mechanism, which is the focus of this talk, because obviously, you know, it was what we published in the context of this special issue. But then we also try to analyze the consequences and of interfering with this process and in terms of a and and and you know, we know that we are affecting mitochondrial transport. We know that we are affecting a suit to suit to levels and and and we know that we are affecting protein aggregates retention and we know that if we over express it to you because we have a reduction of suit to us, we have a rescue, a partial rescue of the phenotype. We know that I also rescuing the mitotic phenotype. We also have sorry, the mitochondrial phenotype. We also have a partial rescue of the but the the the actual mechanism we are still looking into it. So, for example, we know that we we that I mean, it's still not published, but we are looking now into what happens with C2 and we know that C2 is actually not only the global levels. We know we don't think that C2 is is is doing something is specifically at the spina pulvaris. We don't think that the protein itself is localized in there. We don't have reasons to believe that it's localized in there. We believe that is something downstream, but we clearly see that there is not only a global decrease in the levels of C2, but also the pattern of association of C2 with chromatin is clearly different and and and and and and, you know, and that there is a group of of of, you know, of genes that, you know, are affected by by reversing that the process in terms of mitochondria. We also know a transporter that is specifically affected. So maybe, you know, this transporter that needs to be move into the daughter cell. Somehow this needs the intervention of the spina pulvaris to be properly, you know, established. So, you know, we we are looking into the the proper mechanism. But even though we know the pathways that are affected and some of the players still are far from understanding the whole picture. OK, thank you very much, Fernando. Thank you. All right, so I think we can move to the next speaker with Sarah Mouton from the Ereba, I think, in Groningen. So the European Research Institute for the Biology of Aging and the University Medical Center in Groningen in the Netherlands. And the talk of Sarah's the title of Sarah's talk is a physical chemical perspective of aging. Thank you very much, Sarah, for for presenting your work. And I would like to remind everyone to post your question in the chat. And I will leave I will read it once the talk is over. Sarah, the stage is yours. Thank you, Dario. And hello, everyone. Yeah, so to jump right in, in our lab, we are interested in the east of Rumeisen-sur-Visier, which is also known as Baker's East, which, among other cool things, can make delicious bread and beer. Well, more particularly, we are interested in how this single cell organism is aging and what drives aging, the aging process in east. So I'll just briefly introduce you to the east replicative aging model. So when the east cell is born, it starts putting off daughter cells. It will do so approximately every one and a half hours. And with each division, the mother cell completes. It will acquire aging phenotypes, which, however, will not be inherited by the daughter cell. Sorry, can you please share the presentation? Because I think that we are still not I thought I was sharing it. No, we cannot. Oh, I'm very sorry. No problem. Oh, yeah, that's that makes sense. Now we can sit. All right. Oh, sorry. Right. Just move this away. Yes. So replicative aging model. Yeah. So daughter cells will not inherit these aging phenotypes due to the asymmetric division. So sorry, just to get a pointer. Yes. So a mother cell will well, the number of divisions a mother cell completes determines the replicative age. And after approximately 25 divisions, a mother cell will senes and die. On the other hand, daughter cells will just be reset to age zero. And they will be born with a full replicative life potential. So many aging phenotypes have been described in East and most of them are classified under the well known hallmarks of aging. However, how these hallmarks are connected with each other is still not known. So one common factor between aging and longevity associated molecular players is the intracellular environment in which they take they fulfill their function. So we know that there is a physiological optimum at which micro molecules function. And for example, the cytoplasm is packed with micro molecules whose concentration has been proposed to be maintained at the limit of their solubility. Furthermore, we know that in aging, this effective solubility of proteins is decreasing, leading to aggregate formation. However, what is interesting is that old cells do not have increased total protein concentration compared to young cells, which means we need to better understand the physical chemical state of the aging cytoplasm to understand how old cells are different compared to young. And furthermore, we also need more data and more tools to further investigate that. Which brings me to the aim of our paper, which was to construct an initial framework of physical chemical homeostasis of East replicative aging. Now, there are many physical and chemical parameters that determine the intracellular physiology. However, we focused on three, which we were most interested in, which are pH, macro molecular crowding and volume. So we first addressed the pH. And what we saw is that in age cells, the cytosolic pH is decreasing by roughly a half a pH unit or average. And furthermore, what we saw is that there is increased heterogeneity in the old population. When we plotted single cell lines