in running for a while now, aims to give early career researchers and online platform in this crazy time to continue to share their research as an alternative to in-person gatherings. I am Deidler Weigel. I'm Director at the Max Planck Institute in Tübingen and I'm also Deputy Editor at E-Life. I've been with E-Life from the very beginning since 2011. I'm original Developmental Biologist by training, working on Drosophila and later switched to Plants and now work on Plant Evolution and Immunity. So today we'll have three talks again. We'll hear from Enan Morales Navarrete, who is a junior researcher at Pontificia Universidad Catolica de Ecuador in Quito. He'll talk to us about multi-scale analysis of liver tissue organization. He is a recent awardee of one of our travel grants, which he can use at the next in-person opportunity. And meanwhile, he is gonna present the talk to us that he was planning to give at the BioSB20. Next up will be Carolina Makovsky, who recently became a postdoc at UC San Diego in the US. She'll be presenting work from her PhD, which she carried out at McGill in Montreal, Canada. She'll be speaking to us about probing myelin in first episode of Psychosis with MRI, a framework to understand negative symptoms and verbal memory. And this talk again was initially scheduled as part of a symposium at the Society for Biological Psychiatry as OVP Conference in New York. Where Carolina was also to be awarded in Early Career Investigator Award. Hey, hello. And then finally, we'll have Philip Lange, Assistant Professor in Canada Research Chair at the University of British Columbia in Vancouver, Canada. And he'll talk to us about the proteome landscape of pediatric leukemia in patients and xenograft models. So each of the talks is gonna be 10 minutes. And after the 10 minute talks, we'll have five minutes of questions for each speaker. And then we'll move on to the next talk. So to ask a question, any of you participants can type it into the chat on Zoom or directly into the Google document, which is linked in the Zoom chat window. And we are joined today in the background by Miranda, Anya and Naomi from E-Life, who are going to support us. And they'll help me to line up your questions. If you're able to, I'll invite you by name to ask your questions and the team will unmute you so you can do that. Otherwise, I can just read your questions out loud and include your name where possible. And then we'll also have this open notes document which is another place for you to contribute shared public notes. We welcome you to do so and to list yourself as contributor in the list above the speakers for today's webinar. So thanks a lot for doing this. Finally, I'd just like to let you know that we are recording the webinar and also live streaming it on YouTube. And during this live webinar, we ask you that you please be respectful, honest, inclusive, accommodating, appreciative and open to learning from everyone else. Do not attack the mean disrupt harass or threaten others or encourage such behavior. And if you feel uncomfortable or unwelcome on any of these webinars, please contact E-Life by email to events at elivesciences.org. And the inbox is being watched by Anya Starris from E-Life. And of course we reserve the right to ask anyone to leave and or to deny access to a subsequent webinar on Zoom. If you need any help at any point, please send Miranya, Mirando or Anya a chat message directly using Zoom. So in Zoom there's this functionality where you can send private messages. All right. So first we'll hear from Ernan about multi-scale analysis of liver tissue organization over to you, Ernan. Okay, just give me one second. Can you see my screen now? Yeah, here it is. Can you hear me? It is on, perfect. Thank you. Okay, first I want to thank the organizer for giving me the opportunity to present my work today as part of the E-Life online research talks. So now we'll tell you about the multi-scale analysis of liver tissue organization. It was, this was a project I was working on during my stay at the Google Professor Marino Serial at the Max Plan Institute of Molecular Biology and Genetics. So let's start. One of the major challenges in cell and developmental biology is to understand how cells can form complex dimensional tissues. Probably nowadays one of the most powerful to address this question is microscopy. Here I'm showing you an example of a confocal image in three dimensions of liver tissue. As you can see here, we can observe the different cells forming this complex tissue in 3D. But most of the time, just visualizing these components is not enough to understand the structure of the tissue. So we have to go one step forward and we actually have to perform a digital reconstruction of the tissues so that we can perform also a quantitative analysis. So here I'm showing you an example of the reconstruction of the different cells forming the liver tissue in 3D. So once we have the different components, the different cells forming the tissue, so we want to understand what is the actual tissue organization. So what we think is that this problem is like to try to solve a puzzle in 3D. So where we actually have all the pieces, like meaning the cells and the different components of the tissue, we know the shape of the different components. We know the final configuration of the tissue and what we want to do is try to find out where are the rules that we have to follow with all these pieces together to perform a functionally dimensional complex tissue. So during this talk, I will tell you about how we try to apply these concepts into the analysis of a specific tissue that is liver tissue. So for this, let me first give you a very introduction into liver. So liver is a vital organ that is in charge of several functions like bicyclism or metabolism. The minimum structural unit of the liver is called the liver lobbyol. So we can understand the liver lobbyol as a two-pipe system. So on one side, we have the sinusoidal network. So the blood is entering to the liver, to the portal vein, going through the sinusoidal network and going out towards the central vein. So we have another network, the canaliquin network that is transporting bile in the opposite direction towards the bile duct and out of the liver. So between these two networks, we have the parasites. The parasites are epithelial cells that have a very peculiar and unique apical basal polarity. Every single parasite is facing several sinusoids which allows for the proper exchange of nutrients with blood and the apical member of this parasite between the parasite is forming the bicanaliquids which together give rise to the bicanaliquin network. We want to understand how this organization at several levels can give rise to a global tissue organization. For this, as I mentioned before, the first step is try to get the actual shape of the different components. So a few years ago, we developed a pipeline for the imaging and reconstruction of these different components of the tissue and multiple scales. Briefly, so in the first step, we image the tissue, like a big portion of the tissue, meaning around one cubic millimeter of tissue with lower solution. This allow us to reconstruct a big structures like the veins, the central vein here in Cian and the portal vein in orange. So in the second part, we take a smaller piece of the tissue here, these white boxes that we can see here. So if we do the imaging higher solution, we are able to reconstruct a cellular level components like bicanaliquin here in green and the insular network in magenta and also individual cells. And we can do it with such a resolution that we can even identify subcellular components. This is an example of a nepotocyte where we can identify the apical membrane here in green, the basal membrane in magenta and the lateral membrane in gray. So we did this implementation in a software that is called motion tracking, that is free to use. So now let me show you a full reconstruction of the small part of delivery of your, so the central portal vein access. From here to the end of the presentation, you will always have the central vein in Cian on the left side and the portal vein orange on the right side. So this reconstruction of bicanaliquin network, as you can see here, is a complex three-dimensional structure, very similar to the sinusoidal network. These two networks are never touching each other, but they are globally organized between central and portal vein. This is the nuclei of the different cells that are from the tissue and this is the reconstruction of the cell surface of the different parasites. As I mentioned before, every single parasite is in contact with these two networks. As you can see here with the bicanaliquin and the sinusoidal network. And this also defines the apical and basal patches of every single cell. As you can see here in this example, the parasite have what is called a multipolar organization of the apical and basal compartments. So in this project what we want to understand