So today, yeah, I'm going to talk about some insect odent receptor work we're doing and I will say that this is quite a new topic for me and also I think for structural work in general. So there's definitely a lot we still I won't be able to answer and I'm really open to have feedback and suggestions on this work and I think already a lot of the talks today have been really helpful. So just who I am. I'm Cassie. I'm a postdoc who just started Lund University and the new Max Planck Center for next generation insect chemical ecology, which is an absolute mouthful of a name. And yeah, I'll just give you a little introduction as to why we're interested in this work and then move on to what I've been doing so far. So, in sector affection, like, why do we care a lot of people today have talked about sort of medicinal targets and things like that and I think that stuff like what we work on tends to get a little overlooked. And in sector factions really important. As I said in the abstract, I'm going to talk about moths and mosquitoes for two, they're important for two different reasons moths are pests usually of agriculture and we're really interested in controlling them. And mosquitoes are of course pests of humans. So we're interested in them from medicinal kind of human health standpoint and all factions really important as a sense for insects and in fact it's basically the major sense that is used in most insects and already we can use chemical ecology approaches to manage and control insect populations. As a kind of colloquial example, I'm sure many people spray deep onto themselves to avoid getting bitten by mosquitoes. But there are lots of field applications for this work already and we hope to to understand in sector faction to develop better methods in this area and to kind of exploit the chemical ecology of these insects against them. So a little introduction to the system and the proteins we're going to talk about today in the peripheral insect or factory system which happens in the antenna so I'm not thinking about what's going on in like the brain or anything. We have two main groups of proteins these odent binding proteins which are not membrane bound proteins so not of interest to us today. But what is interesting is the odent receptors. And these are heteromeric complexes so they form a complex with something called the odent receptor co receptor or orco. And orco is highly conserved across all insects but you actually only have like one orco per species, whereas you have then a range of odent receptors which have different specificities for different ligands. So it's a really interesting system. There's a tetramer so with the orco subunits, and each subunit is a seven transmembrane domain protein. I think initially they were thought to have a really similar structure to gpcrs, but they actually have an inverted topology to what gpcrs do. So, they're kind of unique, and there is of course limited structural data for these types of proteins. So there's a huge amount of functional studies that show that these receptors vary so much in their function in their specificity for example so you have some receptors which only respond to one ligand, and that's the highly specific. And you have some which are more broadly tuned to a range of ligands so there really must be some differences between them but we don't really understand these. In terms of structural work so far I can't claim to say that I've had any part in any of it but I will give you a rundown of what there is. The first structure was published three years ago now 2018 by Vanessa Reuters lab group who are lab group from America, and they published this cryoem derived structure which was produced in HEC 293 cells. So the HEC cells are used quite commonly for functional assays of these proteins. I can say a little bit about why we choose not to use these for our work in a minute, but this was published as a structure of orco so this is just the coreceptor in a homeomeric complex. Very recently the same lab group has gone on to just published this as a preprint so it's not fully published yet, but you can go check it out if you're interested of a odent receptor but this is again in a homeomeric complex so they chose this odent receptor from an ancient research which I think, and don't quote me on this maybe predates the evolution of the coreceptor of orco so they have this homeomeric complex of an odent receptor which has a binding pocket, and their intention was to look at the binding pocket and how this opens, what is an ion channel that obviously causes a neurological kind of response. So we identified a binding pocket and some of the functionality of this protein, and this is a huge step in the right direction for our work, but unfortunately OR5 is not a specifically tuned receptor. They found that it was quite broadly tuned and they didn't find that much specificity in the pocket. So, we still have a lot of unanswered questions, and they still also haven't done an odent receptor, but they found a coreceptor complex which is how it is in insects themselves so we really are interested in seeing how this functions, because I think it's not quite the same if you have four binding pockets, and you're showing a mechanism of opening versus one binding pocket that should activate the opening. So there's still a lot of work to be done. So our aim was to try and do some structural work with these proteins, but in yeast. Well, a lot of people have talked today about why it's a great system for membrane structure, kind of determinate membrane protein structure determination. We also people have mentioned hexels and I think hexels are really a valid way of doing this for cryo EM. One of the reasons we were interested in not doing. Hexels was one we wanted to create a high throughput system that produces a lot of protein and because we might do crystallography as well we don't know yet. And also because there is actually some inconsistency sometimes with the hexels in terms of functional studies that we do. And you can get some kind of weird results and all consistent with other functional assays so although we don't really know about the functionality in yeast we at least have a fresh start to see how it goes. So I kind of just put on here like an overview of what our process was going to be, but of course this is a lot of work condensed into a very simple diagram. Right now I'm at this point so I decided this project in this year really so at the point where we've transformed some yeast and we're seeing what happens. So in terms of the choice of receptors there are so many to choose from and there this is a list here of the ones we went with. We tended to choose highly specific receptors apart from one on there which has an unusual functionality which I won't talk about too much today but it has some two lig and cell response to. And we also have collaborators and Swedish agricultural university who are interested in mosquito receptors so I can't really tell you detail of why they chose these ones because in this case I'm following instruction rather than coming up with the idea myself. But we decided to go for about 12 just because this seems like a reasonable number to start with, but really this, the reasons why these are on this list there are so many I could have picked and at some point I just have to pick 12 basically so you know that read too much into this. We initially did the cloning. A few people talked about some of the design elements to this today. And we did a multiple site gateway cloning to produce a plasmid which has a ZSN resistance marker. It has our odent receptor co receptor gene and has an EFP fusion odent receptor gene. The reason being that we thought that with the odent receptor it tends to be seen that you don't get functional expression of this without the orco in place the orco has some fun. It has a role to play in expressing that. So we figured that if we saw GFP that the OR would be expressing and therefore the orco as well. Obviously that's an assumption and we will have to check later on that that that's the way it works. They also have constant constitutive promoters, and we didn't, you know, intend to direct them anywhere to express in the yeast because we just wanted to see if we could express them in yeast at all really to begin with. It was a bit of, it's, it's one of those projects where you go in and you have no expectations that anything will work but you've just got to see what happens. So we generated the this these plasmids and in the E. coli and then did the linearization to transform them into yeast. We also generated our transformments with zeosim. After that I've done some additional selection with high zeosim concentrations and also checking fluorescence because it being constitutive we can see expression in just the colonies that we initially get. And of course there will be a lot more optimization steps that will come after this. So far we have four or all the complexes that I believe are successfully cloned and I've got many more to keep trying. How am I checking that it worked PCR sequencing fluorescence, kind of all the standard things but just to let you know that this is what we're doing. Right now I am continuing with checking these and transforming and performing optimization, but I wanted to share we did some initial confocal microscopy pictures of one of the initial transform colonies so after no optimization. And I think we see that there is GFP expressing but as you can see I think it's not in that many of the yeast cells. So this is certainly something we want to try and optimize and this might involve things like fact sorting or, you know, optimizing based on the fluorescence. But I think it looks like the GFP is expressing which was a huge, hugely positive sign for me. And just the kind of close up I guess of that one I am no expert on these kinds of things and certainly this was not an optimized confocal kind of experiment we really just the confocal guide not really even worked with you so much so we were just seeing if we could get any pictures really. But I think it does look as if it is expressing towards the membrane, which I think is again a positive sign because we didn't really target anything or really know what's going to happen. So it's kind of a lot of preliminary work so far. But I have a lot, a lot of time, a lot