from the cytosolic pH versus replicative age, we saw that this heterogeneity mostly stems from the senescent phenotype of these cells because early in the mitotic lifespan, there is a progressive decline in cytosolic pH and somewhere around the senescence entry point, there is a sharp acidification of the cytoplasm. So when we increased our imaging frequency to be able to pinpoint when exactly this acidification happens, we saw that cells first become senescent and only afterwards, the cytosol acidifies significantly, which means that cytosolic acidification most likely is not a driver in senescence, at least in in cells. Now, we were interested also in this early decline in pH because previously described mechanisms could not fully explain why is this happening. So next to proton pumps, metabolites and amino acid side chains, which are on protein services, can suffer, well, can function as a buffering capacity, can function as a buffer, I'm sorry. And they provide buffering capacity against pH changes. And so what we did is we combined several data sets and we estimated the proteome isoelectric point in aging. And what we were surprised to find out is that early pH changes actually follow very nicely the proteome PI. And we think that this decline in buffering capacity of the proteome potentially is at least in part the driver of this early acidification in aging, which also correlates to lifespan. So actually, it's a problem that the cytosolic pH is decreasing and it's a problem in terms of tool usage because if you want to use fluorescent biosensors, then they're sensitive to pH changes and they're also sensitive to division frequencies. So this was also the case with the Golden Standard Fred Fair, which is a CFP and a YFP and which is usually applied for fluorescent biosensors, because this red bear is impacted both by the variable division frequency of these cells and by the pH changes. So what we did is we tested several Fred couples to see what would be the best approach to tackle this problem. And I can't show you the data right now. I don't have time for that. You can read it in the paper. But we found this MEGFP and M Scarlett to be much better Fred bear to withstand the challenging intracellular environment and therefore it provides much better readouts. And furthermore, this optimization, as far as we know, should be applicable to all Fred based sensors. Therefore, being a well ubiquitous aging tool for future aging applications. So now with our optimized tool, what we decided to do is to follow macromolecular crowding in single cell aging. And we were kind of surprised to find out that despite dramatic cell volume changes, there was, well, macromolecular crowding seemed remarkably stable. However, there was a little bit of increased heterogeneity, and we also saw that there is a weak correlation between the ability of longer left cells, well, the ability of cells to maintain the macromolecular crowding and replicative lifespan. So as I mentioned, we were quite surprised to find out that there is such dramatic increase in cell volume, and yet macromolecular crowding is stable. And so what we did is we contacted our collaborators in Yale from the Lost Club because we knew that recently they have obtained images of old and young cells through correlative light and electron microscopy. And so we were interested to follow the cytoplasmic volume, but also other organelle volumes, which we were able to identify in these images. And so what we were able to find out is that in old cells, vacuoles occupy approximately 30 percent more volume relative to the total cell volume versus young cells. And furthermore, the cytoplasm occupies 25 percent less volume compared to in young cells. And we were not able to identify any significant changes in the nuclear volume fraction between young and old cells. So to further follow up on biological consequences from this increased organelle crowding, we estimated interorganel distances between young and old cells. And what we found out is that in young, most common distances are 500 nanometers, while in old cells this is reduced to only 100 nanometers. And assuming that the viscosity of the cytoplasm between young and old cells is the same, we see that a 20 nanometer particle will diffuse from one membrane to another 60 times faster. And for a 40 nanometer particle, it will diffuse while it will encounter a membrane 400 times faster compared to in young cells. And so just to give you a flavor of what cell components are in this size range, these are large molecular complexes such as ribosome, RNA polymerase 2 and also ribosomes. Yeah. So just to conclude, we have taken the first steps to explore and construct a physical chemical roadmap of east replicative aging. And we found that in two out of the three parameters we studied, there is a change. And we see that old cells are crowded with organelles. There is increased membrane surfaces and highly reduced interorganel distances. Despite these dramatic changes, we see that macromolecular crowding on the level of single protein is well maintained in aging, particularly so in longer left cells. And we think that this would suggest that maybe macromolecular crowding is tightly regulated. We don't know that. We don't know how macromolecular crowding is regulated at all in cells. And we think that big changes in macromolecular crowding are most likely not compatible with life. Furthermore, we see that the cytoplasm is acidifying early in aging, and this follows the proteomyzoelectric point. And upon senescence, the cells further acidify dramatically. And finally, we provided a tool, a new optimized tool, which we hope that will help further studies