Everybody forced here ripping 2022 prison football hobby 12 box case number two check out the random results anytime elitonsportscards.com We're getting right to it Back Isn't that just called it like a Jamaican beef patty you put the buns inside the beef Or sorry the that's like a double down, right? The beef inside the buns, and I was really confused. I thought we were having like meet infuse like Donnie's lost I got nothing to say Trying to hang in there It's not work. It's art. We are I don't like that I See we're switching out the style here. Yeah, you know, I just Someone time out Donnie. What did I ask you? You think I remember what I asked people J Robb who's J like James Robinson Silver Julian Love Giants with a Mike Ditka auto to start us off. There you go bears Purple ice to the 225 Daniel Jones Giants They'll sit in an advocate. See I Know Each case would be The case of prison with our pricing be a little over a little under 9k Silver Dante Hall for the chiefs a blue wave to 199 tire Matthew Saints Algier and Ritter This is an interesting signing Rob. That's pretty cheap for a decent running back though. So it's fine Blue wave Daniel Ballinger rookie Giants to 199 George Pickens way mafe Vaughn did which happy birthday to Donnie he remembered Silver fireworks Tom Brady box. Did you guys know that it's Rich's birthday tomorrow? Yeah, Rich and Sean is both their birthdays tomorrow. They're both breaking the moral Bruce Smith on the green scope 42 out of 75 for the bills What could you imagine?  It's not actually his birthday. It's just an inside joke. We always wish Sam and It started with Sam that was Sam and Donnie happy birthday So we got to give rich that treatment Aaron Jones all out for the Packers Orange Trayvon Walker for the Jags numbered 51 of 249 Yeah, get get Sean's something that that would that a five-year-old would enjoy Silver rookie Kenneth Walker the third Seahawks Seahawks hyper Daniel Jones to 175 of the Giants Kenny Pickett Steelers base and a Chris Elave or the Saints We should get Sean a Power Rangers lunchbox nice Red wave Tizen Campbell Jaguars number at 80 of 149 Tyler Gordon or shot white we can't get into a spider-man water bottle, obviously spider-man water bottle and Miami Dolphin's bedsheets. Yes Or like a twin-size bed Silver emerging Kenny Pickett Steelers red wave Theland to 149 for the Vikings Wondale Robinson Trent McDuffie Yeah, get Sean a happy meal, please Silver Zach Hurts for the Cardinals With a David O'Jabbo Ravens purple power rookie auto color match to 49 Ravens and a Kobe Dean and Kyle Phillips. Yeah riches in his 40s Early 40s Purple Jalen Weidemeier Dolphins numbered two or sorry Jalen a while I said why don't my Jalen waddle 14 to 125 dolphins purple James Cook Kyle Hamilton Patriots Weidemeier is on the Colts now Who had bills to pats to Colts the Colts was like December? That took them what one year to get on three games. Yes silver and oa fan Seahawks With a Josh Jacobs silver auto for the rave or for the Raiders I don't know. He's richest exact age. I don't keep track of other people's ages 147 to 249 Davis Mills Texans I know Donnie are the same age, but that's only because it's the same age. Like I don't know anyone else's actual age I won't remember it here. I was gonna say salt kair yellow horse always know my age Yeah, exactly, but like I don't know how old anyone else in this building is I know approximately, but that's it and I think I know that Dylan is 20 No, he's 21 now. He's 21 silver Jonathan Taylor Colts Because Sean was they were talking about celebrating that purple ice John Lynch bucks at 225 Danny Gray Jelani woods. Yeah, it's gonna be 50 years from now Oh Donnie's 80. Hey, what's up, Kenzie? I know that Forrest camo Tyler Algear an ice-card Falcons 12 out of 15 Ryan Robinson jr. George Carloftus Silver hype or shod pavement for the Ravens a blue wave Devin single Terry bills number to 199 Be heavy I say likely We haven't to be fair Donnie and I have not said anything about that this entire break to be fair Yeah, we haven't Silver CD lamb cowboys. So you you hooligans are the ones that are doing this. Yeah A bills rookie auto Khalil Shakir Because Donnie and I are true professionals. Yeah, why would we would never talk? We would not stoop to this this uh This uh mockery of a conversation rookie silver Khalil Shakir bills Josh Prescott, um It's a Kobe Durant You're like Richard Nixon there Yeah, Richard Nixon Hyperjohn hallway to 175 Wrong coast. You think that's Richard Nixon? That's I mean, yeah I don't know water games Does that sound like does that sound like Richard Nixon to you? Yeah All out Jalen Hurts Eagles Red wave fedarian math this commander is numbered one on nine of 149 Yeah, I'm excited. I'm excited to have juju on the team Uh quay walker alec pierce he's a big improvement over Everyone that has been on the page with the last two seasons. So No, I'm not doing burgeon old nixon voice not yet. Anyway, rookie silver josh pascale lions You don't get that instant payoff of me just doing that. All right blue wave jordan poyer bills 10 of 199 Using autotune Richard Nixon autotune would be a No, no the world's not ready quandre digs hyper to 175 seahawks He fits on the team. Well, I think they need to get an outside receiver still But I like the signing as as a whole wasn't too like three years 33 million is like 11 mil aav for a slot receiver That's good silver kupercup brilliance a red wave debo for the niners Number to 149 unfortunately for him if we have the same sort of offense last year no one's getting the ball so But bill o'brien's a better coordinator than maddie p Which I mean it doesn't say much but bill o'brien has had some decent offenses through the years. He's just a bad GM blue ice rookie trail on berks titans 29 of 99. That's a nice berks Raising canes is really good They surely have I mean they have a chicken tender sandwich, but Look at the specify that it has to be like a chicken like fillet, right? Like uh, since you're a pageant fan What uh greens go mickle hardman for the chiefs to 75 Customers all right, right? Yeah, keep keep talking that talk. We'll we'll we'll see we'll see was was the passing issue more on The offense as a whole or was it just maybe mac jones? as good as Like where what's your take on the the zappy mac battle Mac is better. I don't think it's super close. I think zappy's fine Um, I think offense as a whole and the personnel there was just not great last year The o-line took a step back which didn't help mac at all. He was hurt which definitely threw things out of out of loop, but That patricio just kind of kept us down silver hype elie michel hyper daniel jones to 175 giants It's a mere white louis scene Cane sauce is really good. It's very simple though Uh, I don't know if you guys have huma goose where you are. It might be it's a pretty well Huma huma goo sauce is like a better cane sauce. I'd say I think they only have locations Silver court and Sutton broncos tiran davis price to 149 rookie red wave auto for the 49ers Carson's strong jake furgusson i'm pretty sure they have like Best fries is a good one five or six location probably more than that Blue ice