of things to do going forward so of course I need to optimize the growth of these strains that we've generated by monitoring for example biomass or the fluorescence. I mean there are two approaches how can I grow the most and how can I grow the most that's expressing the most. So I'm hoping to sort the cell lines using facts so that we can get rid of those use that aren't expressing. And of course, there's a protein purification and detergent screening steps. And the big question as well for me is that we need to determine that this is a functional protein, and this will certainly be something that's going to need a lot of thought and assay design to figure out a way of doing this. Just say thanks for listening. I'm happy to answer any questions or you can always email me afterwards if you can't think of something now. Just to say thank you to my supervisors Krista and Matt's to Bao Zhang who I think is here and he helped so much with the cloning and help design everything and show me how to do everything. Thank you so much. Thank you to Guppy and Hannah who showed me how to work with yeast. I don't think even mentioned that we're using pickier yeast with X 33 strain if anyone is interested. And of course, Ola who is the microscope person because I can't say the word who helped you with the confocal to begin with and I think will help me going forward to generate some better confocal images. So that's me. Thank you very much. Thank you. You are co expressing the receptor with the this orco code receptor or you call it. Do you have you considered like putting you only have the gfp tag on the receptor. Yeah, have you considered like putting a visual tag on orco just so you can see that you have both. I wanted to think about this but we wanted to reduce the amount of kind of tagging we did just because if this turned out to work we hope that that would help us then, you know, messy further down the line and save us some work. I think it's, it's like there is a chance that the orco will express on its own without the OR, but there is very little chance that the OR will express without the orco so I think the idea of putting it on the OR was that if we see this gfp expression, we have both expressing because this is more likely. Like I said that's that's based on work in other systems so I don't know that that will be the case and certainly that's something we have to look at. Yeah, so speaking about other system would we have a question from Arianna about you if you considered insect cells. Yeah, and we did basically this was the decision between yeast and insect cells, because I think the insect cells are a valuable option for this. It just came down to the fact that we had expertise in the yeast stuff on hand and that we felt that we could do maybe higher throughput assays this way. But the idea is like if this doesn't work we go to insect cells next you know so it's definitely a consideration. And then we have a question about from Martin Caffrey if humans also have orco. Humans do not have orco so human odor receptors are actually RGPCRs I believe they're completely different in mammals from insects we don't have we don't do odor infection in the same way as insects and yeah we don't have orco it's insect specific.


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Boom, howdy fam Anthony Johnson here today CEO of the red man group founder 21 studios 21 convention 22 convention 21 University 10,000 other things on the internet including make women great again today's episode 153 of the red man group I got a special guest today. It's me one-on-one kind of show We will take questions throughout the show from the chat But mostly it's me me and him I've been kind of interview here a conversation about the man is fear about men about masculinity About the frauds these other fucking losers in our space So before we get into that today's sponsor is actually in a movie coming out It's a really exciting movie coming home of theaters very soon 4k ultra HD DVD check it out Fraud's in the hood. I'm really excited for this star as a couple buddies of mine as you know these triplets of idiots and starting on roof rattle simp CEO and the sharp father aka the sharp mama Donovan shart So check it out. It's coming to stores soon. Probably best buy a Walmart and everything like that It's gonna hot movie, you know hottest movie of the year right here at the end of the year here So that further ado though from our sponsor here, please. I'm welcome to the show for the first time Paul from the war room Yep, no holds barred men so Before you even get more into the show man, I want to show off your channel This makes sure everybody subscribes to it. So Paul here runs a channel It's got a thousand of followers and stuffs and blowing up this year and he runs awesome channel I really love reposting his content. He lets us like publish his videos sometimes and then you run great content Man, I love it and I hope everyone watch and subscribe to a channel immediately link in the description all that good stuff. I Appreciate that brother. I appreciate Really appreciate your support man. You've actually helped propel My channel forward quite quite a bit this year. Like you said, it's been it's been a pretty good year and it's it's been For the same reason. I mean you share the videos because we see the same things and yeah Just stamp out some of the same bullshit. So I appreciate you appreciate Yeah, man, the man of spirit to me is a really awesome space. I really love it It's been a massive part of my life.  Yeah getting strikes.  Well, you know the deal with these dudes are a lot of these guys. It's it's When you don't have shit and then you get a little bit of something It's you can tell a lot about what a person is when they get money You know when it or they get cloud or get following or anything like that You can tell a lot about that and a lot of these dudes in this space have taken some work and just Recurgitated and repackaged it and talked to different I guess sections of losers man every every batch of loser talks a different a different tone So you got your donovan sharp followers, and then you got your Tomasi followers and When I look at these dudes man when I first came into the space, it was I mean it was It was healthy for me And it wasn't for all the reasons that these a lot of guys come into it I've I've been the person that screwed my life up I made bad decisions because stuff came to me and so I was like not that I wasn't being who I was supposed to be But I was I mean it was weird It was a weird dynamic. So when I stumbled into the whole man is fear at first It was healthy, but then I found myself getting angry and then some friends of mine were like dude You're getting real salty. You're like do you have a man?  Okay, but how'd you get there? And that was what pulled me out was I didn't I wasn't raised to be a victim It was always hey man, you're here So you must have done something and so when I came to the whole Manusphere it was good to a point But that all women are like that and she's not yours.   I was first I was I kind of was in it You know, I was like, okay, I can feel it, but then it was the same message bro over and over again.   Well, that's what they are They're like with penises man. Yeah The women are saying I mean if you think about it, they're like all the shame and talk Well, what do you plus these do when someone calls you out?  Yeah alpha. I don't want to be alpha That's what alpha is bro. I don't I think that's goofy anyway But if that's what you're striving to be shit count me out, man Let me actually I'm gonna pull that up for the fans because not everyone's seen that so this is really recent So basically I'm gonna pull up Here it is.  It has almost 200,000 views I mean this this channel only has like a hundred thousand subscribers, right? And they even that's brand new That's why is it video here? so these idiots tried striking for Abusing the copyright system on YouTube. They tried striking a bunch of channels big channels with 1.5 Well, you know a million subscribers mid-sized channels like elite attorney They try and they tried they basically tried to delete these channels for anyone watching It doesn't know YouTube like the strike system like me and Paul here have to worry about if you get three strikes Copyright strikes or three community strikes.  They did 20 look at this they did 20 against Abba and preach Yeah, one channel.   I'm gonna tell you my version of it I don't Plus I'm not gonna give those dudes any airtime on my channel It's hard to watch just to look at those dudes and listen to them talk. Yeah, I mean, I can't do it either I pay people to do it.   They're so fat.       They really are man.  Yeah, I don't need I didn't do that with three dollars.   I mean, what's the difference?    That's I used to I've done that before. Yeah It's a waste of money, dude. Hey, you know 500 bucks for a piece of steak. That's two bites I'm gonna I've done that before one.  I don't even think he's banging these chicks.   You can see they they get away from him.   I haven't had no one to do that to me So the eighth grade this dude's a alpha baller and even the chick he's paying.  There's there's some basic honesty. That's why the profession is so old, right?  That's funny thing about it It's paying money to Pretend Yeah, he's getting laid man and making video like every video.  What's up girl? You doing already in my car?   There's I'm afraid. I'm a that's scary to me. That's terrifying to me because you're that is exactly right the dudes Who buy into this stuff that that fund these dudes lifestyles? I'm man. Who are they who are they who raised them? And why isn't someone in their life life going? Hey, bro? Don't do that.   I would I would probably pass out So Dude, you have a word that I love that I had my girl. We were laughing at last night. Everybody loves it It's one of the best words I've ever heard in the manisphere. You call these dudes vaginal These dudes are so vaginal and like I've never described them perfectly all these rods in the manisphere I'm honest man. I stole that from my man patrice o'neill. That's that's patrice o'neill That's that's a hundred percent his his invention, but it's I mean it you take what you you take from people what's awesome That's how we become the characters we are and that way there was when I first heard that I was like, dude That covers a lot of bases. Yeah, that's what they are man. I mean That's everything that these guys do I mean, I'll give you an example that video that you put out the last one when you had rollo Basically crying about that kevin ray wilder dude that See, I don't the only time I speak on these dudes is when they do something that just makes me go I don't have time for that.  