in aging to address other physical chemical parameters. And finally, in the future, we'll be interested to see what are the consequences of these changes and also how they can help to tie together some, at least, of the hallmarks of aging. And with this, I would like to thank the organizers for providing me with the opportunity to present our paper today. Also, my supervisors, Lisbeth Vainhoff and Arnold Borsma, my colleagues from the Vainhoff lab, our collaborators, and funding. And I will be happy to take any questions. Thank you. Thank you, Sarah. Great talk. Thank you also for sticking to the time. And there are a few questions. So the first one is from Craig Walling from Edinburgh. He's a PI. Edinburgh is a PI. So do the cells that show the biggest change in pH show the most aging in other traits? Yeah, this is a good question. So we follow pH independently from the other parameters. Although we can see actually whether these cells have large vacuoles, but it has not come to my attention whether these cells that have large changes in pH they will show other traits. But for sure, these are cells that live shorter and usually they send us faster and also they divide a little bit slower. So indeed they do have some other aging phenotypes there. I hope this answers your question. Thank you. There is another question. This time from Steve Kenham. It is known if, so is it known if the decrease in cytosolic pH is potentially related to leaky lysosomes impaired autophagy in aging, for instance? Yeah, this is a great question. I think I've gotten this question before also on the conference. We have not seen the vacuole rupture. It's possible that this is a very, I would presume if it happens, this is a very quick event. So it could be that with the imaging frequency we had, we have not seen it because we imaged once every 10 hours and later once every one hour. So it could be that just in the 80 cells we analyzed we never encountered this. So it could be happening, but we haven't seen it. We have seen it, I think in the Glam images, but it could be that these are also artifacts from a sample group. All right, thank you. So there is another question from Garda Alsalee. So she says, great talk. Do you think that this inhibits the pH? So this leads to autophagy and lysosome and this leads to protein aggregation. So I guess whether these changes in pH leads to autophagy and changes in protein aggregation directly, causally. Yeah, I don't know anything about autophagy, whether it's promoted or inhibited by changes in cytosolic pH, to be honest. Yeah, I would have to look that up. Whether it leads to protein aggregation, we don't know yet. There was an experiment we wanted to do where we wanted to attack HSP 104 and then fluorescently and then see whether there is like aggregate formation upon cytosolic acidification. And I'm happy to say this experiment is done now, but we haven't analyzed it yet. So I can't tell you the results yet, but I think it's a very valid question and we don't know, but what's interesting is also, you know, if the pH changes and it hits the isoelectric point of certain proteins, then they become less soluble, they can precipitate. So in principle, it is possible, yeah. So there is a question from Martin Bagic. Do we know anything about why the pH, not the PI of the buffering proteins decreases with age? I think it's pH. Is this specific to yeast? Oh, that's a good question actually. I'm not sure whether, we don't know whether this is specific to yeast. I haven't seen any other papers on that. Yeah, and why is it decreasing? I guess the protein composition is changing. I have to say our prediction carries certain, well, some uncertainties related to well, PI predictions and also to aggregate formation that one of our reviewers pointed out actually that proteins that will aggregate will impact differently the proteome isoelectric point. So yeah, we don't know why that is happening. I would presume, yeah. It can be the other way around too. It can be that the cytosol is acidifying and then the proteome is adapting to this new environment that is also possible. Exactly, I was thinking whether actually these responses are compensatory or truly dysfunctional. That's oftentimes the big question in the aging field. Yeah, yeah, we don't know. I actually have a quick question regarding crowding. So do you think that this is actually a big problem with aging also in other systems, in other cell types where the molecular crowding actually leads to traffic jam and to functional impairment. And if the answer to that is maybe or yes, do you think that there are cell types that are more crowded than others and how do you think they solve this issue or this problem has been already tackled by evolution probably? Yeah, so your question is whether there are cell types that have increased crowding and how, yeah, how they tackle this. So that's a good question, I think. For example, it has been proposed that in bacterial cells, crowding is, for example, the highest and then potentially it's reduced in yeast cells and for sure it's known that in mammalian cells, the crowding is even less. So like membrane organizations for sure promote, well, I guess you get more of intercellular organization and this is how it's tackled this problem. Yeah, I don't know. Does that answer your question? Yeah, yeah. Yeah. Okay, thank you very much, Sarah. Thank you. Okay, so we can move to our next speaker who is the last speaker before our first break. And so Dr. Andre Parkitko from the Agent Institute of UPMC at the University of Pittsburgh, the USA. And the title of Andre's talk is tissue-specific manipulations of methionine and tyrosine metabolism extend