larmy tonsil for the texans 33 out of 99 As far as like a big chain, um You bow jangle fries are pretty good. I don't know. I don't know what houston's is I think bill bill is out of touch too. I think that's a big thing He needs to kind of give full control of like an offense to someone that like he needs to have like nothing to do with the offense I think silver mccordy patriots Donald driver to 149. Whoops On the silver signatures or sensational signatures for the Packers I thought uh bellachek was always a defensive guy Yeah, he mainly was orange dalton schultz cowboys to 249. Why would he call offensive plays? He wasn't calling them. I just think he might have had like personnel decisions and things like that It was like with like when mcdaniel's is there. I'm pretty sure it's like all mcdaniel's I think bill check would be like, yeah, let's go for an important down But like mcdaniel's was gotcha like the offensive guy there It's weird because maybe rangers often doesn't bad last year but the rangers team is bad silver z jones for the jags purple ice john elway broncos numbered 17 out of 225 like jalexanders, what's jalexanders? azean pacheco devin loyde I don't know what that is either. I'm sorry Popeye's fries are pretty good I would call Popeye's fries good not great nausea harris hype hyper rookie jeremy ruckert jets 91 of 175 I've been the Popeye's plenty of times. I don't think I've had their fries. They're good Um, I like five guys fries a lot. They're really good silver brilliance jopro bangles They're a little inconsistent. I'd say sometimes they're really too greasy Got red wave larmy tonsil to 149 for the texans Jackson hill is a comma never been never been to white castle jeffrey.  I'm I can't remember it was it was earlier today. It was one of the daytime breaks red wave rookie bellinger for the giants to 199 And I've been on that double today But yeah, we have crystal here. It's um, it's like a step above dog food, I guess Tyler boyd bangles purple numbered 63 of 125 I you know, I actually probably wouldn't feed crystal to my dog Okay, Sam howl rookie base for the commanders We got a rat Would the rat eat it? Probably not. I'm just kidding. No, I've had crustals a few times. I Maybe it's the locations here, but they're just they're like not good like at all lockdown buda baker Got purple iced avante white for the packers numbered to 225 No personals tonight. No personal sign. Unfortunately We were going to have them and uh, I ended up working a double Silver rookie. Eric. He's a con medolphin. So I'm just not gonna stay after the group breaks guys Blue wave chris godwin bucks to 199 Should have more tomorrow night though But I will say they're probably going to be they're probably already full for tomorrow night Just because we're not doing them tonight Yeah, I just not gonna work a like an 18 hour shift hyper lennar williams giants to 175. Sorry 16 hours You might as well just call it even make a 24 Shut it you Yeah, I I I would like to imagine why cast a better than crystals, right My mom said it was a lot better back in the day, which to be fair I mean, I think most fast food places were a lot more a lot better back in the day They used to have like grills and stuff Where now they're just kind of like heating up food. That's already been prepared. Yeah heat lamp or like Like fries are fine. Those are already most places you go fries are already frozen anyway It's different but silver hype divante smith Blue ice pats from andre stevens into 199 George pickens way a mafe How do you go back to new Orleans? I haven't been I wouldn't when I was a teenager so I couldn't do anything like You know hang out on bourbon street and stuff, but silverkirt warner cardinals to 75 cults rookie auto alec pierce nice hit green scope to 75 Go indie wandale robinson trend mcduffy. I would love to go to new Orleans and get like legit like cv That sounds like it would be really good Green scope ryan doc is hey there you go illegal. He's back to calling his back to calling the cards 50 a 75 green scope color match Mysby doc Yeah, that's the one thing I remember when new Orleans and I was a teenager.  Oh, man That's all I think about with new Orleans. I don't even think about a drink I just think about eating food in New Orleans silver tannahill titans A vince young titan silver auto k-dod terry and davis price Yeah, I I just don't have every single time I end up like there's like a vacation time It's I have to go to some wedding or something like I don't get to take a trip for myself Silver key in an allen chargers. I'm just gonna refuse to go to weddings from now on I think that's I think that's the simple answer. It's the easiest one Purple buckner to 125 for the cults. All right. He went to the most important wedding of my life as my own So there you go. I'm out dude. I go to another one New Orleans it is hyper new Kobe Dean rookie eagles number to 171 of 175 That's skymore david ojabo. There's a lot of places. I would love to go Traveling be tough Traveling be tough. It's expensive. Yeah silver rookie emergent george pickings for the stealers purple ice critis samuel commanders Number 61 to 225 weddings. He's around a corner perfect I'll have plenty of extra time. This is not going any Hey, can you come to our wet? Nope No blue wave Trayvon digs cowboys number to 199 Jalen nailer and john mechie the third red wave and mcphearson bangles number 71 of 149 Uh, you could go to disney and wait in the long line spring break is crazy at disney then you go universal Same same thing. Then you go to legality. You have to have to have a child We don't I believe you do lock down trayvon digs for the cowboys that you did Purple power eric kendricks vikings to 49. There's no way that's a rule, right? It might be james cook. I don't think I don't think you can as an adult can go to the lego land alone You're kidding me. Look it up, dude. That's absurd if that's true. I'm not going to look it up I don't want you don't want to get that on the work computer. Is that what you're saying right now? silver rookie evaniel giants I don't want orange jonathan out of two 49 commanders Oh boy, I don't want people to know my search history. I'm only gillish jeremy rucker I get a legality as a single adult I don't know. I don't want to know the truth I can't handle it. You want me to break it your wedding joe? Oh, come on man. I can't even hang out Blueway rookie kyle hamilton ravens. There's not a dj, but there's just a breaking booth. You're there to work to 199 Congratulations lovely couple and that's a joe burrow silver prism Look you're already like halfway there. You just need the yeah, there's no dj.  I'm not doing it red waves the fondigs bills the 149 Vonte white k djork No more manga sin. No more no moss except except coming up soon here. I would think silver and mari cooper for the browns with a romeo dob zirciata for the packers Breaks it weddings. Oh my god. Yeah, I'm sure every single bride would love that. That would be amazing Yeah, you could have kid reported cards instead of rice Oh my god, this sounds like a salt green scope kareem hunt rounds number 1975 Congrats to the happy couple, but you just got shingled Oh, that'd be so amazing forward and crowd If you look under your seats, ladies and gentlemen, there is a special thing down there. You all just got jingled.  