I'm cracking up, but man I'm looking at this. I've had so many dudes who are man rollo would eat you up in a debate And I'm like, what am I going to debate rollo tamasi about? I've screwed I probably and this is I'm not Patting myself on the back of that but I guarantee you up to my sophomore year in college I'll probably eat from not going to stop right there and and still had more successful with women than he did That's right.  That's how you can tell Who a dude really is rollo rollo is like a Karen.  You know what I mean?  He was in my house She's like that dude's like an alien And she doesn't usually talk like that. Like this is it was very candid kind of chick thing Like that dude's like it She just didn't like him.  He's got this weird aura And then when he talks he tries to sound so smart and I'm he might be smart But just because you read some books, dude, we got the Every time I hear him talk, that's what I that's what I see with his baseball cap Hiding his receding airline.  These dudes.  It's like a record.  I bet you don't get it. I hope you don't get it That's what these dudes are. They don't want dudes to be successful. They don't want them to be Right.  He's married because if that dude was in this world this this Piranha world that these women are or do men listen He would be done that dude would be an only fans fan Yes, sir, you know what I mean his lotion.     I was like And you know, this is what messed up Anthony. I'm not even being facetious that she actually looks like that I know and he calls her he calls her he too. He bit you know, it's funny.  She had the restaurant She always orders more food and she's a fatty basically So just like miss the actual character miss piggy.   I hope not. I don't want I don't want that bad to happen I just like make a fun of her because she's in on the scam too And that's what I'm saying if she wasn't aiding the bedding this Food cake dude didn't be different if she was an innocent bystander, but she's not She's yeah, dude, Rolo's I've met Rolo's wife.  That's fun.  He's a predator Oh, for sure But his wife is just some random woman and Donovan's wife is not she's definitely in on the scam She pushes that she she basically is the main scammer. She's the brains of the operation Right, uh, Donovan's like a frontman basically like a beard That's six men You see and that makes sense and that's why that's why I don't feel sorry for I don't feel sorry I won't say anything about Rolo's wife because you don't see her You don't see or you don't hear about her and it's you know, and that's but Rolo Rolo's his own problem by himself. That's right. That's right He's read my book. Read the book.   Anthony.  Come on You've never been around women longer to your wife and whoever's in your house And what you heard and what you read in the book But this idea that it's just this, you know, a woman's always gonna monkey branch and all the stuff I get it. It happens. But what I look at it is this There's no talk about the man. There's no talk about what the man does And that's what I I know that I've dogged out a lot of good women and good girls when I was young because Honestly, dude, as an as a grown man, I can say it just because they came with a lot of responsibility You know, I mean dating a good woman a good girl who had her dad in her life There was structure there And when you went over there that dude was standing there and you had to stand up That's shit scary, man. I would rather go find the girl whose dad left the mom and she can sneak out the window You know what I mean?  You know what I mean?  Now, I know that happens, but I think it happens because it wasn't proper vetting We fall in love with their representative and not the person And so when you read that book get that kind of advice from someone It it it almost calcifies you and makes you harden and angry and bitter and suspicious I'm like, dude, if you walk around like that, you might as well again You might as well just go harm it up with migtau dudes and play marbles and Show each other your toys and I'm not banging those dudes because I think that's a good thing to do But celebrating it as a way for life. I mean to me, yeah beta Yeah Yeah, with with rollo's book I do want to uh make it know because not everyone in the monastery knows this a lot of rollo's work in general But particularly the first book it's plagiarism, right and guys guys every Yeah, the couple forms So suave a little bit, but that's really old at this point a lot of it actually comes from the red pill on reddit I was trying to pull it up, but it's quarantine. So I can't uh, I make it show you guys what's going on here You have to like log into your main account and I don't uh, I'm not logged in right now to it But basically there's a whole community bot on this page reddit.com slash the red pill And that has like 300,000 guys in it before reddit quarantine its censorship. They'll share it They killed off the migtail one too. They just deleted that one. They deleted the donald one a bunch of these things, right?  He calls it his own version of the red pill That's why I call it the rollo pill and I call it the rollo sphere that wing is super toxic and retarded But he steals but these guys on that form these The guys who post a lot moderators should like that A couple of them have reached out to me citing the specific posts that rollo stole And where he reposted them on his blog and then how they went into his book They actually track how he does it to that because it pissed them off This is from like shit from like 2012 and then like six months later rollo posted on his blog with a few tweaks to it He does us a lot and so basically these guys in the manisphere particularly like let's say the fresh and fit Fans they're at the bottom of a manisphere bukkake of fraud I'm serious. It's trickled down like trickled down economics.           This guy ain't getting laid for free.  You have to ask yourself. Why am I celebrating this?  Why does that make me feel good? She didn't do anything to me Yeah, and what these dudes are doing they're living vicariously through these bullies these guys are are Yeah, I wish I could have told the girl who took my credit card and who took my kids All the bullshit.  No, really? Oh, yeah for real. He told he said this in a speech at 21 years ago He had for for about 30 seconds.  Yeah, he's the one that sells you the car. It'll be some other dude. I said roll over break your legs So we bring it you brought up a really good point Um, I want to get into it some more here with that little I put this out in our community tab a couple days ago So you were talking about basically these guys are living vicariously particularly through fresh and fit a little bit of donovan too And it's when they're lashing out these these skank. Yes, ma'am.   I don't like those dudes. They make me sick, you know Girl, not they're hustled.   I'll get this rhino 7 000 You can't get mad at the people who make rhino 7 000 for taking 10 bucks apart from these guys. I mean I'm not mad at them.    Yeah, and where we used to be productive We used to be producers at one point in time America had a different set of values We had a different belief system at all.   I feel like I feel like a dinosaur now because Where I come from and what I believe in doing these kids it's so foreign to them And they think I'm a guru at my job because I make shit happen.    Oh, you know what?    I don't follow women.    Oh, that's not pc. I'm like, what are you talking about?  I thought they were talking about me Like no political correct I'm not political correct. So that's not something I would say and then it just kept changing and so our culture Is so sick kids run the parents now.  Who are you be somebody and be that dude?  It's only you're not staring anywhere else It's over get under the deck as a matter of fact. You don't even need to see where we're going You probably saw my make woman great again hats, right? Like a couple back there.   Oh my god. Yeah, man. I'm 51 and I was telling my buddies the other day, man. I am Maybe two or three years away from being that old man that has zero filter. They're like, you've got a filter now I'm nice still but man. I'm so close to just Letting it go in public weapons free man, and I can't wait.    And so I try to I try to drop them now plus, you know They were I think I was going too Too hard at first, you know, I was saying a lot of the words that I didn't know would get you Get you cog blocked.   I don't concern myself with that stuff I mean if I can if I can make money off of youtube bad ass, but I ain't trying to Yeah, I was trying to I would do the same content these dudes are doing Tell dudes don't worry about it. They're all like that.  I found everybody.  Let's go do the work, bro Yeah, man. What is the work? Tell me what the work is man. Yeah, you know what I mean?   Thanks, fam Like I'll thank I got a super chat. I got to get to here in a minute You know, I'm thankful that he gave it, but I don't know this dude. I never met you I don't really care about you because I don't really know you. I'm not going to pretend to be your friend Even if including in even if you give me money. I don't care.  There's 20 more dollars man. There you go.  It's like a female. It's vaginal It totally yields, man and these dudes that are paying these guys are are A lot of them are doing it simply to get that validation, man That my man said and so these dudes are treating them. They're they're finessing them.     