is how this multipolar organization that looks a bit complicated to explain at the cellular level can give rise to two networks that are functional at a global level, meaning a tissue level between central and portal vein. To do so, the first thing that we have to do is to describe mathematically and the polarity of the parasites. So for this we had a collaboration with Benjamin Friedrich and Frank Hewley here from the Max Plan Institute of Physics of Complex Systems. So with them we use tools from the physics to mathematically describe the polarity in the case of cells with the multipolar organization. So just let me briefly explain you. In the case of simple polarity, imagine that you have one cell where you have only one patch of any market could be the apical market, for example. And then mathematically it can be easily described with a vector. So it's going from the center of the cell towards the place where you have the maximum accumulation of the market. In the case of cells where you have not only one patch of the market but several ones like in a parasite, we cannot describe this anymore with a vector. We have to use something more complicated. We use in this case what is called a tensor. So a tensor can be described graphically with two axes. So one axis is going to describe the orientation where we have the maximum accumulation of the market and the second axis where we have the minimum accumulation. So let me show you two extreme cases. So imagine that you have a cell where you have only two patches. So the first axis that we are going to call it bipolar axis or A1 is showing you the direction where you have the maximum accumulation of the market. Another example, so imagine that you have not two patches but a ring-like structure over the cell. So A1 cannot be defined because you have all over the surface. But you can define a second axis that is called A2 that we call ring-like axis, which give you the direction where you don't have accumulation of the market or the minimum accumulation. In the real case, like in a parasite, these two axes are uniquely defined. So it means that we can define the bipolar axis and the ring-like axis in a unique way for every single cell. In this case, for the organization of the apical membrane, we can do the same for the basal membrane as well. So for the cell that we saw before, so the bipolar axis is showing the maximum accumulation of the apical surface. And the ring-like axis is showing where we basically don't have apical surface. So once we have the mathematical description of the polarity that we call biaxial-cell polarity because we describe the polarity with by two axes, so we wanted to see how this is organized over the liberal of yield. So here, we just plot the biaxial in one of these axes, the bipolar axis, so you have the same membrane, the portal membrane, and every single axis represents one aparocyte. So from this picture, it's still difficult to see if there is some pattern of organization. But actually, if you only do some local averaging, meaning that you remove the local noise, we could already observe the underlying pattern of organization. It's actually not that there is no organization at all, but there is a pattern behind the organization of the aparocyte polarity. And if you can see, if you see to this organization here, through the mines to a flux line that are going from one side to the other. So we can quantify this. So for this, we simulate the flux, our flux process from center to portal vein, and then we can measure how well these axes are organized, aligned with this. So this is shown here. So this is the quantification. This is another parameter. Negative values mean they're perpendicular. Positive values are parallel. So to sum up, we found the polarity is aligned with the flux of this diffusive process. And this is very similar. So we can do something similar to the Neville, for example, a sinusoidal Neville, and we found a similar result. So it means that both the circularity were parasites on the sinusoidal Neville, a couple, and probably locally organized. So sum up. So today, I show you how we are starting from microscopy images, in particular confocal microscopy images. We can reconstruct the different components of the tissue. And we can use this information to derive some principles behind the organization of the tissue. In this case, we talk about multi-scale organization because we see at cell level, a cellular level, bi-axial polarization of these cells. So at the multi-cell level, we have a coordination between the polarity, the circularity, and the organization of the sinusoidal Neville to different cell types, which at tissue level will serve like a long range organization. In terms of physics, it is called a liquid crystal organization. It's not a perfect organization like your crystal, but it's not totally dissolved like in a liquid. We have something in the middle. We have local noise, but we have some long range organization. So with this, I just want to say my former lab and Marino with Marino Serial and my collaborators at BKS and Pinnacle University of Tennessee. And now I'm happy to take any questions from you. Great. Thank you very much, Anand. Awesome. All right. Let me ask the first question. So this is really amazing what you can do with modern microscopy and reconstructing these networks and the cell surfaces and whatnot. So you did this with hepatocytes. Could you easily transfer this to other tissues as well? Yes. So actually, so in the beginning, we applied this only for the reconstruction of liver tissue, but it also worked for the nicest of kidney tissue or lung. That's something that we are very proud of. So the methods are very general, so it can be easily translated to any other tissue. And so I guess the answer to the other question is chicken and egg type of problem. So you have this sinusoidal network, and then you have the cells. So what comes first is that this sinusoidal network that forms the scaffold structure that then guides hepatocyte polarity or is it the other way around that the hepatocytes control the organization of this network? So that is actually a very good question. So what is describing the literature is that the sinusoidal are making like this scaffold in the beginning. So because they are transporting blood and doing development there, they want to form first. And then they guide the polarization of the parasite. However, we found that it's actually not only one direction, but it's a two-direction organization. So to analyze a little bit more, what we did is we added one protein that is Integrin Beta 1. So Integrin Beta 1 is basically in the cellular matrix. So we did the unknown down the nanoparticles in the parasite. And surprisingly, what we found is that the polarity is still establishing a parasite. However, this liquid crystal organization is gone. So here we see an example of the knockdown of the Integrin Beta 1. And we also observed that the organization of the sinusoid is gone after this knockdown. So since the knockdown was specifically for the parasite, so it means that also the polarization of the parasite is altering the organization of the sinusoidal network. So we think that it's a two-way interaction. We have a certain communication between the two types of cells. Is there precedence in any other tissue where you have a similar relationship between the individual cells and then some supracellular network? So I find that no nothing. But I don't know. OK, all right, cool. All right, great. Wonderful. Let me check. I think that those were all the questions. All right, awesome. Thank you very much, Annan. Thank you, thank you very much. All right, so we have Carolinas slides up. So Carolina already introduced her from UC San Diego. San Diego has a special place in my heart. I was a professor there for 10 years. So I really love San Diego. Carolina, please take it away. Excellent. Let me just share my screen here. Can everyone see this? And can everyone hear me? Excellent. So thank you so much, Dietle, for the introduction and Eli for allowing me this opportunity to share my research in these crazy times. But it's really great to be able to connect virtually. So yes, San Diego is amazing. Montreal is as well. This is where this work was actually conducted since I just actually moved to San Diego. So we're going to shift still looking at imaging techniques. But now in humans, in a psychiatric application. So I'm working here with first episode of psychosis patients. And we want to look at neuroanatomy, particularly myelin, to better understand predictors of poor functional outcome in psychosis. Let's make sure I can advance perfect. So I want you to go back way back into time and think back to when you were a late adolescent. So when you were a teenager and to early adulthood, you can appreciate that this is a time of immense growth.  