That's amazing too. I love that Oh boy rookie silver alante tailor for the saints an orange brand and cooked at 249 texans freeze-haul rookie and a kairi elam You may now kiss the black prism Rain Charizard Oh Hyper rookie eagles jordan davis number 143 of 175 Danny gray and jelani woods donnie can't do unless we pull us late to speak We should know the rules right now the gift bag is scourge themed. Jesus Dude, I got I got a cool idea sliver fireworks rustle welson broncos. There's just like a blue ice rich gannon to uh 99 for the raiders Brian Robbins a junior george carloftice There's just a um Everyone gets like a like a gift thing and it's like sound bites of like sam just going like We got london on the track and uh confidant Purple power alex smith cheaps to 49 12 out of 49 alex smith bailey zappy Worst wedding ever Yeah, we are we are literally making a list of how to make the wedding the worst it's ever been Purple ice justin tucker ravens or the best 154 225 Pascal de coppie de rant We may now place the one of one diamond card certification in the brides Silver michael thomas saints A dante colpepper silver auto for the vikings Oh my god, what their uh, what was the marriage certification was a redemption? Oh Marriage certificate is redemption. Oh You may get married in six to 12 months silver. Khalil mac for the chargers purple the forest buckner colts Oh, no donny 21 of 125 Ha ha ha same williams kenneth walker the third Repaint we're placing the bride and groom on the on the wedding cake with salacious be crumb instead I mean i'm doing that i'm gonna do that mojo come on.  Today, but yeah, I don't know bernie cosar hyper for the browns number to 175 It's probably that. Yeah, that's probably it We're going off of coffee and and uh and willpower determination No blood sweat tears amount of sweat dehydrated Silver brilliance mohomes chiefs Blue ice andre johnson's a 99 for the texans. He has a tears to cry James cook Italy night italy moved on mario and louise you're moving on the world baseball classic Is it mario or louise? Yeah, we were joking You weren't there forest camo treylon berks Seven of 15 titans Nice That is awesome Tennessee this goes out to greg. There you go, buddy Greg m Congrats on the berks. That is really sick 7 out of 15 going into mag Malik behind it ruckert Oh, I like that shane and a mod sauce garter. Oh, look at look at shane. Dude. That was good Forest camo christ johnson now for the titans back-to-back forest camo packs titans three of 15 Orlando legends christ johnson. Hey, that's my camo right there. Yeah, that's crazy. What about a third one here Silver donna the mcnaab eagles donnie mcnaab mj d auto for the jags Go jags quay walker and alec pierce Back-to-back forest camos on the same team is If it were different teams, it would be crazy if it's two if it's the same team that is kind of outrageous That's unreal odds right there. You probably want to see that again in prison this year silver herbert for the chargers and orange cooper cup rams number to 249 Jalen nailer john mecci my thumbs are starting to hurt from opening so many packs today My fingers hurt. What did you say after that? Black prism One out of one giants one dale robinson Nice, we got one Let's go Going to the new york football giants jeffrey h Wow, what a box Wow, what we what could you imagine if I if I was doing a A wedding ceremony. I did that that'd be unreal Wow filth filth Yeah, kentucky legend wandale robinson silver fireworks erin rogers packers of purple ice to forest buckner cults number to 225 The forest siren Whoo Turn on your siren The silver ray lewis ravens with a sam howl rookie auto now this box is crazy grads commanders We gotta keep this this this this box this box is uh, it is a good one So let's see what the last three packs have sam howls a good rookie auto too We are doing well Blue wave dj more panthers color match 175 out of 199 Skymore ojabo all out michael gallup for the cults and orange sam howl commanders number 79 up to 49 Breeze hall chair elam silver rookie trestin ebner for the bears hyper javon holland dolvins to 175 rookies dany gray and jelani woods are halfway through. Let me get a sip of water. That's a lot of odds Sin let me get this sweet mama jamma right here. I don't know. I don't know a mama jamma, but you know That's just how it goes sometimes What that has to do with it, I don't know Yeah, I I skipped the water Silver height mac jones gonna be break hung over tomorrow You go patriots blue wave deck for the cowboys the 199. I need to get one of those camel back Backpacks, so there's no need. I just I just keep going dude breaking machine I'll be like, uh Got silver javante williams broncos with a bailey zappy rookie auto forest camo 15 of 25 Wow Goodness gracious the forest camo in this case Brad our bigpats hit We will recappy That zappy chrysalave Charles cross Yes We will Maggie the zappy. It didn't work. Let's try hyper steve young to 175 for the niners Since I'm enough of it can he pick it? Kenny I'm like Oprah. I'm giving everybody's getting forest camo here all out erin jones for the for the packers orange christian watson packers This break just keeps going man 204 249 It's the break that keeps giving it really is Happy holidays everybody. Happy birthday. Donnie as well. Whoo. Isn't st. Patrick's day like this week. Yeah Okay Anyway, when's the next real holiday? silver rookie bailey zappy patriots Nice zappy purple kyle pits falcons number eight of 125. Yeah, that is a jersey number pits It's true the rumors are true Christian watson jordan davis Thanks, carl What up carl Tattooing while watching prism I assume Blue eyes michael thomas for the saints to 99 bonito mantra washington I haven't I'm not gonna talk about using the restroom, but silver rookie emergent trayvon walker jags I'm just a weird thing to talk about orange, uh johnathan taylor colt's number 238 to 249 It's it's literal. It's a literal Sauce gardener. No, I was just curious why you even said anything. Um carl like so when you're you know When you have this on we got a manga Okay, I'll wait hold that thought donnie Hold that thought. I don't know. It's like a teal. It could be like a dolphin. I guess.  That's awesome dude. No way Carla Get the camera Dude That's something else right there. Yeah That is ridiculous Oh fire dude in a pacheco purple ice right behind it to 225 for the chiefs Desmond Ritter good god, man I'll put that one on this thing by the way. Okay, so this he uh, justin herbert is now the raiden of football He is the raiden purple ice jack Lambert 225 for these Steelers Mortal combat champion. He just needs one of those hats that raiden wears. I don't know what they're called Yep, pacheco color too. Good lord, man The last two boxes have been kind of insane silver no a fan for the seahawks A james connor 1099 blue ice signatures for the cardinals there McDuffie and pickens. Well higgly the way it's going it might happen What might happen? What's gonna happen? Uh, we pulled a one-on-one mate right I mean, hey It could happen silver zay jones jaguars Blue wave jj watts 199 cardinals Shane, i'm just gonna go ahead and do that Blue wave mantra washington rookie till 199 for the broncos We got like uh three three more boxes in case we need to I run on the one mags. No, we can't run the one mags around here. We will not allow it I forbid it All right, james, that's a disturbing name for that Silver rookie jahan dotson commanders fireworks. Yo, what up yogi? Well danian salmonson purple ice chargers numbered 75 out of 225 yogi. What's up, dude? christian watson No, i'm not gonna say that word. I'm not here. It's not a bad word.  