It's the same business model. They say we're crazy It's it's the same they're doing the same thing to these dudes as the girls on only fans are doing As the girls who are having them take them out for dinners all that stuff And i'm not saying that it's that it's wrong for i mean if you want to do a super chat and you know Donate to the person but what I look at it is this what are they doing with that money? You know, what are they doing? It's none of my business, but if you're here to help men Every any penny that I get in anything that I come if somebody sends if somebody was to send me money You know because of what I do here It's going right back into one of my programs. It's helps them because like we do a lot and that's one of the We do a lot of shit here with my program with veterans with children that don't have fathers Trying to get involved in the school and help them with the bullying program and all that shit and I don't Dude, I don't really talk about that a lot because I'm not here.  Oh, look at mine I got 15 screens back here and look at all the pictures. I put of myself on the wall These dudes are selling A character. It's a show. It's entertainment.   They actually believe this. This is fake. This is not true.  You're gonna save yourself, right? If you save yourself using someone else's tools That's fine, but they didn't save you you save yourself Right unless unless someone physically pulls your acid from drowning.  Yeah, but if he taught you how to swim a long time ago, he didn't save your life Yeah, you say you saved yourself, but they but you're right though They're finessing them. They're grooming them on purpose because it it puts them It facilitates that same addiction of attention and validation from a new guru, right instead of the woman guru Now it's a male guru. They're simping. They're simping for women or the simping for men It's sick either way and they're doing they do it such a good job at it I mean do think about think about if a woman could do that Think about if a woman could do that without giving you any booty, man You do no pussy from her. So all she does is convinces you that all other women are bad They're all that way. They're all gonna leave you.  Let me show you the statistic for marriage, man And it's this that and the other yeah, you can if you're a good debater If you're good at that you can make somebody you can argue a point and have a motherfucker go.  That's a good debater That's a scary human being and that's what these dudes what they're doing is they've gotten it to the point where these dudes They have convinced these men that the best thing to do is to play with themselves invest in Vaseline and Kleenex and Whatever all the shit and just be happy You know smoke cigars and written boats and take selfies like dude, you are being like bitches.    They're not good for the show either They want these dumb skank. She's low IQ trashy skanks with no class no manners.  Yeah, I got a pause real quick I got to take uh, this guy did donate a super chat and I appreciate that I do invest it back into the company heavily You know my company makes a lot of money every a couple hundred thousand dollars And I invest virtually all of it into the events into filming and photography and videography To actually reach and impact men and make change And I don't brag about that a lot.  Anyway, lord hybrid.  I got a trump 24 a hat that I was gonna wear, but it's kind of hot in here.     They got a little bit of money I think way less than what they brag about anytime these these guys are liars every time they say something They're exact donovan does this all the time.   It's all shady.  They're abusing the copyright system They're basically weaponizing copyright stuff to fight defamation which youtube tells you not to do their policy on defamation is hands off 100 They say we're not here to determine what's true and what's false, which is what would be considered defamation, right?    They run from challenges. They run from being exposed They run from the truth.      This is not only funny. It's it's a real reflection of who they are. It's really on point That's why that's why it's so funny.  They cost themselves a lot of money They're frauds by their own actions.  I think than faking fraud.   How does a woman allow it and stay?   Wow, you gotta start a gym You gotta open a fight gym tough guy Hey, let's Let's take a step back because we're doing something that I think no one else does Unfortunately, even the guys who criticize fresh and fraud faking fraud So me and you both have been we're going after the fans and I think rightfully so a lot of guys they they draw the line Now they're like, oh, we don't want to do that. You know their fans Come over to their channel.  I think you're just you're They basically it's birds of a feather flock together, right?  I don't need you.  I can ban you.  They want to get lied to they know they're getting a lot of them at this point I would say like more than half of them unless they're haters.  They're being lied to I don't know that they I don't know that they actually watch the videos. I think that they read the uh They read the the thumbnail They read the title and then they get the channel and their comments go.  Listen.  I would Oh, they're all they're all millionaires, right? They're all millionaires with anonymous youtube accounts.  I don't think those dudes are dumb.  So, you know when you have Let's just say computer nerds.   And their alpha their leader the dude that they it'd be steve jobs Steve jobs to those dudes is more dope than the rock, you know what i'm saying?  They're all of that These dudes have put themselves on top of them and they're manipulating them But the thing that they're not telling them is i'm teaching you how to be the strongest version of a bitch If they say that would be cool I think what they're really doing is they're basically faking this journey from beta to alpha and their own terminology and stuff And these guys buy it just because they say it They're I don't know what the exact phenomena term is there's like a term for it Like I think it's first mover principle or something.  Oh, I heard I heard color written house was a white supremacist. Yeah How do you know that?  That's the same principle of play.   I don't know any guys who can get six girls around table like that. So it gotta be alpha Hey, I thought it was skanky.   It's monkey see monkey do it's beta see beta do but it's You're right. It is it's not they're in my view. They are dumb.     These kids are lost They have no real dad.  I know donovan didn't Donovan was raised by a toxic single mom.         I mean it was hard.  So I get that part of it But it stopped being a problem by the time I got into college.   Yeah, man, you know, I was raised by this My mom was an alcoholic. My dad was abusive. So that's why I drink and beat people. No fucker That's why you're inclined to do it.  I got just skits of fun.  Like it's okay.  It's you I want to pause here and a point you just made me think of with these these little in particular the fans, right? I I want to put it out there so everyone understands my position on at least and I want to see what you think about it too I think the reason that these guys who follow them suck with women Is directly linked to why they even follow those guys in the first place And I think you would agree with that, right?  She sniffs.  Yeah, exactly. Exactly.  I mean, that's exactly what they portray Yeah, if you know and that's what luckily for me when I was When I was being negatively affected by the stuff that I was watching because you know, coach gray guy Adams I like coach gray. I think coach gray Adams is he's kind of funny.  They're gonna find you you got hundreds if not thousands of videos So they can scroll back and you know get your She's not yours. It's just your term video.  You know what I mean? Like This isn't gonna work anyway, bro.  I'm gonna put the effort for I know what I mean Like that is to me.      Well, I can do it Man, it's hard to do it It is hard to do I can disagree somewhat here because I learned how to do that the hard way by doing it Like I was it when I was it's being it's it's good for confidence though You know, you're scared and timid. That's good to break that shit Yeah, the idea that you've got to go smash up on a thousand girls and do that I look at that.  That's kind of weird, bro Yeah, no, that's that's trauma playing out.  That's crazy.  He's just living out life But he's not a weirdo.     Like I race BMX professionally. So that was super easy to do it was we'd go out of town and It was just easy to do it.     You got to be a man.   I was afraid of The responsibility that was gonna come and maybe I wasn't afraid of success. Maybe I was afraid to fail again You know what I mean?     It's not worth it. I'm like dude. Listen.  They have fun when we party.      It's better.  This here's a large woman. I mean, there's a big woman Yeah, she's I mean dude, that's And again, it wouldn't be bad if he didn't tell everybody she was a dying piece. That's right I wouldn't I would be way I'd be 10 times less intense or not She said she's a negative four and but she's a good girl.  Yeah, homie. At least you got a good girl Or he just says I love her like hey, I love her. She's kind of big whatever. I would leave it alone, man Straight up just hey listen. I know you guys might not you know, everybody's got their own flavor of kool-aid This is delicious to me and then leave it alone man You told both of her she was a nine and a 10 like on whose scale Dude on death row. Maybe a dude on death row that hadn't seen a woman in a minute.  So he's really good though with photo. He's my main guy for creative stuff my creative director So videography video editing photo editing photo shop. It takes time. I mean, dude, this meme This took probably like almost two hours to make this meme That's pretty cool. Yeah, I want to learn how to do that But that it's I've watched some videos.  This is your business like yeah Getting rid of that trash is important for your work. You know, and it's me. It's not I I mean, I really appreciate what you do because you I mean even if even if people find you abrasive and all that Shit, you're honest man. And what you really you really want to help men.  This is too much. This is too much work. And there's too much pushback. There's too much Of the cult the counterculture from the 60s has become our culture now and now we're the counterculture Now men who believe in men men who believe in traditional women And and things being the way that they're supposed to be now we're the counterculture and that's hard to do But even even within the monastery man when I push back on these frauds This this cost me money when I do this. I could have had donovan be a speaker.  