drosophila lifespan. As I said before, please, if you have questions, do ask in the chat, do write it down in the chat. You can add your affiliation as well and I will read them after Andre's presentation. Andre. Thank you. Thank you for organizing this symposium. It's really great and thank you for giving me this opportunity to present my data today. So different labs, we use variety of omics approaches to study agent. So I'm very interested in how metabolism changes with age and where we can target these changes, and the changes to extend health and lifespan. And to do this, I decided to use drosophila as a model system because we can quickly manipulate different metabolic pathways there. And to start with, I decided to use a model from Trudy Makai Laboratory. So we developed flies that were selected for exceptional longevity. And so I used these flies to do metabolomics at different ages. And so when we compared how like metabas... Like general metabolism changes in control flies and in flies with these exceptional longevity to see where we can see differentially affected metabolic pathways. And so when we started with the first metabolic pathway that appeared in our analysis was metanin metabolism. And I'm not going to spend a lot of time on this just because we published this before. I just wanted to discuss our models so I can move forward. So metanin that we get either from food or from our microbiota. So half of these would be used for protein synthesis but half of these would be used for metabolic needs. And so metanin will be converted into essential metanin which is or SEM, which is the major methyl donor in our cells. And we have a few hundreds of methyl transferases that use this methyl group from SEM. And after it donates methyl group, it will be converted to essential homocysteine or SEM which actually can competitively bind to these methyl transferases and then give it to them. So it is very important for cells to keep appropriate amount of essential homocysteine in our cells and convert it to homocysteine and back to metanin. And so what we found that when flies age they will accumulate amount of SEM and homocysteine. And so if we can find a way how we can reduce these H dependent accumulation, for example, they can do it the metanin restriction will be reduced amount of metanin or the activation of the flux via metanin metabolism, the activation of this enzyme, which is called H13 or sec in humans. So when they can extend health and lifespan. But another interesting question actually is can we define a specific organ where metanin metabolism is important for aging and for regulation of lifespan? And we cannot do it simply by reducing the amount of metanin in flight food just because when we reduce the amount of metanin food we will reduce amount of metanin in all different organs. And actually it would be by different extents in different organs. So what we decided to do is I wanted to use this enzyme called metaninase. So it doesn't exist in humans or in flies but it exists in different bacteria. And so this enzyme will degrade metanin to free volatile substances that can be easily excreted from blood or from hemorrhage. And so we created a transgenic flies where we can induce expression of this enzyme, metaninase. And so we analyze these flies, I will quickly walk you through this slide. So here in red, you can see flies like regular flies which are maintained on regular food. And in light blue, you can see flies that are maintained on the chemically defined food where we know each component in this fly food. And that has one millimolar metanin, sorry, in dark blue. And you can see that these flies have the same amount of metanin as flies in the control diet. So meaning that one millimolar metanin is what we have in control food. But when we either completely move metanin from this food or we express this enzyme metaninase, we can almost completely wipe out metanin from our flies or we can, for example, change amount of metanin, like slightly decrease it and we can cause metanin restriction. So it's interesting when we increase amount of metanin. So the level of metanin itself would not increase, but when you analyze downstream metabolites, I don't show them here. So they will sky up meaning that cells they have keep a proper amount of metanin. And so when we analyze downstream metabolites like I showed here Sam, but we analyzed metabolites. So we will follow the same pattern as we observe for metanin. So meaning that these enzyme metaninase could be a very efficient tool to deplete metanin in a tissue specific manner. And so we started to analyze which, like using different tissue specific inducible drivers to see which tissue would be responsible to depletion of metanin and extension of lifespan. And here I just wanted to show you that when we deplete metanin in intestine cells, so where the flies actually live much longer than control flies and they live over a hundred days, which is quite long for flies. And I also want to mention that these recombinant form of this in time has been used in different cancer studies. So we can actually like give it to humans and to reduce plasma metanin in humans potentially we can translate our studies into mammalian systems. Okay, but the big question is can we identify other metabolites that are responsible for aging and can we manipulate these pathways to increase lifespan? And here I want to show the heat map of all different metabolites that change differently with age in control and lonely flies. And so many of them actually you would recognize as very well-known players in aging, for example, NADP, but fundamental light that attracted out attention and also in the acids