Yeah, it's not fun joe burrow flashback prism bangles Love flashbacks hyper miles garret to 175 browns. Yeah flashbacks are Alger Ritter they have they have lower number parallels on those two. Yeah, you can get like mojo on those.  What are those vending machines decided to rise up? This pan's done for orange hunter renford a 249 raiders James cuck but they have cool things to the vending machines. They're like whole meals in their vending machines They have ps5s in their vending machines We have We have expired chips and soda in our vending machines Merrick purple ice lt for the giants Number 77 out of 225 or yeah, it's 225. Malik willis Rocker dude like I don't know if they're like in retail stores or whatever, but I'm pretty sure like japan and china Legit like bringing robots and like customer service silver kid on diners Pretty crazy. We're he mustered auto for the dolphins Still gonna be a dolphin a couple more years Yeah Is what is at this point on it? What are we gonna do? Our robots coming true.  It's a lot of work That's that's blue wave deontay johnson stealers leave me my Hogwarts legacy alone to 199 Okay, walker alec pierce All gotta be like will smith and iron robot right against me Blue wave pierce strong jr. Patriots. What if I want to be on their side since they're gonna win 138 to 199 Um, you know what that's a good point. I would they're gonna win. I'll be on their side. It's true I'll just act like a robot Hello You're doing a good job, brother. Awesome. Oh, yeah Robots ever take over I'll just be like Hello bleep blurb Just like stereotypical like 1950s era like Just just murder me right away new recruit tricky silver chrysalovay saints purple ice michael gallop to 225 for the cowboys Robot breakers. Yeah, we get robot breakers. Now functions are slamming cards everywhere Start shooting cards out One of one Brock Hardy He just like shoots it into the wall silver nick chub browns A tarik woollen on the silver rookie out of the seahawks. Nice woollen James cook chrysalovay Blue wave george kittle to 199 49ers Malik willis Jeremy ruckett Apparently magnets are the weapon used to go through love silver derrius slave for the eagles A lane johnson silverado for the eagles sauce gardener nick bonito silver jerry alexander packers hyperkirk cousins vikings to 175 dulcich aviketts. Yeah, how long until uh There's place cousins Five more years. I don't even know Who's like a guess purple ice rookie cardinals tray mcbryde? That one's numbered 123 of 225 Algear and ritter my buddy's hoping He's a minnesota fan, but he's hoping that minnesota goes after uh Wait, that's the running back that's the running back for the browns a little while ago Brilliant silver derrick Henry titans purple de force buckner to 125 with the cults. What's the florida quarterback? Started the a Caleb Williams is the usc one. Yeah, orange diante johnson steelers numbered to 249 Jordan davis typhon thorton purple ice tony pollard numbered to 225 cowboys Yeah, no, it really is it really is tint christian watson. Yeah, it's a lot better a lot more consistent, too Anthony Richardson the oh this year. I'm sorry. I'm sorry. I'm sorry. No, I meant this year I couldn't think it was locked down hayward for the saints Yeah, thank you. Uh, yeah, thank you guys and switch hyper lane johnson and 175 for the eagles I thought Donnie was talking about next year. No, I meant in this next draft silver rookie pierre strung jr. Patriots the red wave two up to 199 dolphins Sorry to 149 the 149 in the red wave Orange rookie lewis scene for the Vikings numbered 45 out of 249. Can he pick it stealers rookie base? USA They determine who's on what team so I don't understand about this these games because like What is it like the mezzan cardinals play today, right? Like We're none of those players like potentially on the us team or Columbia team, I don't understand how they draft like the players Or whatever What's that for these games? It's like world cup kind of in a way. We got a gold prism coming up What pierce should be this way all the parallels are inverted Gold looks like a vet Khalila Herbert bears gold prism Number one out of 10 barbie one Rock party James Williams All out quarter all Patterson for the Falcons orange jerry judy Broncos number to 249 Calvin Austin the third jack Jones silver rookie trey mcduffy for the chiefs purple ice ryan tana hill titans numbered to 225 17 of 225 jerseys and ever tana hill Spiller and Logan. Uh, what's going on? Hey, I don't know. What is your role making? Oh, we're almost we're almost we're gonna get started on it soon. Actually Red wave Daniel Ballinger for the Giants number to 149 Drake London equino Just an original manager. I'm the um I'm like a temp. I think I think I'm a temp right in the town Rookie new recruits silver Bailey's abbey for the pats blue wave mclauren to 199 commanders Alante Taylor Malcolm Rodriguez Hey, Joe, how did you like it? Steve young hyper to 175 for the 49ers Johan Dotson Derek Stingley jr James I'm pretty jealous now that james has my jorts. I just have regular athletic shorts on right now I'm not doing anything athletic either Breeze Hall blue ice and 99 emergent for the for the Jets nice rookie there red wave divante Wyatt to 149 packers rookie Dotson and Stingley little do you know the best athletes start out breaking? I'm sure I'm sure purple rookie drake london very nice eight of 125 falcons very nice london Traylon berks, you think j jettos became the best fighters you were not breaking red wave john elway to 149 Broncos drake jackson scholar thompson purple on the track Yeah, london on a track silver marcus allen raiders orange brian berns panthers are 249 evan neil pierre strong jr hyper sauce gardener Here we go jets collar match 167 and 175 silver rookie fireworks drake london falcons green scope christian kirk jaguars number 30 out of 75 To reek wolden carcin strong rpa coming peace can silver vine mellard bills And a alex pierce 59 out of 99 rookie jersey auto for the Colts Very nice hit for indy david valgic one brisker two more boxes everybody in the case Illegal Silver brilliance down and cook for the vikings blue wave andre johnson a 199 taxons algear ridder james must be the must be like amazing at like bar trivia We got some skin coming should be this way banners trey landsnake skin That's awesome Congrats niner's had a numbered party this break too, right? Got all your got all your bases covered for this prism There we go. We'll get that mag 49ers rookie orange garret wilson jets to 249 141 of 249 Yeah, pickings poyamafe Who's aliyah aliyah Oh a zekel aliyah I don't know hyperfletcher cox eagles number two 175 Probably like it's gonna go to the c-dragons to reunite with the new chi They're gonna fall out home doesn't get me Sorry, I'm not used to this. I've been here all day. So like any news that happened today It's not really registering yet. Like it's not going to register guys. I'm sorry Silver Jarvis Landry for the saints. Like if you asked me who was equal I played for I would have Told you he was released, but like none of it's registering really A devon mccordy silver auto for the pats legend right there retired now. So I can call them the legends hamilton and phillips You probably end up somewhere like the cardinals or like the raiders or like the rams No, I know I'm just saying like it's gonna be a team. Like that's not good, you know silver chacoby meyers pats right wave hayward stealers to 149 James cook nicobi dean the goat.  