I live in Orlando a couple hours from them, right?  That's right. That's right.    Do you wouldn't believe? But because it shows you who people are man when that when push comes to shove when you get even a little bit of money I think it becomes more than that, right?  And it's it's it's I learned I know dude. It's great. Like That that made me dude. Tony knows what i'm talking about man.    It makes me sick. I it's unethical. They broke They have huge followings too. That's and I'm not hating dude.  I have a problem with that. I don't I just don't like it and That's exactly what I mean. You've got these dudes Playing it. They're paper tigers, dude They're paper tigers playing king of the jungle and it's because there's so many sheep out there I mean that's that's a That's Yeah, no mercy, man. I got no mercy Rollo was another one man rich cooper this big channel this big this canadian pussy, right?    That's what that's where the memes come from man Oh wait, that's the wrong that's behind the thing but Yeah, I love Because this latest fresh and fit shit going on I've left, you know, I've been I've made my videos and moved on and all that But what they're doing now them going after people I got something to say about it, you know And I figure if I got 4,000 from smashing them before maybe I can give another four and You know get convert some of their dummies over a little bit because there's a lot of people who used to listen to them Out of that 4,000 of people that came over There was a lot of dudes that didn't know and when they when they saw that I mean, there was a mass exit is there's a lot of channels that that blew up because of that Yeah, but those channels i'm watching them.   Yeah, I cut off for a second. I think you're back.  Yeah, I'm here.  Oh for sure Yeah, he would exactly he was trying to kill my company man. He wanted to steal it Yeah, when I say though it cost me over a hundred grand. I don't even mean I'm getting some nuts back now Uh, I don't even mean it just cost me like opportunity a hundred k. Oh, no, no, no, no I didn't even mention and that's it. This is like intentional fraud and deception Rolo basically pushed like $50,000 in charge backs and caused all these other damages. This was like hard dollars Not opportunity cost it costs like some serious money, man I mean tickets and stuff that never got sold who cares that that's an opportunity thing But that is what it is that just stacks on top of the actual hard dollars it cost And then you froze up. Uh, can you hear me? Make sure that's not my internet Hey, can you guys hear me on the chat? Yeah, I'm in it's working fine. It looks like I think uh, he froze up. Oh, he's back. Hey there. Yeah, I just rebooted. I didn't know if it was my fault That's okay. Yeah, you're good now. I'm gonna kick your other cover one out Good stuff Cool And I know what I was saying. I don't know if you heard it, but that hundred came man That wasn't like opportunity cost with tickets.     Do you man?   That's right. If you're if he's no longer with you then he's Against you. That's the way that I mean, that's the way that See, that's the one that's what I was trying to make. I was gonna make a fresh and fit Off the Napoleon dynamite when they go to the MMA gym I was gonna make one. I need to figure out how to make a deep fake and put the faces on that to where they're real That would be dope. I try to do it. Yes. I'm like, I don't I don't have time I won't make it in time for tomorrow. Yeah, it's tough, man.     That's awesome. That's good stuff I really enjoy it too. Like I really I really hate fraud So it's not a personal thing. Like, you know fresh and fake, uh, whatever They've never like attacked me people think there's beef and there's no beef. I don't have any There's nothing personal between me and that at least right donovan.  He lied to my he lied to my audience He lied to his own audience. He lies to the manisphere.    Do you see that dude? Uh, oh my god.   You know what I mean?    That's what these dudes are doing. They're Not only are they doing the same type of things that we're trying to do They're they're Directing men to go the other way like a grain of what it means to be a man That's not what manhood is and if that's what you're here for You gotta be called out.       Yeah. Yeah, because they've tried that. That's what these dummies, but they they're dummy fans They believe it when they when they apologize. This is not an apology. This is pandering. This is political propaganda. This is like a politician Oh, I got caught banging hookers and snorting coke as a congressman. I I apologize to my constituent That's not what we're doing america and I will not do it again Yeah, you got three hookers with cocaine on their titties in the hotel room waiting for you That's why I made fun of uh, you know, you remember andrew gillum in florida that guy who's running for governor Do I have a thing of him there? Let me see. No, I don't got any pictures and he pulled up Anyway, the black guy, yeah, dude.  I think all that. Yeah, like that that's what these guys I tried with donovan. I made this a big point on my look when donovan being donovan's a fraud until a lot of guys This was a shock when it came out, right?  He's the same thing though Almost every public figure not all of them But probably like 80 of them politicians celebrities athletes anybody who tries to especially the woke people, right?  And that's what all these public figures do whether it's donovan rollo I don't mean I don't know if he's putting their their dicks in men's booty holes, but But it's the concept is the concept is there But you know myron myron. No myron. Oh, yeah. Oh, yeah He looks suspect to me for sure.  You can tell But myron doesn't like women.  I don't know.   Hey, there's women around Then women started cussing, you know what I mean? But there were women started cussing because men allowed it You know what I mean? And that's that's the whole this this whole The whole thing that we have but myron when I see dudes like myron I had a buddy of mine named brian when we were young dudes And brian was an athletic dude man, and he didn't we didn't know he was gay He didn't know he was gay.  I don't remember.  He was just like dude, but he punched like like this like a girl So it didn't it was splashed It wasn't real brutal, but it sounded bad. He was just pop up and we went out there We broke it up. Well about three years later. I run into one of my buddies that was in that crew He's like, you know brian's gay, right? Like wow that explains a lot because he used to he used to get so mad at I mean he was like myron as we said anything that was weird.  So when I look at my when I look at myron The way he moves and tries to bully chicks.  I had the thought about this I knew myron is kind of bi or something. That's what he looks like to me Uh gay or gay or something, but I think the psychology of it. I think you're breaking it down pretty well that makes a lot of sense It's weird bro. It's it's we I've never seen An adult straight man do that.   That comes about you know, you get that but he's too old for that He's too old for that to be the case Yeah, myron's my age.  Like he don't just yell at him He's square like he toes the line.  What are you gonna do? You know throw a left hook? I mean, what are you doing? What is the thing you're doing? You know tower no means a tall dude All of those things when I look at that.   And so Yeah, maybe that's it, you know, and if that's the case we've got to kind of feel sorry for the dude because he's trapped in the wrong body And I I think he would be if he comes out as gay and he comes to Comes to grips with it. I think he would a lot of that anger would subside, right? Yeah, I would I would guess that would go away because he wouldn't be a war with himself It's self hatred, you know, and you know what and here's the crazy part about anthony If he did that then he could give Then he could give women sound advice about how to Be with the men that they like because he's You can't help me a woman can't tell a man how to be a man So he's sitting there trying to tell men how to be men based off of his perception He's telling men how to be the man. He would like to date He's he's still like listen, man If I could be free if I could do what I wanted to do I'd be this kind of man This is my boyfriend right here But once he if him when he comes out and says, you know, I'm gonna own my flag then Him and the little troll they can have a real conversation But the troll has to say I'm a trick. All I am is a john for these hoes.  Yeah Yeah, yeah, yeah, that's what's a rollo. We got a rollo one of those two rollo looks like smeagol too with the hair and I'll do this a little bit and got a tan man If they were just honest, that's all they need to do is just be honest, dude be honest, uh, one of them say he's a Decepticon in transition and the other one.  I mean, let me be clear about that. She's a 10 on a scale of 1 to 100 Right. Yeah, she's a she a 10. You just gotta understand the scale Hard yeah, hard.  So She should have let him lie.     Oh, you know, I got this girl.  Well, what's interesting I don't I don't know if you know this maybe you do but let's talk about this for a second Then I want to shift gears a little bit to like blm and should like that Because you you've done videos on them too, but yeah donovan is actually a sub. He's a submissive and she's She's the dom in the relationship. She actually runs Like miss piggy, right running kermit the frog. We're trying to she actually runs donovan like edwin hardcore She runs the business. She makes all the decisions. She makes she manages the relationship She blueballs him when he is disobedient. He jokes about the son's patreon. I'm dead serious Now the joke is the truth. That's just telling the truth. Exactly.  That's all like I said in my documentary It's like a batman persona that he's crafted that's completely fake, right? Wow And in the reality is the opposite.     It's sick This is this is my uh honest observation and opinion And evaluation of what that relationship really goes on He really is a blue pill beta male mama's boy Simpanzi who goes on the internet and role plays that he's some alpha And in real life.   It's you know, I think of that's one of the things, you know I was talking to one of my buddies about this very thing. They're like, man, how are you putting your dirt?   