There's a lot going on at this time that can really set the stage for the rest of your life as well. And this unfortunately is actually when psychosis tends to have its onset. And when people think of psychosis, they often think of positive symptoms. So these are symptoms that are kind of a blurring of what's real and what's not, things like hallucinations and delusions. And these are treated quite effectively with anti-psychotic medication. But there are other factors that are, I don't want to say ignored, but we don't have great treatments for. And these include things like negative symptoms, so like a lack of motivation, social withdrawal, as well as cognitive deficits. And in particular, and what's sort of an enigma in psychiatry is that verbal memory deficits tend to have even more pronounced deficits in patients with psychosis compared to other cognitive domains. So these are the factors that I really want to focus on in this talk. Now, when it comes to the anatomy, every single region has basically been implicated in psychosis. It is a disorder of disconnectivity. But the hippocampus stands out as potentially a tangible marker that we could use for therapeutic purposes. This is actually a figure from Carol Tominga in 2010, which showed that the hippocampus might be central to psychotic symptoms, so positive symptoms. And she basically showed that there's specific lesions within the circuit that can lead to those positive symptoms. And when we look at imaging studies, again, the hippocampus kind of comes out at the forefront where there's the largest effect sizes with volumetric differences. So lower hippocampal volumes in both patients with schizophrenia and bipolar disorder. So there's something to be said about the hippocampus. So for this talk, there's two primary aims. One is the hippocampus connected to other cortical abnormalities. So we wanted to do this by probing intercortical myelin, which I'll show in a moment. And if the hippocampus indeed is central to some of these cortical abnormalities, is it also related to verbal memory and negative symptoms? These predictors of poor functional outcome. So there's lots in the cortex that we can look at. And I'm really gonna be focusing on compartment A here. So these are intercortical myelin fibers. And the reason being is this is a highly plastic compartment of the brain. And also when we look at the proportion of these fibers in the brain compared to other long range white matter fibers, they're actually very, very highly expressed in the brain. So there's more intercortical myelin fibers than say long range fibers. So as I mentioned, this was a study done in Montreal. So we are taking these first episode of psychosis patients from a clinic called PEP Montreal. This is a longitudinal data set, which is really, really valuable and very hard to get in first episode of psychosis. So we're tracking these patients. I have 27 patients and 29 controls with really high resolution imaging. And we track them for about 18 month time period after their entry to the clinic. So the bottom here, you have clinical time points after their first episode of psychosis. And then we have imaging time points currently at the top. And then we also get our clinical and cognitive assessments. So you can see there, we have pretty closely spaced time points to really be able to characterize fine changes in neuroanatomy as well as cognition and symptoms. And these are patients that are between the ages of 18 to 35. So that early adulthood time period I was saying. Okay, so how are we gonna get at myelin deficits in vivo in patients noninvasively? So for this, we turned to a MRI or magnetic resonance imaging technique called quantitative T1 mapping. Where essentially each of these pixels or they're actually in 3D. So each boxel in the slice actually holds a relaxation time in milliseconds. I won't get into the physics here, but all you need to know is that the shorter the relaxation time, it's more likely that there's more fat or white matter in the signals. This is why it comes up really dark on this image. And the longer relaxation times tend to have more gray matter. And it's actually been shown that a large contributor to this T-run relaxation time is myelin content. So whenever you see QT1 on my slides, I'm just gonna interpret it from just for clarity as myelin content. Okay, so we, as I mentioned, we really wanna understand hippocampal centrality in relation to intercortical myelin. So to do this, we have a really high resolution T2-weighted image here that really lets us gauge hippocampal subfields, but we also had an atlas that allowed us to resolve the white matter that is output from the hippocampus. So this includes things like the alveus, fimbria, fornix and mammillary body. And at the level of the cortex, we can probe intercortical myelin layers across many, many portions of the brain, but just for simplicity, we then reduce the data to 62 cortical regions. So we can get these myelin content for cortical and hippocampal regions. So we have longitudinal data. So in order to handle this, what we did is we really first wanted to see is the hippocampal central to cortical abnormalities. So we adapted a method from Ellen Evans Group at McGill University, where we can assess between two regions of interest, this similarity or dissimilarity in their trajectory of myelin across time. So you can imagine ROI is subfield CA1, and this is prefrontal cortex. How similar or dissimilar are the myelin changes across time? And in this way, we can actually get for each person or each participant a subject-specific matrix when we look at all pairwise comparisons. And once we got these matrices, we calculated something called the participation coefficient. And in graph theory terms, this is a measure of centrality. So just to give you kind of a visual of this, this is obviously airport activity pre-coronavirus times, but for those that are familiar, Chicago, here is a very, very popular, well-connected airport. So you can argue that has very high centrality. It'll get you anywhere you want. Whereas an airport such as Missoula International, it has very low centrality. So we can kind of adopt this approach to the brain and we can say, okay, this is our hippocampus with all of its regions, and how is it highly connected or not as connected to these other cortical networks in the brain? So we actually did indeed find lower centrality in first episodes of psychosis patients in this hippocampal unit compared to other cortical networks. And when we looked post-talk, it was really driven by these output structures of the hippocampus, including CA1, alveosporinics, and the millery body, bilaterally, as well as a little bit more left hemisphere representation. Okay, so that's all fine. In Dandy, we have the centrality measure, but I really wanted to bring it back to clinical outcome. So at the behavioral level, we knew from our data and also previously that negative symptoms and verbal memory deficits are related. So having a worsening verbal memory trajectory over time is associated with worsening negative symptoms. And what's really interesting is that lower hippocampal centrality is also actually significantly associated with worsening negative symptoms and worsening verbal memory over time. And once again, when we look post-talk, these results are really driven by centrality of the left CA1, alveosporinics, millery body, and right fibria. Once again, these output structures, mild and rich regions of the hippocampus. And just to bring this all together in a model, we actually found that verbal memory or changes in verbal memory over time mediated this association between hippocampal centrality and changes in negative symptoms. So to sum up, we found that there's disturbed covariance between the hippocampus and the cortex and this might actually stem from mild and rich output regions of the hippocampus in these first episodes, psychosis patients. And we are showing here for the first time, not only is the hippocampus central to positive symptoms as has been believed before, but it's also central to negative symptoms and verbal memory might help to explain this relationship. And the hippocampus, as we all know, is highly plastic. So it positions it well as a potential therapeutic target. And I don't want to get into every single future direction here, but what made me really excited is that we know that hippocampus, for instance, can actually change with exercise. So we know that exercise can actually increase neurogenesis in the hippocampus. So perhaps this could be a really beneficial treatment avenue for these patients with negative symptoms since antipsychotic medications aren't helping them. And finally, there is transdiagnostic potential of this work. You may have noticed I really didn't use any diagnostic categories here. We can actually see verbal memory deficits and negative symptoms in many psychiatric and neurological disorders like epilepsy, Alzheimer's disorder, obsessive-compulsive disorder, just to name a few. So with that, I will wrap up. I'd like to thank you for your attention. This was work supervised