That's that's that fits my criteria No, we need brian robinson's Right, but like he can still be like the backup. No, I need silver prison break trellenberg's titans blue ice to 99 Mr. Brandon cooks Number to 99 Mr. Brandon cooks Number to 99 Number to not number put over here Quit it. Can he pick it chrysalovay? Probably the biggest hit all day right there brandy cooks to 99 Pats backup. No, I don't I don't think it'd be a pass back. Oh, we didn't have jeremy hill one here silver trevon digs for the cowboys To 149 a drake london red wave rookie auto There will be no london on this track 102 of 149 congrats falcons Sweet hit atlanta chrysalovay is your wealth too. We'll be a london in this bag There'll be a london in this mag. Yes I'll be here all night, you know what it's because y'all said that i'm staying it's like Forest camo again a herald andry guess which team titans One out of 15. That's this is getting a little weird all the titans navy camo A little weird. Maybe the titans are recruiting you as a fan.  It was cj2k. It was berks and it was oh No, like titans It was berks. Oh, and then it was um. It was zappy. Zappy was the no there was another titans navy, uh Oh cj2k. It was it's landry and it's a berks Trayvon digs silver lockdown Godboy screenscopo jabbo ravens to 75 That's getting strange christian watson Forest come on. You know what? I've had with y'all Coming after me First of all, how dare you silver rookie trend mcduffie for the chiefs orange gabriel davis at 249 per buffalo Sauce gardener No prism has no on-card autos. I don't know prism. I think prism may be one here at on-card autos, but every other year They just don't really do signings on these chrome cards. It doesn't end well purple ice titans ryan tana held a 225 It's all such an epikete one year. They did yeah one year. They definitely did but it was literally just one year, I think isn't isn't uh Gilded on-card, but are they just not pro me? It must be different. It must be like different printing. I honestly have no idea Maybe they take more time to sign With puny dust things is different. Oh, yeah I don't know but that's how it always has no prism silver tristan wereps of the bucks Hey mike all stop 39 of 149 flashback autobuccaneers a train Purple power kittle the 49 49ers seven of 49 You're strong roger mccreary Yeah, new england's it wouldn't make sense for zeke. I mean they just signed jr out But even before that it wouldn't make sense. They like writing backs. They're a little dynamic and zeke is He's not gonna play special teams like he's not gonna start over Paris or stephenson.  Because yeah, the first two the first two seasons of that show are are pretty fantastic Very enjoyable purple power rookie jag straight on walker One out of 49 jags for a first overall pick the number one right there Mcbride and ebbner What is this midi-werbin nigger man jensen Silver brilliance jesson herbert chargers purple ice judy for the broncos number to 225 Traylon berks I know this herbert is from the current break 12 box full case number two Silver can enters and mangos. I only display cards from the current break up here Brian cook chiefs on the rookie auto Brian cook collial Shakir For this hat. All right. I'll give you my venmo I'll give you all the hats in the building for for a million dollars Here take my hat. Yeah, seriously donnie can have some of the money too 20 bucks and then Blue wave tray andrickson bangles to 199 donnie's like, you know, i'm your dad I'm gonna hang on to this one actually eight hutchinson average hr everyone in chat I'll say Well, it's a school night. It's a school night. Oh no, it's spring break rookie flashback chrysalovay sainz red wave fedarian matthas for the commanders to 149 rookie. I would say 31 Ogan hall cabin austin Oh, yeah, you're right, johnny Sorry, johnny been here since 9 a.m. So i'm a little uh Little brain dead essentially rookie silver germane johnson the second for the jet spruce smith purple ice bills to 225 Of course, I am I'm just gonna watch spongebob all day tomorrow money hyper john alway broncos number 2175 Malcolm rodriguez drake london I could think of 10 good reasons to never let go of a dime silver prison break jaylen waddle Dolphins out of a prison break for you purple joe name it jets to 125 stingly allante taylor average age in chat I'm gonna say 40 purple rookie garret wilson jets Very nice 53 of 125 we can get this one Congrats to the jets scholar thompson johan dotson should call the dark side Wash everybody blue eyes debo Seven of 99 for the 49ers I don't have much in power Don't know some good impressions kaleb pulling radian charizard slay just be crumb and anakin after he kills mace windew No, he's not dead. You don't know if he's dead yet. Yeah, true at star wars. Do we see his do we see him get absolutely murdered? They're gonna bring jango fet back somehow. Oh, yeah, no, they're gonna do that book of book of jango fet It's just it's just episode two from his perspective Donnie would enjoy that and watch that All right, let's do a recap I would say between 35 and 40 is average chat age I think we it would be different if it were like 6 p.m. I felt like there'd be younger kids watching Like teenagers or something teenagers got video games at night though. So yeah Illegal is definitely I know who the Eagles age, but I'm not gonna say it gold clear Herbert bears Oh, no, it was bryan cook devon mccordy jersey auto alec pierce lane johnson tarik woollen rihim moester james connor sam howell orish jones drew dante cull pepper Romeo dobs vince young alec pierce Donald driver tearian davis price khalil shakir josh jacob's david ojabo and mike didka coach He was number one Then 15 I think you might be wrong Donnie Anyway, here's all our mags purple garret wilson. Congrats jets to 125 rookie forest camo trail on berks to 15 titans red wave rookie auto drake london falcons to 149 snakeskin trey lance for the 49ers rookie orange kenny picket a 249 stealers forest camo rookie auto bailey zappy patriots to 25 purple ice rock party to 225 for the 49ers niners did well with the uh lance and the uh party there and then these two beauties one of one rookie black finite prism wandale robinson Wish I was 26 my back would hurt less and This thing is just filth It's hard to imagine a cooler manga than this one right here We'll have to see them all but man justin herbert chargers manga ssp That's fire. Thanks again everybody one more case for tonight