I'm I am who I am I'm not an alpha. I'm not any of that.  It's just it's disheartening because The the men that they're leading straight Are part of this this population only fans.  Why?   I have an other there's a saying I like from one of our speakers He's a medical doctor in South Carolina.  It's simple. You come to a conference.  Woman ease woman ease woman ease Volume volume 25.   I'm a broken wounded man Man, it's sad. It's sad It's very very very sad man. It's it's very sad because You have all these guys doing this that our culture is still fucked.   If you've been doing it that long you've influenced all these men You should I mean my goal in my channel is this I'm not trying to be anybody's Gurus all my I don't want any of that. I'm trying to help you see what you can't see And then go do what I'm doing. You know what I mean?    There's no I started to do patreon and all that. I'm like dude. I'm not trying to squeeze anybody from I don't need anyone to give me money Because I'm talking to him. This is free.   You know what I mean?  You catch that shot. You go.    So when they're doing this on tv eventually, I got I got mad.  At a bar like I was it was 3 in the morning. Anyway, so it's kind of really tired The filters are going away. I'm like, all right now.       I hated that show.   That's right. It's a it's a weird It's a weird deal.  Hey, let me put What about now? Can you see me now? That's to me.  Because chances are that fat girl might think she's better than you Anyway, with the way the dynamics go today with with tender and all that She might she's it.  She might be dating Donovan and she thinks she's a dime Yeah Hey, let's uh, we've been going for almost two hours. So before we wrap up, I want to shift gears a little bit I'll talk about bigger cultural issues. You've been pretty critical of blm Uh, but you know, what is it burn burn loot murder? Black lives matter Talking to talking about your views on black lives matter and what you think about particularly with 2020 What we saw with that when that really got big and there's the riots and all that shit man, you know that Being my the age that I am now I've seen this this Cycle this is a site.  I saw the results of it when I was a child.   I made a video about it called nigger and it was uh Basically talking about it. It was pretty good dude.   Well, you know, why can't I do this? Why can't these kids play with me?    It's just they play They're playing chess with people in the court system. That's all it is This lawyer's got five dudes.  So he'll sacrifice the other four to let the other dude It's all they're just a buddy system But when you have a high population, you know, the print entry is a lot blacks and mexicans.      I mean, that's what we did as children.   He can't be here We were children.  We'll pay we'll pay your rent section eight. We'll got you covered. Don't worry about it.  I think they're a terrorist group And they should be treated as such.  They're burning down police stations, man.          Yeah, you do a lot of videos in a channel I've seen about hard times that are coming and and all that stuff and I love it man. I agree. I'm out of Florida I love it. I'm really glad I live here. I was born here. I was raised here But I watched the rest of america in particular and it's uh, it's really sad man.  We got a lot of special forces vets here Yeah, this is where I want to be, you know, if shit's gonna hit the fan Die right here on this hill Do it man. So it's a good place.  It's not it's not They change they're changing definitions man, you know what I mean like If I'm gonna change the I'm gonna change the definition of drunk to happy and then just tell the cops I'm just happy right now. Let me drive man.  It just makes it comfortable.  It's not about health If it felt we wouldn't be where we are with the food and shit So yeah, uh It's a real it's there's gonna be rough times for sure and we have it coming You know what I mean? We've got it coming as a country.    It's how it's gonna crash Right, is it gonna be in is it gonna be a kind of a you know a decent landing where people survive? Or does it is it crashed?    You know what I mean?  I mean and that's what you said earlier about money Yeah, when it comes when it comes quick it goes quick, but normally when it goes it hurts when it goes Yeah, and that's where you know You when I think about It's pulled out that you fall further than where you came from. That's right That's what you mentioned with fresh and fit.    Oh my god.  Uh ran into a bunch of clout and you know popularity real fast They didn't really earn it. It just kind of got dumped on their lap, right? That happens It is what it is on youtube.   Yeah, I mean nobody did anything to them.  It's time to go You know what I mean?   And they have nothing that's that's what happens.     You know what I mean?   What are we gonna do?   Like they got I wouldn't be surprised if they already got that shit now it's why they're flexing on us but We need to get men.      It's too perfect And we're having a lot and it's celebrating this shit, dude Can't wait to get the new thing in my hand.   I grew up most in the 90s and even that feels alien compared to what we have now Oh, yeah, I mean you had what different time now Yeah, we're dinosaurs We're just dinosaurs and you're young, dude. You're younger.  You're a young dude, man, and it's That that you've seen it.    There was no social media There there was not even like a while.    You know what I mean? That's scary They're gonna be they're gonna be judges and shit man judges Policy, I mean Yeah, we need a hard reset and not by someone else's hand It needs to be by the man in this in this culture And it's I mean, I think if we don't have a reset, you know by the men and like you're saying by the men in our culture and Real real, you know authentic masculine stuff like that I think the elites want and this will be people say it's conspiracy theory.  It could just be famine.  That's what the chinese did. How'd they kill? That's what the russians did bro. And that's what I made a couple of videos ago I told people look up in the zeno island.        That's how that's an empty balls, man That's from the environment. You know what I mean? So you got empty balls.  There is a Calling going on for sure. It's it's undeniable and they put up the agenda 21 That was a world forum thing.  So you can look at that go. Yeah, it's just conspiracy That's what you're supposed to think.  It was frequent. Yeah Yeah, it's frequent major genocide. Oh, man.  That's what's going on with the weakers.  It doesn't matter like Okay What happens over there? It happens here Oh, the ogre the muslims. Yeah in china. Yeah, it's sick. Yeah. Yeah.     Yeah It is it's it is I made maybe it's a willful ignorance or whatever. But again, it's the school system People don't I didn't know what I know now until I actually started researching myself I've always been fascinated with nazi germany.   And then at the end of it I was like, well, I asked my teacher after a couple We had I don't know how many classes we were going through this part of the textbook, right? I'm like, I get why the germans were these bad guys in world war two Why were they the bad guys in world war one?  Yeah, I'm like, there's there's there's no explanation You haven't given one and and in the book there ain't nothing Why did this war happen and why were the germans the bad guys?  It was russia Basically, soviet soviet union basically won They expanded so much land mm-hmm Hitler made a mistake going into soviet into russia in the winter.  They got bogged down Had blitzkrieg worked it had been totally different and there's no telling what the world would have been because Like the people who run our world now you have to when you want to know Who the real bad guys are i think you have to look at who can we not criticize?   I might get mad too, bro I might retaliate too and then you start to see all the lies.   Yeah, I mean it doesn't make any sense.  It might not it would be a truth It'd be a different story though. It'd be completely different. And so now what we've got is we've got the same Faction pulling the strings doing the same thing because the same the same way that america's fallen They did the same thing to germany, bro same thing to germany germans were proud After world war two after world war one. I think it was berlin It's one of their major cities. I think it was berlin. I can't remember Exactly what it was.  It's the same thing. We're doing now Everything's good.  Yes.   That's a that's full stop. It's time to stop It's time to stop I think I think this is also like just the downfall of civilizations.  We're fucked.  But what we are going forward.  Yeah, you're back. Yeah We're basically in the purge if you look at what they're allowing.  It's already ruined. What's so Kyle? That's how Kyle written house. They were saying that self-defense itself was on trial Which is correct.  Man, if that would have happened, I would have been afraid I would have been really afraid if he would have gotten if he would have gotten convicted for that because there was nothing There was nothing wrong with anything he did.  That's a high stress situation. That's not training.   Let's enjoy the Marines, bro.    I've been arrested a lot And guess how many times I've been killed? You know what I'm saying? Never even been shot. I never even been roughed up by the cops I got slam one time because I tried to run but when it's over it's over.   I mean, what are you saying that word for you supposed to be? I mean you don't you don't get to say that I think the word itself is dumb.  No one has a background Yeah, no one has a backbone.  It's supposed to be community of men pretty much is right There's not really any women and it's very very few that have any engagement with it And yet there's no culture of honor. There's no culture of checking each other, right? Now men like me and you do that, but that is heavily frowned upon.    It's pathetic It is it really is and it's it's sad to watch but Unfortunately, it is where we are and we let it get here You know, I mean we let it get here I I've said it many times. I've been guilty of all of the things that are wrong, dude All of the girls being whores dog and chicks out turning them bad I did all that I've been a part of it. So I'm part of the problem too. You know what I mean?  