by Allen Evans and Matan Lepage in Montreal. You can see all my co-authors there in this paper. The revisions were just submitted last week to hippocampus. So hopefully we'll see a good outcome and I'm happy to take questions. Thanks so much. Great. Thank you. Thank you, Carolina. Thank you for being very much on time as well. So these patients, are they medicated? Yeah. So what's really unique about the clinic that we worked with is in order to meet criteria for a clinic, they had to be relatively anti-psychotic medication naive. So over the course of their life, they could only have a cumulative dose of one month of anti-psychotic medication. So this means that when we did take patients to the clinic and started scanning them, we could really rule out anti-psychotic confounds. Now, of course, once they had their first psychotic episode, they were put on anti-psychotic medication. It's a naturalistic study design. So it's not like they're just randomly, or they're not assigned to a particular medication. But because we worked so closely with clinicians, we could really track down their dosage, their medication adherence. So we actually did do a supplementary analysis controlling for their anti-psychotic medication dosage, which we had very well recorded. For the purposes of the hippocampal centrality analysis, it did not impact our centrality measure at all. But there are some studies that have suggested that a psychotic medication can have an impact on intracortical myelin. But it didn't seem to impact this study. So, you know, being far away, so you say psychosis, is that just one disease? Or isn't it many diseases, psychosis? Yeah, so psychotic disorders definitely have, they're quite heterogeneous. There's a wide spectrum. So most patients will go on to have a diagnosis of schizophrenia. Others will have bipolar disorder. There's also, some will have a diagnosis of major depression with psychotic features. And then others actually have a first episode and they're okay for the large part. So it was just this transient first. So, you know, sometimes it's just the first six months and then they're put on a psychotic medication and maybe there's also some other factors in their life that just required more clinical attention. And, you know, that's, I wish it was more of a common case, but about, I'd say about 15 to 20% of patients actually have a first episode that resolves itself with, at least with early intervention. And I think that's also a really big plus of this clinic is that we are catching cases early. But yeah, absolutely. There's many psychiatric diagnostic categories here, but I think the field is trying to shift away from looking at these diagnostic categories and instead looking at severity of symptoms and how it's related in a more dimensional manner to the brain and outcomes. So from your work, does that speak to, you know, how important is how early this is caught basically? So how early you got diagnosed? Yeah, I think that principle really stands for many disorders, you know, early detection is really key for better outcomes and for more successful interventions. So absolutely the same stance here and it has been actually shown that having these first episodes, psychosis clinics in place, there's quite a few actually in Canada does indeed actually reduce the severity of psychotic symptoms and relapses in the future. So this is a really important part. And now there's studies that are being designed such as the one I'm working with right now actually called the ABCD studies. So this is adolescent brain cognitive development in the United States, tracking 11,000 kids and their families across the country for 10 years. And the idea is we're tracking them from nine to 21. And there's gonna be unfortunately all sorts of, there's gonna be some kids that do develop psychiatric symptoms, including psychosis during that time, but then we might be able to really find early markers, both at the level of the brain, also in terms of behavior, school performance, et cetera, that we can help catch these disorders earlier. We have one last question obviously from somebody who understands this in more detail than I do. And question is, could there be some influence of verbal memory on social function that could contribute to motivational deficits and other negative symptoms? Absolutely. And I think that's why we are seeing that verbal memory negative symptoms are linked. So, and it's not clear if one comes before the other, but you can imagine that if someone has verbal memory problems, they might not be able to express their thoughts as well. And they might be a motivated to actually engage in social interactions because of that, or they might be embarrassed so they don't talk as much. And then that leads to some negative symptoms as well. Sort of looking at the literature, you do find that before the onset of psychosis, so in clinical high risk, you see verbal memory deficits already. So it's possible that verbal memory might be the one that comes before. You also see verbal memory slightly to a lower extent, but lower verbal memory deficits even in individuals or family members or first degree relatives of patients with schizophrenia or psychosis. So it could be that, yeah, exactly, verbal memory might influence negative symptoms, but absolutely it is very much intertwined with social interaction and communication. We need to kind of disentangle those pieces a bit further. Okay, great. Well, thank you again. Thank you again, Carolina, wonderful. Thank you. All right, our final speaker today is Philip. And he'll talk to us about proteome landscape of pediatric cancer in patients and xenograft models. Philip, over to you. Thank you, Detlef and the fabulous team for having me here and Carolina and Hanan for the fantastic, interesting talks for me. And so we'll switch gear a little bit, but we stay with the scheme of trying to identify problems earlier. And so we'll now look a bit at cancer, specifically cancer in kids and proteomics. And so you probably all heard about the new idea of precision medicine or by now I'm not so new anymore where you find a genetic mutation. It's usually genetic mutation and match a specific drug to it. Previously, what we usually did is we found a common phenotype. It's a cancer or it's a cancer in the lung or it's a cancer in bone. And that's the best treatment we can do for it. Now we can do a bit better and find a mutation and find a drug that might hit this susceptibility. That's the idea of it. It's a nice idea. The question is how well does it work? Well, the positive news here is that in the adults and even in the kids, we're doing very well at finding such mutations that we could potentially target with a drug. That's what we call actionable. But then when it comes to how many of these we can actually treat with these drugs, it drops dramatically. So only 23% of the adult ones that have a drug identified get treated and only 8% of the kids get treated. And if we look at the outcome in this MITA study, we don't have data for the kids yet, but for the adults, really only 7% show a positive response. And now this has a number of reasons but one of the reason is really that we look at this late. Usually it's in the relapse, it's in the high risk patients. And at the time when we find this mutation, this susceptibility, they are already in a very dire state and oftentimes don't make it to the time when we can actually give them the drug regulatory slowdowns there and the disease is progressing rapidly. So we're late. And in this cycle that I've painted here, where we go from a diagnose, we can treat fairly well initially in a monitor and then we relapse eventually. We also have a psychological problem where you have this constant fear of, it all starts all over again, the cycle starts all over again. And at BC Jones Hospital in Vancouver, Canada, we started initiative to think about breaking this and really switch this to preparing for a relapse. So understanding the kids cancer before it comes back, coming up with an idea about the relapse before. And what we do for this is, well, we look, we enroll all kids at diagnosis and look at diagnosis biopsies and remission biopsies, try to find on genomic level and now also on proteomic level, specific differences in these tumors that make them susceptible. And then also expand these cells. We're only doing this for leukemias so far, we expand these cells in a xenograft model in a mouse model where we get more of these cells because one of the problems really with kids is that you don't get much material that you can study. And then we can study the biology of each kid and we can study susceptibility and really treat them with drugs in the lab and we will be eventually ready for a relapse. Now, this is a setting that sounds extremely costly and implausible, but luckily, even though childhood cancer is a huge problem for the ones who get it, it's still a rare disease. So number-wise, it's actually something that you can do. And