So, good morning everyone. So, it's my great pleasure to talk about, you know, lipid membranes or cell surfaces and membrane mimics at this interesting sweetness course. And before I start talking about science, I would like to briefly introduce you myself because I'm not a Swedish professor. Yeah. So, as Tommy mentioned, I was born and grown up in Kyoto and got my PhD in Kyoto University. But during my PhD, I spent some time in Helsinki and also in Copenhagen, precisely speaking, in detail. After my PhD, I moved to Germany 23 years ago and kept my career here mainly. So, I started as a postdoc in the physics department in Munich under the guidance of Erich Sackmann. He was one of the pioneers of membrane physics in Germany and Europe. Then later on, I got an independent grant to run my team myself for five years in Munich. Then made my habilitation degrees in physics at Munich. Right afterwards, I got promoted as a full professor of chemistry and later on physics at Heideberg. Yeah, then since 2013, I revived my activities after 15 years of absence in Kyoto, leading a team there. So, the reason why I'm here is because of your academic coordinator, yeah, Tommy. So, he mentioned already that we met for the first time, I think 15, 16 years ago. And afterwards, I don't know how many different places and occasions we met and had beer and talking about science in different cities and countries. So, we're here, Tommy and me. Yeah. Yeah. One was the summer school held in the island, Uito, 10 years ago. And the second one I showed you is the conference that I organized in Kyoto, where also Harbour is in the middle. So, in my team, I have three scientific pillars. I'm a sophomore physicist by training. So, the main pillar is dealing with physics or biological soft interfaces, where I set light on structure, mechanics and dynamics using both real space techniques as well as reciprocal space techniques, like scattering. This stems out into two directions. The light left one is suited for Heideberg, which is strongly in biomedicine, where I study the non equilibrium physics of life, especially focusing on the physics of diseases and development. Another direction that I'm working still on is to generate biosimical data hybrid materials, such as depositing membrane mimics on the surface of gallium nitride quantum nodes or two dimensional nuclear gases and quantum wires to detect the smart bio functions as an active optorextric system. So, of course, today, I would like to focus sharply on my main field, physics of biological interfaces. So, that's the reason why I chose this or I like this topic to talk. So, to start from, you know, let me start from a historical question about molecules and interface. In 1774, Benjamin Franklin made a very short report at the Royal Society meeting. He reported that when the tin spoonful of olive oil was spread on the pond, it stills away. But most importantly, the effect reached up to one quarter acre, but not more. So, that's a very short message and you may ask yourself what one can learn out of it. And of course, this is not an experiment recommended nowadays for the sake of environment. When you pour oil on the water surface of the pond, I think some people will call the police. The answer is given by Lord Rayleigh, another famous guy about 100 years later. What he did was he just divide volume by area and gained the thickness of the film of olive oil being one nanometers. Now we know that this is a very simple calculation, but giving us a right guess about the size or thickness of monolayer of fatty acids. This is so simple, but we tend to forget that the interface is not just a boundary that separates two bulk phases, but they have structures as well as functions. This is manifested in biological systems. So we can learn why does interface matter by visiting nature again. So I took a very famous cartoon from Strayer's textbook. It's showing that in biological cells, many key reactions are confined near the interface, which is nothing but the membrane separating interior and exterior worlds. So one can think of the importance of membrane from the economical viewpoint. So we can start from a classical, classical, most costly equation of diffusion and calculate. The diffusion time required for a particle collide with another one located at the distance B. We do it in 2D and 3D spaces. This was firstly shown by Stefan Hart in Weizmann in the late 70s, and the solution shows that the dependence of tau on 2 and 3D is quite different. So the tau, at least the diffusion time, in 2D scales with natural lock of R, while the one in 3D scales with 1 over R, clearly indicating that the dependence of diffusion time on R is minor in 2D. And this explains indeed the confinement of reaction in or near 2D membrane is very much an economically clever solution to run the reaction involving biomacromolecules such as proteins and enzymatic complexes. As you learn already from Ilpo's lecture this morning, membranes are not just boundary of all, but membranes know actively control the biological function, such as protein recombinitions, receptor activation, and so on, so forth. So here I show you one of one picture I presented in one of the most recent review together with my teacher, Erik Sakman, where we showed the proteins involved in cell migration, which are shown in this cartoon. If you zoom into the region near the spreading front of a migrating cell, you find many protein machineries involved. But all these proteins are initially swimming in such a specific space and they are not active unless they are recruited to the membrane surface. And binding of the PI3 kinase is indeed the master switch producing a lot of PIP3 lipids, so phosphorynostero 3-phosphate lipids, that connects proteins to the membrane. Also, if you see from the mechanical viewpoint, cell membranes possess very unique mechanical properties which are playing instrumental roles in sustaining our life. As I show here with the scheme, membrane consists of consisting of amphiphilic lipid molecules, and lipid itself has a polar head and hydrophobic tails, sharing common features with symmetric liquid crystals. So, symmetric liquid crystal phase is the leftmost phase on the right panel. Actually, in 70s, many physicists working on liquid crystals become aware of unique mechanics of lip membranes and they actually build up the fundament of membrane biophysics. So, in this slide, I show you three principle mechanical parameters describing the mechanical properties of lipid membranes. The first mode is an isotropic extension and contraction, and the middle one is a sharing deformation, which is a deformation which is not accompanied by the change of the area. And the third one is the bending deformation, which comes from the asymmetric tension balance between interior, inside, and outside of the field. So, everything can be represented by the Hukian energy representation I show in the lower