It's I've got all these scars.  Okay. I own all this I maybe I didn't do this to myself, but I allowed it. So what what can I fix? That's helped me a lot and all the things that I've gone through When I was younger, I didn't understand it now. I know why the suffering happened I know why all the things that I've said why me god No, I know why because someone else needs help I went through the shit because I wasn't in a break and I didn't and now where we are I'm looking and I'm seeing people Like outright mislead motherfuckers for money And where they're mislead them is going to cause me harm when I finish getting old if I get blessed like that I don't want pacified men.  That's where we're headed.    Oh, yes. He was the ceo of theranos Mega fraud mega fraud competing competing with like bernie madoff.  I mean that's gonna happen most of the time either fraud or it falls apart Because these chicks are either bullshitters or they're just gonna be incompetent It's not what they're designed to do like biologically and within nature They're not they're not leaders like that. Women are meant to lead children. Not men Not businesses.  Not a religious zealot But there are certain things that work and the divine order call it what you want I'll just say the universe the universe over man man over woman woman over kids jessy, what's his name? by Jesse Peterson He's he's the right on and until we get that order back and you don't have to be I think people that are bible bible thumpers are dumbasses anyway. I mean that's It's a good book to it's a good instruction manual But it was never supposed to be compiled in a book like that and once you understand all of the history of Constantine and all the romans Dude, it's one of those bruce lee things. You got to take what you what's useful and then You know, some of it might not be for you.  What else can a woman want?   Well, we've broken it at every at every part of the chain There's no chain. There's just a bunch of links on the floor now Yep, I mean there's none even it seems like there's not a way to put them back together But that's where men come in we need to pick up the links and go okay We need to do some cutting and some some welding here. It's going to be It's going to be some work.   They're going to have to learn to Say no to themselves. They're going to have to learn how to tamper and temper their lust and their desires That's hard It's hard.  Okay Do what you want to if it feels good Now you got preachers telling you that So you got you got woman preachers, man. You got female preachers, man Not crazy I I was with jesse league. Listen.  And jesse he was one of the speakers. He's one of the pastors. There's a couple pastors Couple men. I'd like him.  We'll kind of wrap up But it's been a fun show So I'm really kind of free-wheeling the different topics and issues and history and feminism and the frauds obviously I'm really excited about the new movie. We'll do a quick, uh, shout out again. Make sure you pick it up on amazon your local stores frauds in the hood Really excited for this one. I heard it's amazing. It's gonna win an oscar probably So I really picked this up. It's stars three mega stars here these guys The sharp mama on the left here is sharp father i'm rufraudal and simp ceo Really good movie excellent excellent acting All right Sorry about that And no problem. I was just showing them on the the movie again that uh, it's coming out soon, man I'm really excited. I heard it's gonna be a winner, you know I And then we've been going for almost three hours, so we're gonna kind of wind down here I really appreciate your time today man has been a lot of fun A lot of guys a lot of guys were hitting me up in comments And they were excited for the show because they love, uh, they see you on our channel I want to put your stuff out, but they don't like know who you are sometimes And they wanted to learn more about you. It's been good Everyone make sure you go to go to his channel the war room Uh, there's a link in the description and it's just youtube.com slash the war room We'll pull it up too So check out his channel subscribe Uh, I really appreciate your time again today man. It's been really good really good stuff I appreciate you having me on anthony again. I appreciate everything that you do and i'm and i'm glad i'm on your side Dude, you're you're doing a lot of good shit and if I was going to be on any side nest This is where I want to be so it's an honor Thanks, man. I appreciate that Yeah, it's uh, we're in the trenches man fighting for fighting a good fight. Fuck. Yeah, let's get it All right, everyone else. Thanks for tuning in today. I will see you probably next saturday another episode 154 Of the red man group Thanks for tuning in hit the like button leave a comment all that algorithm stuff I hope I appreciate it helps the video helps the war room all that stuff. Thank you. Peace out


ign. Many of them will likely gonna be based on personalized medicine and genetics, but that's kind of gonna be your job the next 10 years to solve this problem. We also, yes, I think I have 10 minutes in my hands. I'll see if I can go through the last few slides. So now we talked about pure beta sheets proteins and pure alpha helical proteins, but they're also mixed ones. And the mixed ones, I kind of like the mixed ones. They're beautiful. You can't mix a single beta strand directly with alpha helices, why? So this beta strand would have to make hydrogen bonds with something, right? So a single beta strand can't make hydrogen bonds with the helices. So then there are gonna be two ways of doing this. You could either have alpha, beta, alpha, beta, alpha, beta. So you alternate them back and forth or you could have a larger part of a protein where one, say the first part is only alpha helix and the second part is only beta sheets. You probably spoke about this a little bit in bioinformatics and classification, did you? Well, okay, nope, then I'll... So the simplest one are the alternating ones. And one way of doing that is that, in particular we wanna use parallel beta sheets. So here you have the beta sheets on the inside and the alpha helices on the outside. So what you do there is that you have one beta sheet going up and there you go on the outside and have a helix going down. And then you go back and you can now have a second beta sheet that goes parallel to the first one. It goes up again. So by alternating them, you're using the alpha helices to get back to the first start of the beta sheets. This is called a Tim barrel and the barrel is the inside. Yeah, oops, I shouldn't throw that. This is another structure where slightly... So here it's more complicated. So here we have one structure. Here you have sheets helix, sheets helix. So you end up with some sort of sheet stack between two sets of helices. But on the other part of the structure you have a pure beta sheet domain, right? Yes. I would say there are coils. Because when you say turn, I would usually reserve... I might very well be sloppy and say turn at some point. But the real turns are these super tight turns you have in beta sheets where you just need two residues to reset and go back in the opposite direction. So here I would call them coils or loops. So even between... Parallel beta sheets. No, but if you parallel beta sheets you would need some sort of coil. But typically, having that long coil is bad, right? So you're typically gonna have some sort of... Anytime you see parallel beta sheets you typically see these structures. You have one strand and then you need a helix to go back and then you have one strand and then you need a helix to go back. Since it's Friday, I think that some of you...  You know what this is? This is what you do to break down alcohol in your liver. And I would suggest that you do some experiments on this purely in the interest of science, of course. So there are some... If you look in the worldwide population there are actually some genetic differences here. So in the part of the world, in particular in Asia the... Parts of the population at least have a deficiency in alcohol dehydrogenase. I guess it's good if you want to get drunk because you don't need to drink as much. For some reason... For some reasons, in the Nordics it's the opposite. So in the Nordics you have this vodka belt in Northern Europe and everything and people are frequently have lots of alcohol dehydrogenase. Why? I have no idea. Normally it's like... Alcohol is not typically something you feed from. So you could argue that it's not something humans should need. But I guess for historical reasons we have... Alcohol is a very important part of culture and being able to use crops that have been fermented or something. It's just a fluke of nature. Yeah. And there are... I don't know. That's a separate chapter. I'll come back to that. We talked about membrane proteins. Alcohol is an amazing molecule just from a cultural perspective. There are 4,000 years of human culture related to ethanol both use and abuse. So there are a couple of these faults that I think is good to have heard. Rosman fault is this... You saw the Rosman fault on the last page but I didn't recall that. So Rosman fault is really this concept when you alternate between sheets and helices, sheets and helices, so that all the beta strands are parallel. And then you get one very nice beta sheet between two layers of helices. And it was Mike Rosman who found it. This is very common in binding sites, partly for nucleotides and everything. And one of the reasons is that up here you can have very lots... First of all, both the helices and sheets. So you can have fairly nice polar regions up here. So here right at the end of the beta sheet is typically a very, very nice way to create a binding site. It's revolution. So when you see anything like that, if you see any structure like that and I'll ask you to predict where is the binding site at the end of the sheets or the end of the helices, it's usually a very good guess. The likelihood that the binding site is there is zero. So I would say that there you could have a binding site or you could have a binding site up here or you could have a binding site up here. Because that means that unpaired hydrogen bonds that's also frequently going to be good. Yes. So I think