you have to remember that the cost occurred if a kid has to live with the late effects of a cancer treatment for decades on is actually not a small one. So the question for this talk today really is that we have to answer how well do these mice that carry the kids cancer cells reflect this cell biology that it has in the body? It's sitting in a very different host in a very different environment. So how much has changed? We know that the mutations are retained. We know that the general drug susceptibility is retained, but if we want to study more of it, we need to know do they actually have the same makeup? And the critical component in its biology or what we use as a proxy here is the protein makeup. So what we did here is we studied 10 BALL patients and 10 T cell leukemia, four T cell leukemia patients expanded them in several mice. And then we looked at the protein. We also looked at post-sensational modification, namely phosphorylation, most will be familiar with that. And we looked at protein proteolysis cleavage by proteases. And I want to segue into that for a second here. So proteins can be cleaved by proteases. This can have huge functional impact. And to monitor this, because most won't be familiar with that, what you can actually do is you can look at the newly created start of a protein. So if you cleave a protein, you will create a new immunoterminous. And now we developed a new method that is very sensitive at enriching these N-terminal parts. We call them N-termini of proteins. And so we can now get thousands of those out of very small amounts of material. This is 10,000 T cells or respectively a few leukemic cells from a patient biopsy for example. And we can do this with a nice small coefficient of variation, which is of course always helpful. So if we now look at this from a high level perspective and just look at the protein, quantified protein of a cell, a kid's cell out of the patient and out of the mouse, do they align or are more the cells coming out of the mouse group together versus the cells coming out of the patient group together? What we see here in high dimensional reduction is that all the diagnostic BLL samples fall together or most of them, most of the BLL relapse samples fall together, the T cell leukemias fall together. And this is regardless if they come out of a human or if they come out of a mouse. So really on the global perspective, we retain the features of these cells very well. We see that cell lines are actually very poor model for these disease, they are far out from everything. And we also see if we look at normal blood monocytes and these leukemia examples here that had a very small blast count, so cancer cell count, they all fall together with normals. That is a nice control. So now the next question we wanted to ask was, do they retain features that determine or differentiate between a primary or a sample and a relapse sample? And so we looked at what's significantly different between a primary and a kid and a relapse and a kid and then looked if the ones in the mouse on the protein level cluster together in the same way. And you see here these purple boxes are always a match patient and xenograft model sample and they all fall nicely together, which is very encouraging and these are primaries and relapses. Now there are some differences that we found though between patients and xenograft expanded ones and this is primarily in response to the immune system, which is not surprising or is actually expected because I didn't tell you that these mice are immunocompromised so that they don't reject these cells. So you would absolutely expect a cell to respond differently to anything that's coming from an immune stimulus essentially. But interestingly, we also found that we have increased cell cycle and probably more expansion, more growth of these cells in the mouse versus the patient. And we confirmed this in a pairwise manner using a number of the classical proliferative markers. Now, looking at prosensitization modification, we say essentially the same thing. They also retain specific features. We lose a bit of the patient specificity here and I can talk about this more. Now, what is interesting, what we see is that many of the proteins that don't change.  Many of the, oh, I have to hurry up here a bit. Many of the proteins that don't change actually change on the post-sensational level. And this is probably because a change on a protein can lead in a cascade down to changes on the modification. So if you knock out an enzyme, this has a massive effect on the proteins it modifies or even a ripple down effect if we hit an inhibitor. And now we can of course also use this to study the drug effect, not only by seeing if a cell dies or doesn't die, but also by looking at which pathways like phosphorylation signaling here is now activated or deactivated in a specific patient. So to better understand possible compensation mechanisms that might occur in the relapse and prepare for that. And with that, I would like to summarize that essentially we have shown that mouse models of pediatric leukemic BL and TLL are accurate models with a few differences but by large, they are very nice models to really study a patient specific model off a pediatric leukemia. And with that, I would like to thank everybody who did that work and would also like to let you know that Vancouver is a beautiful place and there are positions available. Thank you, happy to take questions. Awesome, thank you, thank you, Phillip. So it's comforting that these Xenografts and the patient samples overall are quite similar but you do see these differences. What about using humanized mice? Would they be useful at all? So do you think it's an interaction between the mouse immune system and the Xenografts that make them diverge from the patient samples? In some of the ways it's absolutely the lack of the immune system or remaining mouse immune system that makes this difference. And using humanized mice would probably change this. It is possible to do this for pediatric leukemias. It's not really necessary and adds an extra level essentially. Our collaborators on this, Dr. Gregory is doing that for other cancers now so that we can expand other cancers because most of the others don't actually even want to grow in mice properly. So for some of these having the humanized system in place will not only be beneficial but also give you the more accurate picture, absolutely. So are there Xenografts that fail? I assume they don't always work. And if that's the case, do you think there's some interesting biology basically? There are definitely some that fail and there seems to be some indication that maybe the rate also of the growth is somewhat correlated to the aggressiveness of a particular clone, which would then in turn mean that the ones that don't grow don't grow are problematic. But it's also always a question that I would think at what time you actually get this biopsy and so you have, to imagine that you are getting this out of the bone and it's a very crowded situation usually by the time you diagnose these kids and it's very packed with blasts. And that's probably the reason why they stopped proliferating at this point when you actually biopsy them while they're still growing in the mouse where you're hitting them at a different time point. And so some are probably just not in a state to really make it through that process of biopsy and keeping them moving them into a mouse quickly enough. That's cool. So figuring that out what is actually a clonal effect and what is actually the effect of timing is probably difficult. So we have a question from Hossain. I'm trying to see like an unmute him. I asked, let me see, do I see him? Attendees, okay, okay, okay, okay. I think I'm almost there, okay. So let's see, does this work? No, I think I'm, because I'm not the organizer, I think I cannot unmute him. So I'm sorry Hossain, so I'll ask your question. So when you do this phosphor proteome analysis, so the proteome changes and then phosphorylation changes. So do you normalize phosphorylation changes by protein change or how do you do this? We don't strictly normalize it by protein change but we control for protein change. So we essentially primarily focus on the ones that are not changed on the protein level and then change on the phosphorylation level. And that's of course an important thing to look at and not get fooled by. At the end of the day, it's not always a easy answer which one is the more meaningful one because even if a phosphorylation is up and the protein is up, this might have the same effect as if only the phosphorylation were up. So if it comes down to the fact how much of that phosphorylated site is there, the way how you get there is maybe not important. So just normalizing it is, I think not always the best answer but you have to look at it. Yeah, got it, got it. Yeah, yeah. So it gets it, yeah, it's also a function of whether the two forms compete with each other in one form. So, okay. I was saying I