part, which is even by one half times the modulus times the strain to the power of two. So, this is exactly what you learn in high school about the Hukian spring. So, as one of the prominent examples of biological membranes mechanics, I selected one very well-known example, human red blood cells. Here, I show you some statistics. About 10 to the power of 13 cells are running inside our body, and actually, when I was a student, I was astonished to see the production rate. One million cells per second are produced in our bone marrow, and they are traveling over 400 kilometer distance in 12 days, keeping finite volume of area in order to avoid the clotting. So, they are traveling long distance, patterned through many narrow capillaries. So, what I show here is a movie taken by my student showing healthy red blood cells passing through a constriction, which has the cross-section of 3 by 4 micrometers, meaning the capillaries. What is intuitive is the healthy guys are able to go through these narrow channels and easily recover their original shapes. But the disease cells as well as aged cells cause stiffer than the healthy one, and they are often rupture when they are encountering through this kind of constriction. In fact, the membrane has extremely low bending modulus, center power minus 20 joule, and low shear modulus, center power minus 6 Newton per meter, which are like 1,200,000 times softer compared to polyethylene fumes, which has a similar thickness. So, compared to plastic forms, they are 1,200,000 times softer, and this is a secret why they are able to keep their structure integrity against the strong shear stresses. So, now we look into the multicellular systems, and the first question that I can ask myself is how are these cell-cell and cell tissue contacts are precisely controlled? And when we look into the biological systems, we find that many cell-cell or cell tissue contacts keep a very finite and well-controlled spacing to avoid non-specific contact with an aid of many biopolymers. What I show here on the left is taken from Albert showing two plant cells, two neighboring plant cells, whose contact is mediated by a 200 nanometer thick cellulose layer. So, if I draw a cartoon showing this, the skin looks like on the right. In fact, many cell-cell contacts are mediated by biopolymers containing sugars, and they are controlling the generic and specific interaction balances in order to find and adjust the sensing contact. Actually, from the viewpoint of physics, cell-cell contacts can be treated as a certain property of working physics. There are two important points for us to keep in our mind. The first point is the stratified layers, namely layer by layer structure, like you saw in the previous slide. So, phytosodic polymer and membrane, excessibular polymer, membrane and cytosol. They are stable only if the complete wetting conditions are fulfilled at the interface, namely the presence of an additional layer. For instance, in this cartoon, the additional layer means lipid membrane. This must result in the gain of the surface free energy, which is characterized by the spreading coefficient as being positive, which was described by Dijon and Francois Rochauer quite some time ago. Second, when you look at the vertical force interactions, to keep a finite inter-cellular distance of typically tens to hundreds of nanometers, the interaction potential at the interface should be kept weakly repulsive. So, if you think about the interfacial forces contributing to these biological interfaces, you can name some, like ecostatic interactions, van der Waals, hydration repulsion and entropic forces generated by conformational fluctuation of polymers, as well as thermal fluctuation of membranes. But if you think about cell-cell contacts, it's very difficult or almost impossible to dissect each force contributor. Therefore, we can grab the net force active per unit area, which is nothing but the discerning pressure. This is a concept written by Teriagin from Russia for quite some time ago. And from the dynamic viewpoint, the discerning pressure is nothing but the first derivative of the work. You have to invest to change the distance between two membranes with respect to the distance. As you can imagine, the minimum can be found. So, corresponding to the equilibrium corresponds to the discerning pressure equals zero can be found only if the second derivative goes to positive. On the other hand, if you have negative discerning pressure, this causes the continuous thinning of the interlayer, which eventually results in the dissipation or rupture. And this is nothing but the scenario of wetting-dewetting transitions. So, therefore, in order to understand the physics behind, the design of defined cell membrane models with less complexity would be helpful to reveal how biopolymer modulate interfacial interactions. So, now we come to see the very important model of cell surfaces called supported membranes. There was a very historically important, I think, review published by Eric Suckman 25 years ago in Science. Entitled Supported Membranes. This is now cited over 2,000 times, and now it's really widely spread all over the world. So, the first report came from Adrian Bryan and Harden McConnell from Stanford 36 years ago. The systems look like that. You have a planar lipid bilayer deposited on planar substrate. And the membrane is separated from the substrate with a very thin water reservoir, which has a thickness of 5 to 10 nonsomes. Compared to the other membrane models, like, you know, black lipid membrane, liposomes, you know, the advantages of the supported membrane is, first, they can stably coat macroscopically large surfaces, which can go easily beyond tens of square centimeters. And also, you can study the structure and function using various surface-tensive techniques, such as AFM, you know, because of the planar geometry of the sample. Actually, in the review written in 1996, Eric already mentioned that this model of dark membrane that allowed the application of a manifold of surface-tensive techniques. And they form versatile models of low-dimensionality complex roots, which can be used to study interfacial forces and waiting for pneumonia and adhesion. And even he started talking about practical applications for, towards a design of biosensors on electro-optical devices. So about 30 years ago, related to neutral science, there are two important papers published by Swedish, British, and German physicists. The first paper I picked was published by Adrian Reigning, who was, at that time, ILL, and both Thomas in Oxford and Eric Sackman from Munich. They did the speculative neutral reflectivity of lipid bilayer deposited on quartz substrate using ILL D17, which is still one of the top runners in the field of neutral reflectability. The second paper I picked was also published by these people. Well, they were very active in this year, obviously, where they performed the new speculative neutral reflectivity of lipid monolayer at the outer interface. There they used crisp beam line at ISIS, the Rutherford-Uppleton Laboratory. So these systems have been used like a gold standard, and still we are using