is tyrosine. So you tend to decrease in control flies with age, but when you look on the lonely flies to different species like lice or lonely flies, it would actually significantly increase with age. And just to remind you again, tyrosine like part of tyrosine will be used for protein synthesis. But so the main role of tyrosine in our cells is to produce tyrosine derived neurotransmitters like dopamine, octopamine, and tyramine. And octopamine and tyramine are analogous to epinephrine and norepinephrine in humans. I told naturally, tyrosine can be degraded via the tyrosine degradation pathway to produce acetylacetate and fumarates so it can feed into the T-cell cycle and play an aperitif problem. Because we found that the level of tyrosine changes with age, so we asked the question, so the levels of enzymes in tyrosine degradation pathway which would control tyrosine degradation would also change with age. And we found that several enzymes transcriptionally upregulated with age in puncture flies. And so we used JP trap for the first and greatly emitting enzyme in the tyrosine degradation pathway to transmit the transferase. And we found the protein level of this enzyme also is increased with age. So interestingly what we found, so if you take young flies and actually disrupt mitochondrial function in these young flies, they're using RNA against mitochondrial electron transport chain. So that would dramatically upregulate the level of tyrosine transferase and cause these upregulation of tyrosine degradation pathway in young flies like phenocopyne agent. So when the event proved where these tyrosine transferase is really required for tyrosine degradation. So what we did, we used CRISPR-Cas9 approach and we generated null flies for tyrosine transferase. And like if you like absorb the lifespan for just for a month, so the lifespan will be similar. And so when you apply excess of tyrosine in fly food, so wild type or heterozygous flies, they would not care about this, but homozygous flies, they will start acutely dying just in a couple of days. So meaning that this enzyme is really important for degrading X amounts of tyrosine in normal situation. So because the level of tyrosine tend to decrease in control flies, it is high in lonely flies. And because levels of enzymes and tyrosine degradation pathway also increase with age. So we wanted to ask a question. So what happens if we are suppressed with enzymes? So how does it affect lifespan? So we down degraded tyrosine immunotransferase with V and strong RNA in whole body. And we have sort that these flies have extended lifespan. And similarly, if we down-regulated to other enzymes like downstream enzymes in the tyrosine degradation pathway, we also can extend lifespan. So meaning that if you suppress tyrosine degradation pathway, we can extend just for a lifespan. So we then ask a question. So where is the specific tissue which is responsible for this regulation of lifespan? So we down-regulated tyrosine immunotransferase in different tissues. And we found if you use a neuro-specific Genswich driver to suppress tyrosine immunotransferase, so when it actually extends lifespan even more efficiently when you suppress it in whole flies. So meaning that it has own pros and necks to suppress it in whole flies probably like it is required for normal aging in other tissues, but it is specifically detrimental when it is up-regulated in neuronal tissues. So as I mentioned to you before, like one of the main roles of tyrosine is production of tyrosine-derived neurotransmitters. So these vile type and homozygous flies that we generated to test the levels of different tyrosine-derived neurotransmitters in collaboration with D.V. Ramesh and Axel Rockman in Bangalore. And as expected, when you suppress tyrosine-degradation pathway, the level of tyrosine itself is going up and we found that the levels of different neurotransmitters it was also up-regulated. And here you can see the levels of dopamine and tyramine and we found the same for octopamine. And interestingly, when we compare just the level of neurotransmitters that are not derived from tyrosine so the levels don't change. So it is not just general response for different neurotransmitters, it is specific for tyrosine-derived neurotransmitters. Okay, and just to understand a little bit the mechanism of how we study the pathway is responsible for regulation of flyspan. So we use several reporters for processes that are known to be relevant for aging. And we found that the level of GCD-JFP, which reflects the amount of oxidative stress would be increased in controlled flies which would be expected. But when we suppress tyrosine-degradation the transferase, so we suppress this accumulation. And actually, so when we serve like consistently we spend the feed flies to the far apart so we can increase the resistance to oxidative stress. So potentially one of the potential mechanism how this inhibition of tyrosine-degradation pathway would extend lifespan is increasing the resistance to oxidative stress. And finally, just to finalize our data, so we believe that in young flies, majority of tyrosine actually is used for the production of dopamine, tyromine and octopamine. And so when flies age or like somehow they have mitochondrial dysfunction, so cells, they try to compensate loss of mitochondrial function and they reroute tyrosine from making neurotransmitters into the TCA cycle. So that actually would suppress production of neurotransmitters. And actually we know that from different model organism and from human studies that their levels would be degrees of age and it would power aggravate the mitochondrial function. And I just want to mention