hope I asked that correctly so I unfortunately cannot unmute you if you can maybe unmute yourself. I hope I did, he says thanks. Okay, so that was good. All right, so I'm looking at the questions. I think that's about it. Okay, so thank you again, all three of you and then Carolina, Phillip. Thank you, awesome. Thank you for sharing your work with us today and thank you to all the attendees for listening and asking questions. So we have our next slide come up. Yep, next slide is coming up. So this brings our scheduled online research talk series to a close. I actually shared the first one I think and the last one now. It's clear that lots of colleagues really do appreciate the chance both to present and listen to talks in line instead of in person. That's really I think something that will stay with us. Although I do have to say I've given a couple of online talks myself. I've been just as nervous as I am in person. So that hasn't changed. So we definitely plan to continue these. And if any of you listeners and in person talk canceled or postponed or do not have an opportunity to share it online elsewhere, we welcome for you to register your interest in being a speaker using the link on our screen right now eliveside.org wait list. And it's also posted into the chat, I think. And to learn about the next online talks, please follow elivecommunity on Twitter and sign up for our early career researcher ECR newsletter. And for updates, you can also follow elivecommunity at elivecommunity on Twitter. Final thank you. We can thank each other. And again, once more, thank you very, very much. So we're keeping this open this Zoom for 20 more minutes to give you a chance to chat directly with the speakers. We'll see who is staying with us. If you'd like to stay, please do so. Everyone else, thank you for joining us. You're welcome to sign off and we'll be ending the YouTube live stream now. All right, great. Hey, thank you very much, guys. Were you guys nervous? I...


. He's too fast. Hey, look at that. I did it again. I did it again. I did it Back to back trading baby. I'm not good. Yeah. Give me a second one editor Let me add them. Let me add them. Let me get in there. You think you wait. Hold on I was gonna say no you got Goku and Vegeta It's the Goku it was the Goku and Vegeta that's been cooking me spoiler alert in the other one I'm glad I had to do the last round, bro. You enjoy. They had tree yourself, right? You know, whenever we do these three in this video So we always do all the different rounds in each other's parts You just hadn't done part three yet or round three yet. Humble these guys rhyme humble them Got him looking like Wilbur the pig out here, bro Why are those that haven't read charlotte's web?  All right Can't share a little litter.  You're not good.  Okay as long as Vegeta doesn't round start I know widow peak ain't laughing. I'm about to beat his Why'd you bet? No, no, no No, you got a little carried away there, man. A little too Why you gotta end up in the intro No, he's gonna edit in the music we're good. I know every time Dino that's added Man to start making noise He's gonna be so far off of what the actual song I'm gonna make it look easy Pretty easy No, let me give you some of my confidence, bro. You got this.  Yeah, I don't need it I don't need any that I need a little bit. I need a little bit. Vegeta get out of here next Hey, my bad chief you got it Hey, so I got a fight. I'll fight these two for real. You know what? No, no, no, no, no Hold on. I'm confident dotto tag win frame one. Are you sure about this because me and dea discuss this in every other video You kind of built like this bow. No, no You know, these guys ain't really fans. All right, bro. All right. Well, I hope it goes well for you, man Oh, so far so good. Good start. Good start. Good start. It's all up from here. It's it's all down from here Oh This was not a part of the deal dea never agreed to risk it's teamwork It's team effort Well, he just got team efforted in the face. No, actually, it was a low starter. So I got team efforted.  I got this. I got this What dude, bro, he did not like that. I'm gonna grab him by his saiyan scrub. Are you going? Let's go with you. Go on. Are you going? There you go. There you go. Go on trying to run from the truth, bro Do you and feel some type of way about not being invited into the next dragon ball super move? Well, no, what you guys don't know is is when your team dying is here. Or maybe a flop.  What is going on? What is going on? There's no way I'm plus on that one. You just denied frame data. Frame data. Hey, run that back All right, I'll do respect but my frame data says that I should have won that last match And I'm going to beat up on these two to prove it. All right. All right. Now, well not. Are you willing to put up a steak on that? You know what wait, what's this? I don't know what an appropriate bet would be.    I need this. I need this. I need this. All right. This is my call out I think you get past Goku, but I think Vegeta going beach. Oh as all right now. I would definitely uh A double perfect.  Come here. All right It's about to fall apart. It's about to fall apart. It's about to fall. It's about to fall apart I said, I told you I'm just an astrodamus over here, bro What I want to know is why does VL want us to fail? I don't I just knew Vegeta was gonna beat his ass, bro I just noticed and this keeps happening where you keep predicting what's gonna happen and then it happened pretty good Right, you know, I got you. I got you more. It's a really good stuff. I don't you know It's about to happen.  Let's go That's the kind of prediction. I love you.  No, I told you people predict good stuff. I told you. Brian. How do I start the next round? Uh, well, he's gonna about say you better make it to the next round He's gonna command grab so just 2h. So I know I should 2s because 2s is cooler 2s. Yeah 2s 2s 2s He did it. He did it go for the command. He did it. He did it. He did it late though. I did it super hard.  It's kind of looking goofy shout out Kingdom Hearts 4 dude.  There's so many of me. There's so many No Dion can you take the challenge and actually go in the next round without getting cooked because I think that's Well, I mean you have me on your side So clearly you can put your controller down and I'm just gonna I'm putting a control down. I drop my controller loud as hell in the mic I want people to know that I'm confident drop your control like I gotta No, I gotta awkwardly pick it up again. I gotta awkwardly pick it up again. Hit x All right, I dropped it.  We don't have the same type of controllers. Ours are heavy weighted, bro Just like in the anime just like piccolo has heavy weighted clothing. We use heavy weighted controllers Imagine if we had light controls.  He's confident. Oh, right. Oh, right. Hold on this time All right, it's he's got enough code. I know I'm confident. I'm confident 5m.  It means deon's in his peak form deon's gaming. He is getting yeah. Yeah Yeah, yeah, hold on. Hold on. He's good. He knows everything about gochita's combos We're gonna get confidence and predict that deon's about to cook.  That's the problem. My confidence remains unshaken.  I just need a level one I just needed a small damage boost on this Nice block. He doesn't he doesn't even see defense the same as us. My god. He's a different beat I know you ain't laughing for either.  That's a lot of help All right. All right. Just only because you're spitting facts. Hey, come here. I wish they were smart enough to be like You did meet the quorum I got it bro. You can't lose here. I got I got I got the Losing here would be the most embarrassing thing. It'd be really embarrassing because none of us got beyond me. Just wait I'm just going too well. I'm gonna just to him. It's fine. No Face your demons.  Oh, let's go. I'm fine. You're better. You're better. Yep. Yep. You got a level two You're better. You're better. You're better. You're better. You're better. You're better What is one of you're better? You're still better. You're still better. You're still better. You're better All right, who wants dibs on goku?  We could still beat universe seven in the tournament of power Let me get into I'll weaken them. Why are you going first? I'll weaken them. Just trust me I know you kind of spin for real if we could just get down to business and to beat goku Keep singing keep singing. It's far me up. It's far me up. No, we need music bro And we got music in the background. Anyway, be goku.  Yo, it's just a mouth.  It's over. It's over. You stop singing. I'm sorry, bro. We're gonna make it to stage seven I promise it's a promise. I'll I tend to keep. Let's go Tend to keep Bro the dragon balls if needed. Oh, right, right, right, right. I should just I should just That's what I wanted to do.  