we can show... Yes, I'm sorry. So what you frequently end up having here that you first have either helices or sheets, right? So that if you have a binding site, you're going to need some sort of flexibility in the structure. So the mere fact that you have loops that you can somehow change the structure a little bit off, that means that you have some free amino acids that are not necessarily bound inside the secondary structure. And these amino acids, depending on what type of amino acids you have here, you can create something that has good binding properties to whatever you want to bind. And the way you see this structure, if you want your amino acids to be free, right? You're going to need to have it at the edges of your secondary structure. In theory, well, the same thing here. Well, actually, no, this is just beta sheets. If you look at helices, remember the thing I told you about the ion channels that because you have this dipole moments in the helices, say at the end of the helix, something happens. Once you're in the middle of a helix to actually interact with the helix, you would typically have to break its secondary structure and helices don't like that. No secondary structure would like to be broken apart. So the other structure that you saw in the ADH is that you can have one part with just helices and another part with just sheets. And then you would typically have a few alpha helices, few beta sheets, few alpha helices, few beta sheets. They also occur. And when it comes to protein structure prediction, we typically classify proteins in a few very large classes that I think are nowhere through. So you typically can see something is purely alpha helical or you can say that something is pure beta sheets or there could be these two ways of mixing helices and sheets. So the reason I bring up, do you see the hierarchy again? So now we're even large, now we're just not even looking at super secondary structure. Now we start to look at folds. We have multiple helices and sheets organizing, but these are still just complicated blobs of atoms. But as we keep moving up, the hierarchies keep coming back and by organizing things in hierarchy, it makes it easier for us to understand. These occur in one very common place that I will deliberately speak a little bit about later in the course. Last year the students felt that they would have liked more about DNA. These are typically very useful for binding DNA. So that in particular these beta sheets, they tie into bind very beautifully in this ridges in the DNA. The sink finger is one of them and there's this tata binding protein, that's another one. And the reason why I call them tata is literally T-A-T-A is not really more complicated than that.  Why would you need to bind to DNA? Initiate transcription, right? Could you imagine any other reason to bind to DNA? Could you imagine any other way to bind to DNA? Or prevent transcription, right? Because you can also say, this is a gene you would no longer like to express. And then by having, not the tata binding protein then, but having something else bind there, you could prevent it from expressing. So that's also something you could use, say in cancer. If some genes are expressed too much, could we somehow block those genes so they're not expressed so much? And that's whether you do it for the protein or something else matters, but somehow you're going to need to find a way to interact with the protein. And whatever you want to create to bind here would have to bind better than this protein. Because if you don't bind better than this protein, this protein is going to knock out your molecule. So you're going to need to find something that has lower free energy of binding. And now this is a pretty complicated large binding surface, right? So if you would like to create an antibody or something that binds better than this, it's not entirely trivial. I will have three more slides, so I'll do those so that you can have a weekend and then I'll finish it. Some of these beta sheets, can at first sight appear to have quite irregular structures. I showed you this last week, I spoke about these knots, sort of cysteine knots, right? Where you have the disulfide bridges that created small and very regular structures. Not all proteins look like that. These are some neurotoxins that they literally have an unstructured long loop there. Why on earth would nature have a long loop there? Well, but if the loop is not important, right? Nature would just cut out the loop and produce a smaller protein. So these things, there's a lot of research where you talk about disordered proteins today. So what are disordered proteins? But that's kind of strange, right? There is no point in just carrying around amino acids that aren't used. Exactly on that. And now we're very close to the research front. So just the last five, six years, people are increasingly, what happens with most of these irregular disordered proteins is that they become ordered when they bind something else. So if you just look at the neurotoxin itself, it's going to appear to be very disordered. And they're typically so disordered, you can't even determine their structure. You can have an NMR structure and have a very rough model. But what then happens when this binds to something else in the body that is targeting, suddenly this might form stable beta sheets or something. And that's why you need the residues. So I'll just finish off with one thing. I've showed you a couple of folds, right? And so how many of these folds do you think there are in nature? If you look at small parts of structure, proteins or something. So there was a very famous paper that I think we have a link to in Mondo, otherwise I'll make sure that by Cyrus Schroetja from Cambridge. And the title was A Thousand Folds for the Molecular Biologists. And Cyrus predicted there are, there is only in the ballpark of 1,000 different folds. Despite all, you can imagine the diversity we have in pepta and amino acid sequence, right? But all these amino acids, for whatever reason, there are only roughly 1,000 folds that nature tends to reuse for all of them. And there are many reasons that that we will get back to discussing later. But one of them is likely that there aren't really that many ways, if you consider 1,000 to be limited, that you can form stable small structural building blocks or relatively large structural building blocks. Now, Mike Levitt wrote a paper in P&S almost 10 years ago, it is 10 years ago, where we actually followed up on this. And again, it's like 30 years later, right? No, 20 years later. And this appears to be to not hold. There is more than, depending on how we classify folds, I would think there are probably 1,500 to 2,000 folds today. But the point is that there are not 100,000. And we probably know at least a factor of 100 more proteins today. So the number of new folds we discover is not really growing that much. There are a few of new folds every year. And that's extremely relevant to the bioinformatics course that you went through. Because that means that if there are, we certainly discover new protein structures, right? But what we are now increasingly seeing, that most new proteins we find are similar to something we already knew. So that the white, there are fewer and fewer white spots on the map. And that's really how we're going to be able to design proteins in general. There are, most things are no longer white spots on the map. So that, I think we'll finish there. This is one cool thing. If you start to design proteins, you would imagine that the sec, well, you know that the structure is encoded for in the sequence. But at some point, if I change one amino, if you take a large alpha helical structure and change one amino acid, that's not going to turn into a beta sheet, right? But if I take, if I change 100% of the residues into something that's stable as beta sheet, then it should be a beta sheet. So at some point, there's going to need to be a crossover here. And Lynn Regan at Yale University at the time showed that for a small protein, they managed to find one single protein where they changed less than 50% of residues. This probably doesn't sound that impressive to you, that by changing half the residues, they could turn this into something else. So they started with this protein and then by changing less than half the residues, they could turn, let's see here. Yes, they could turn the partly beta sheet protein into a four helical bundle. No, of course, they changed it in various places. They carefully picked the residues, they changed. But if you, based on what you know about bioinformatics, if two proteins share 50% sequence identity, that's extremely high, right? That's each my left true territory. So if I had ever asked, if I have ever given you this sequence and said there is a protein that looks like that and I have a second protein that shares 50% sequence identity to that one, every single one of you should have said, based on your bioinformatics knowledge, it's obvious has the same structure. It doesn't. So there is no rule in biology without exceptions that structure is sensitive. If you pick your residues carefully, just because in general, on average, you would assume that they had the same structure, there are cases where relatively small differences or at least smaller difference that you would expect can lead to completely changed structure. Yes. So you're of course right in the sense that evolution typically doesn't work that way. But again, there are plenty of cases where you've changed more than 50% of the residues in a protein and they're still homologous. So the reason why evolution survives is typically because we need to reuse things, right? And it's unlikely that that protein would have exactly the same function as that protein. But I think we'll come back to that when we talk more about evolution later because this starts to influence what function does. We have, there are a number of chapters booked, sorry, study questions that you can go through. I will come back and talk more about protein structure next week because we're going to become more and more biological. Björn and Dori have a third lab for you this afternoon that I think still builds on statistical mechanics, but they are gradually going to catch up and have you work with real proteins there too. So have a nice weekend.