that there are two different FDA approved drugs. One is called nitizinone which is an FDA approved drug and what would suppress tyrosine-degradation. And another drug which is called tiger-cycline but in gibbets mitochondrial translation, I didn't show you this data, but when you give it mitochondrial translation, you can suppress this mitochondrial dysfunction and use a regulation of the resin amino transferase. And at the end, I just would like to thank Norbert Perimont, my former mentor at the Heart Medical School and other people in Norbert's lab who helped me with this project, my collaborators on this project and our funding. Thank you. Thank you, Andrei. Great talk. We have several questions. So the first one is what is the concentration of RU-486 use with the tick smells and when is the inducer applied? Yes, it's a great question. So we only apply RU-486 in adult flies usually starts in the day seven. So when all development is already finished and we have adult flies. So I believe we use 150 micromolar concentration of RU-486, like as most other people in flight would use for these genetic drivers. Thank you. This question was, by the way, by Michael Riva. So what happens if you suppress tyrosine degradation later on in life? It's a great question. So we never tested it by ourselves. So it would be like great to test if we can make more transitional conclusion and suppress tyrosine degradation pathway with R&I or even with drug in older flies. I think sometimes the problem, so we haven't tested it because we'll need a lot of flies and we haven't done it. But also some people argue about invisibility and tissue specificity of gene switch system in older flies. So, and I don't think it could be a problem. So I haven't tested it, but it would be great to do. Sorry, maybe I missed this, but do you see different effects in males and females? Yes. So we see different effects in males and females. So for example, sorry, it's a bit complicated question. So for example, when we use neuro specific driver and express against tyrosine in the transferase, we see extension only in females. But then you can argue that females actually eat more food and so they consume more RQ486 from the food. So you don't know where the males, so we don't see extension of flyspin in males because they eat less and they get less in user and you know, legs down regulation or it's just because it's male to female specific fashion. And I think it's an amazing fashion. So we see a lot of sex specificity and it is something that we would like to address in the future. Cool, thanks. This is a question from Sarah Hack. Would suppression of tyrosine also be expected to extend the lifespan in humans or maybe associated with more healthy states or organs in humans? Yeah, it's a great question. So what we are doing right now, so we got a pilot fund from the paper center. And so we are going to take old mice which are really old and you know, feed them because I'm interested in tyrosine and titanium tyrosine. So we are going to target both titanium and tyrosine metabolism in really old mice and see whether it would suppress like felt index and you know, different phenotypes associated with aging. And I should mention that again, both metaninitrizes and tyrosinitrizes you can target if you know, I've a FDA approved drugs or drugs that have been used in humans like recombinant metaninase. So I think if you can see a good effect in mice then you know, we should think about how we can translate it into humans. There is one more question from Kurshid Wani. Have you specifically tested the role of neurotransmitters or the neurons that produce these neurotransmitters? This is from UMass Medical. Yeah, so it's again a very interesting question. So what we tried, we took just wild type flies and feed them with you know, octopamine, tyramine and dopamine at the concentration that have been shown before in flies to rescue, you know, these like deficiencies of these neurotransmitters like different genetic models. So we found a slight extension of lifespan and so like again, but it wasn't as great as the absorb the down-regradation of tyrosine and neurotransferase. And again, there are multiple explanation for these it could be just because when you feed neurotransmitters you don't see, you know, distribution of these neurotransmitters like to different neurons. We never tried, we never located, you know, specific neurons which are responsible for these phenotypes and again it would be great to test, but I also want to mention that there was a recently very interesting paper and cell reports when people showed that if you feed octopamine, it would actually phenocopy effect of dietary restriction without, oh, sorry, effect of exercising without exercising. So it's, so I think it's, so it would be interesting to like, you know, cover tests, you know, interaction between your neurotransmitters and, you know, trying to, are the tyrosine levels in long-lived mutant flies or in flies that undergo DR dramatically affected? In DR? Yeah, and in long, also in long-lived mutant, you know, classical long-lived mutant. So we only tested, so we, you know, selected flies and we got a lot of species in them because, you know, these flies, it's the claspin is not the only trait but it's different with these flies. But, you know, by also, so we couldn't measure, you know, tyrosine using like, you know, biochemical kids. So we only relied on metabolomics and so we haven't tested, you know, other mutant, like long-lived mutant. So it would be great to do, but we haven't done it yet. Right. Thank you so much, Andrei. And I'd like to thank also all the other speakers of this first part of the symposium.