So there you go. And then auto combo. Okay. Oh, I guess you're right But I'm just saying like after the sea assist auto come that's two dragon balls for one Oh brother. I know why he's scared me for real. Yeah, he's pretty low though. He's pretty low Do you just got to face it? No, don't say that to me as long as it's a spark Krillin and tn, I I literally don't care about these two the commentary is going to be so dry These two literally we've beaten them a million times. Let me let me break it down for the viewers out All right, go ahead right down while we're scruggling they start. No, not why we're struggling yet, bro That's a later. That's that's a later. No, why you got an aura.  Yeah, because I am cool. These rates start really easy, uh, but things get very bad very All right, I Do that one time in the first like five Yeah, we might as well bring it home by the 17th rate start easy rate get hard Yeah, and the the ending of this raid is the hardest raid I've ever seen bro because we can't do it 100 we can't do it with no power Friction fist boom friction key blast Honestly, I don't call it what you want bro, it's the friction of your fist and you gotta see this attack, bro So I get it I get it. I'm just saying hold on does that mean hit technically as friction fist when he does his sea assist I think so, bro.  He got folded like a scorpion.     Shut up It's funny because you keep saying that as if you like want to add that into your title or something I'm just trying to keep it topical But if you can't tell we haven't stopped recording these videos since the first one we recorded so it's been a couple hours now Yeah, what in the nice Oh my god That may be the brother. Please die. I'm sorry. I've made mistakes. Just die. Please. Why are you trying to mix it?  All right, but I just in my head bro.  All right. Well, why do you say it then? You can't say that why do you say it then either go back? I thought he was gonna dp me then go. I'm just happy to be here, bro I ain't gonna lie. Y'all got cooked, bro. We'll see him in his new starring role in the dragon ball super superhero Oh, it's funny because the way you say I just imagine like they actually have a red carpet event We're gohan the like like there's a universe outside of the dragon ball, but it's still in dragon ball You know what I mean?  I play the role of gohan my real name is shingeki nokiochi Yes, we're just so excited this project finally got out after the big delay from the hacking scandal But me and tyler tyler's piccolo are just so happy Brenda did a great job in fans Oh Who named the kid run to that should be a crime Scarlett does marvelous my wife Adele We fit for quarter for five hours Oh, you get me I got made by the androids.  It's in silent.    I feel like for the rest of this video. I got to talk like this I'm done in bro.    All right.  Hey, you got this. Yeah, this video.  I'm good. I'm good. I'm good.  This is video was six Video six consecutively. I and I did two videos before we did this. So this is my eighth video Yeah, I might need a part three y'all's part two is way funnier than my part Yes part two is like we're going to get to stage seven My part two my part two is gonna be linking to you guys as part two Go watch those one. Oh my god. Oh, what? Let's do you a fight. Yeah, this five this five. Oh brother guys We're still recording. You guys seem a little quiet, brother. What am I gonna care? Yeah, wasn't even that funny and plus you got to get to stage seven bro. Get up. Come on. You know what you're here for Somebody's a super serious out there. Didn't you guys start recording this video? There you go Working into the man who got mixed by the Oh It was by the first five ten seconds Dino didn't even know It was like I I can't even look at a picture of Android I'm free to come back in here Lock-in we got another match after this man I'm happy to be unexperienced a laugh attack that we experienced in silence Finish the job go on I put my controller down necessary Oh No, no, no, yeah, I'll save it for go on I'll get the meter back real quick Honestly, it's not even like me trying to avoid like mixing him or whatever.  All right. Here's the question. Can we make it through his Goku?  All right Look like Elmer's blue I'm sorry It's not let's get the stage seven Just don't don't let us die without using spark friction fist Oh my god, I actually am so confident that you wouldn't use for some reason. I don't I'm not at your confidence Friendship fist, please maybe just he just business casual disaster. Hello there go-go. Yeah, it is I go-go I'm you but different. Oh, you had it. Okay. You're gonna reset. I'm not in things. It's going to get so I love that no matter what you keep doing you You got to be kidding me no Goku's the hardest great enemy I've ever seen in this video game just plays out to play plays out you're in the end This is the power that's even greater One of the best mechanics ever added to Dragon Ball fighters were the raid battles And now it is finally time to take on another five-star iteration of these impossible What he meant to say is when you think of raid battles Let me introduce the boss fight today a five-star Beerus, but if you look on the x-ray, hold on bring up the x-ray machine You can clearly see he's got that dog in them and we're gonna Cats versus dog Beerus one dude.  Me and tag me My combo this was supposed to be my perfect my perfect victory Anyway, normally I wouldn't say it like this after you got hit But um, yeah, the reason why this starts super easy and no one gets hit is because it's super easy And you should never get hit, but as you can see that just was not the case. Yeah me and yeah I'm personally gonna go to it.   Hold on. Hold on. Hey Beerus Beerus.   Nah cuz I gotta get my licks back Then you better stop is it air start is it air start look at damn.  I literally can't win at anything today.  I don't need anyone but my own damn self I'm already the combination of Goku and Vegeta.  I was real. He doesn't even know it's gonna be the top one.   There's no way that combos Oh, okay, I think the only way you lose if you have I'm three now. Yeah, we're good. We're good.  I'm gonna work with go Jita, which rounds have you not taken off? God damn, are you cooking me bro?  Good job. Hold on.  I didn't even feel it. Maybe if it was 100.   Okay, that's fine. That's fine. That's this is this is okay.  Let him get away with that. Are you kicking the screen, bro? There you go. Look at that changes. You notice it now. You can get to drag balls right here if you want it use my sis Okay Oh my god, he leaned that all together What damn What do we have what do we have six out of seven so all you need is one auto cup His game I'm literally just pressing square. Let's see what happens nice backflip. Okay. Okay. Maybe not literally you got it You got it. You got it. You got it. You got it. I'm back, baby. All beers.    It's look. It's literally monomy model one-on-one. Oh, no, you know, so you're not using our assists Not no, no actually no Yeah, no, I'm not saying put it down or anything like that, but I'll handle it. He's fast Well, I don't really one straight keep last hippie. That was it. Good. Bye. Don't worry. I got I got I got I got I don't know God damn.  Yeah, I know D. I knew D. I would be No, you're wasting he's at war I got a countered it. This is bad stuff with this counter.  I'll just say it now during this loading screen on the way back to where we are It's gonna be all me all the time. No assists do a little montage as I prove myself worthy beer is I've come for you There's no shot.   Get out of here. All right.  Let's talk about it. I can't restore the cop. I did build confidence It's not a blue combo that's the first don't talk to me see how I smack them anyway though I was teaching a lesson. Let's go. Okay. You're wasting our spark and now you compromise What no, no, no, I got this I got this I got this You have He shouldn't why did he dunked there? Nobody does Kind of schmicks What did I tell you get in there You got to let him heal us You've got to let him heal.  You still got this, right?  Oh you dropped it.   She's up. Okay. I don't need body up though. Do you want to heal? Do you want to bring me in so I'll let you know oh Noice confidence and dishwash confidence because you've got to be tucked away by me in this next match that cats dishes me Dino you don't have to go first. No. No. No. No. Yes. We do. Yes. We do. Yes We do editor. Let's do a cool transition into battle six put a sponge across the screen and wipe into the next one Watch watch watch. Okay, bye. He's up. Nice read.  He does have three bars though.  I'll do it. I might slide in am I different? Hold on I think he's awoken. I think Donald has awoken. He's never mind. It's I'm going back to bed. It's still this guy level seven He's only on level one. Oh, but be afraid. Was that a hint of fear? All right. No, you got you got you got you got it. Whoa air conversions. Okay, you got it You got it. Oh my god. Oh my god. Oh my god. Oh my god. Oh my god. Oh my god. Oh my god. Oh my god He can't do a level seven. Yeah, just kill him. You're different bro. You are he's going for it. Oh my god But maybe he actually is different.  I got to apologize ahead of time for I mean simply carrying this and just winning the whole thing But you pulled that I didn't no problem.  There you go my assist. There you go. Oh my god. That's scary. It's getting scary Just don't die Dino. If you want you can tag me and I'll try to If you don't Okay, it's fine. It's fine. It's fine. Am I going dragon balls or what am I doing here? I might just try level seven all three of them at the same time. Yes You have to win the game now.  Oh, that's it Oh These do it to beers And I'm bringing my friends back. Yes. Yes. Yes, bring me back. Please said Rod.   All right. Well, it's worth it though to end with friendship get it Key charge. Oh, yeah, you're right.  Goodbye. Bye. That was not normally My boy We have fought every raid battle in Dragon Ball fighters so far What of the hardest was the very beginning with Dragon Ball Super Broly the video that started at all the first three idiots video And now look at us. We're all playing fusions, but Nothing's really changed. We've just gotten stupider over Describe the entire metal fighters online But we back bro, look at us. Look at us all these years later y'all know the first video we did was in 2019 I can't even remember 2019. That was three years ago, bro. That's crazy Is it the three idiots? Three it is three year anniversary. I needed to say it. Go ahead. Yes, December 9 2019 That's our three-year anniversary. So what are we gonna do? Are we all gonna like meet up and fight? Meet up and just like we should come to me.  Yeah, I'm playing games weeks.   He thought he had me too. What's up? Hold on. I got you know what? I got I got to end it with the move that everybody knows and loves Why cannot what's the move? That's the one move Bro, take somebody's And another one rainbow ally That's gonna kill because it's pure heart Well, what about that? It is pure heart fucking stopping me into the chest Hold on don't you know how you and I always make fun of like dragon ball theories and stuff Yeah, I saw what I'm taking out was like the reason why soul punner she didn't work on Broly It's because it's pure heart What do you mean picture for the movie bro is screaming in pain Yeah, right Throw us on the verge of saying tears Let me add them. Yeah, too bad. Oh, he already level 60. You sure you want this.  Mine was red bro because I got the fire in my heart That's really a Broly's pure of heart.  I'm going to kick you very hard.  I want to see Broly start fight I remember round one in 2019 Broly used to be different I remember we were we all pretty new to holding the controller on our hands And deans video you was beating our ass. What happened? Hey, bro took a big day. Did you not send the many the settings to men? No, it's on hard difficulty bro. I didn't change anything this on max five star Ah I can't have any fun in this house. Please bro. Why are you doing a zigzag? I didn't do that much damage broly fights like how most people run away from alligators I wonder if he sparks here now. He ain't gonna spark.   He was he did a little more than I expected. He broke my left arm, but it's fine Broke broke it at multiple places editor and play that noise bag.  I'm gonna ask a random request look up Look up putting funny moment and go to page five on the youtube search results Sort by new and play that video right now.  Yeah, that's all We beat him with the character. I haven't played in over six months. Go ahead, bro. You got it.    I gotta roast you Hey, bro, you're not fusing it. Do you notice the video and we said you'll show. Yeah. Yeah, you remember that Yeah, yeah, I don't want to Why is that I don't want to like that.    Why are we just man handling bro?  He's got command grabs. That's the scariest part about this. All right. Yeah My else a little too low to just be thrown around like that. I gotta remind you my sis don't do anything other than just get me hit Don't worry. I just wouldn't be here Like use my sis now in the middle of the combo, bro. Not not in neutral. I am going to punch him really hard All right. He didn't listen to any of that. I'm dead. No, I don't need your help. I need your help I'm trying to help you. I'm trying to help you Nice reflect day and you could have won the game and now you won the game I'm sorry. What nothing nothing now you got results. You clearly got results Just make sure you because you were you were like back seating me Valorant style I'm not back seating but I'm trying to protect myself.  What's going on? Bro because I'm I'm the limit breaker bro. Watch this All right, enough of the solo act boys. We're actually a team on this one. That means we're tagging in I'm going to use you guys as like actual teammates now Okay, so like that means just don't use me in a compromising situation Yes. Yes. Yes. I'm looking out for you like you would look out for me Well, no, hold on.  There you go. There you go That's how you do it. We're level seven. Oh sure.  We a team Okay Well worked out bro. So just don't even worry about it. Yeah, you're fine, bro He can sense your fear. Just don't panic. There we go. Oh level seven Oh the Broly gonna Hate this, you know the beam actually didn't kill him because she lie was pure of heart This is a lot of super dashing Right now That's it. Winning Two eight. No way seven. 2007? FTW No, five. What's the total five? What's the total five? Switching to Dion? Is he at five? No, he's at five. No, I'm at four. I'm at four. Give me one more at this rate XD Rar, bro I'm sorry It was like slightly mis-time Oh Like okay It's the tag got it's the tag. It's the tag out. Are you sure? I didn't agree I know. No, I know. You're good. You're good. You're good. You're good. You're good. I never agreed.mi Well, that's a rain video Why don't you stop that like that? Well, I got I got I got it. Well, I was Oh Did he just convert Venice to Venice? I think he did. I think it was Gargantz for the Venice or something. Oh! What?  You know what to do, Dion? I'm not Dion.  I got him! I got him! I got him! I got him! I got him!  I can't just Dion. I can't see him yet. Finish him off. Oh, you gotta be real pure a heart.  I hope you bulletproof. I hope you bulletproof.  Hey, we a girlfriend that, bro. That's my girlfriend now. That's our girlfriend, bro. Imagine you got one bruise on that fat-ass forehead of yours. This is what we're gonna do.  And this time, don't fail us, bro. Bro, just give me the level 5 and preferably have Freeze out there, and then I'll get to level 7. I was gonna say, Dino, get Freeze out here. Let's style on Freeze. I'm sorry. What'd you just call me? No. I just, I don't know.  What are you talking about, Rhyme? The gaslight ain't gonna learn it. Dude, bro, Rhyme is going crazy right now. Dino kind of snapped on this one. Damn, he gotta play in the game for real. For those who don't understand, I normally always call him Dino, and he always gets called Rhyme by Dotto. So we're flipping it. He's different.  That was very, very close. It's a game of inches. Very average. Oh, alright. Disco hurt. But you lived this. Go, Gino!  They're capable. Alright. How they can finish a combo, but they can convert any straight B into a table 3? Goddamn! Dion, we have to be the heroes of this video. I got an idea. I got an idea. I got an idea. It just works, bro. Get Freeze out here and just get my level... Alright, alright. Well, here comes the giant fist. Alright, you just have to carry it. That's all. I mean, that's pretty easy, honestly. He has nothing that can beat my 5M round starts. Alright, bye guys.   This run back about to hit Bear is broly, because he didn't even got us like that for real. Editor, wait, what is that? Oh my God, is that a cartoonish mop sliding across the screen transitioning us back into when the real video starts again?    I was really waiting for you to say something creative. Why is the screen so wet?  Why is the screen so wet? What do you mean you don't have any money? This is a bathtub. This is a bathtub. Stop! He didn't like the joke. Daggisid, Daggisid, Daggisid, Daggisid. Hold on, is that Dottles pride kicking in? No, it's not. No, it's not. What? Dottles!  Dottles! Oh my God, Dottles! Okay, okay. That second half was your fault. But Dottles!  Why are you fighting for Dottles? Thank you! You better get your girlfriend on the line, bro. As you run away. Because I'm scared! Goddamn, bro!  I didn't know that would beat that! That's an answer here? Hello? Did y'all know at one point there was a debate whether to pick it up here or not? In my video? God, well, gladly did. Super Dash! Nice.  Nah, bro. He different when he fight me, bro. Bro, cut this man in twain. Bro, grab me out of my super.  I already know I'm the intro, too. That makes me sad. Well... Goddamn it, Dottles!  Alright, but I got a level two. I think I just easily killed him. Dottles, don't lose here. Dottles! We're done. We're done. We're done. We just got back. Oh!


But it's that gumption and that single-mindedness that I think is common, I think, among most successful entrepreneurs.