this lipid monolayer and supported membranes as a cell surface mimics. But when you want to go towards more biological questions, there is a fundamental problem, especially for supported lipid membranes, which is a direct protein substrate contact. The drawback of the supported membrane is when you incorporate transmembrane proteins, they are sticking out of the membrane typically by 10 nanometers. So when you deposit them on solid substrate, they always encounter the risk of protein denaturation, and also ill-defined density and orientation of membrane proteins. On the right, I show one of the data presented in my old old paper, where we stained platelet integrin alpha to be beta-3 and incorporate them into supported membranes and deposited on quartz substrates. As you can see, the picture looks very ugly, where the protein distribution is very homogeneous, and when we performed the fluorescence recovery after photobleaching experiments, we found that almost all proteins are completely pink. So to overcome this problem, we developed new models of membrane called polymer-supported membranes. So in this field, I published a review in Nature 15-16 years ago together with Eric, which was a time when I graduated from his school and moved to Heidelberg. And this morning, I checked and found that this paper was now cited more than a thousand times. In this review, we focused on the influence of soft interlayers and wedding interactions, as well as in the typical technological applications. So what is important is this polymer-supported membrane can be considered as a half model of cell-cell contact. So again, I showed this cartoon of cell-cell contact mediated by polymers, and if you cut in the middle plane and deposited on solid support, you find polymer-supported membranes. You can consider this solid substrate as another membrane, or you can consider that this is a half model. In 2007, I published quite a heavy paper in physical review letters, short, where we control the density and the thickness of the polymer layers and measured the diffusion of the integrin protein, which I showed in the previous slides. Now you see the proteins are completely uniformly distributed, unlike the one I showed here. So they are the same proteins, but now they are deposited on 10 nanometer 6-cellulose fields. So starting from the classical Einstein's equation modified by introducing the two-dimensional viscosity, as well as a dimensionless particle radius, epsilon. One can actually precisely calculate the frictional coefficient exerted on proteins, as well as the interlayer viscosity, which is regulated by the density and the thickness of the polymer interlayers. In the following accounts, we demonstrated that actually about 10s of nanometer 6 ultra thin polymer interlayers enables the maintenance of that little diffusivity, as well as the receptor functions for various transmembrane proteins. So now we come to examining the structure aspects of these polymer-supported membranes, and here we use, of course, neutral reflectivity. So what I show here in panel A and B are taken from our previous paper, showing the reflectivity of SOPCs, so sterile, oleo, and phosphatidic colline membrane deposited on thin and 6-cellulose fields. So the advantage of our cellulose is we deposit them layer by layer, and each layer has a thickness of five angstroms in dry state. So we know exactly or we can control the thickness by the layer numbers. As you can see with lines presented in each panel, the data will fit it with the slab models, and the neutral reflectivity of membrane exhibited very clear fringes in Fresnel plot, where we plotted the reflectivity multiplied by Q to the power of 4, indicating the formation of a layer structure with clear SND contrast, scattering density contrast. This is a very good sign of the formation of high-quality membrane, because we did the experiment in D2O, and since the hydrophobic interior of liquid membrane now hates water, so this gives us a clear SND contrast. Also what we found was a Gaussian roughness of membrane is much higher compared to that on solid supports, indicating the fluctuation of membrane under shallower confinement, which means indeed the theoretical prediction made by Draynkowski and Leibler in the 80s. So to see confirm that the answers or best fit results we got are unique and solid, you know, we performed also specular x-ray reflectivity. So here, you know, you note that the specular x-ray reflectivity of systems under water is in principle not possible, because unless you use an high-energy x-ray, so in these experiments we used a high-energy x-ray at ESR ref, now using the beam with an energy of 22 kilo-electron volt, which is far beyond of the elastic absorption peak or absorption band of water existing at around 8 kilo-electron volt. For the use of high-energy x-ray guarantees the high enough transmittance of beam through bulk water, the advantage we're using, say, or the difference from the neutron reflectivity is that for x-ray the contrast of the scattering length density is much poorer between the elements existing in biological systems, but the resolution is much higher. So if you compare the Q-range, you know, presented in this and the previous slide, you see that in the previous slides, you know, with neutrons you can go at most up to 0.3 reciprocal constants typically, but with x-ray you can go up to 0.5 with no problems of the counting statistics. So despite of the fact that they give different resolution and the contrast, the best fit results converged each other confirming that, you know, our solution was unique. So this brought us to the point to nail down the physical roles of biopolymers in fine adjustment of interfacial interactions. Again, you know, the disioning pressure equals zero is found at the equilibrium cell substrate distance. Since SOPC, so phosphatidicolin is petylionic and cellulose is neutrally charged, so we ignore the electrostatic interactions and focus on three major force contributors. Fundamental walls, hydration repulsion, and the hair free repulsion, which was originating from, which is originating from the thermally activated membrane fluctuations. So the important thing is all these three major forces can be quantitatively calculated. I show you two examples because van der Waas is far much complicated. So I show you the case of hair free and hydration repulsion. So hair free repulsion was described by Wolfgang Heflich, a German theorist in 1978, you know, which indicates the scaling of the repulsive pressure, you know, which is proportional to 1 over D to the power of 3. So what you can plug, what you should plug in is only the copper. This is a bending rigidity, which I mentioned, you know. This is measurable and also you can get easily from the literature in the range of 20, 30 kT. The hydration repulsion is a bit more tricky because you need to get two numbers, the offset pressure as well as the scaling. The scaling is very simple, it's an exponential decay function. These two values required to quantify the hydration repulsion can be calculated from the measurement of the film thickness as a function of osmotic pressure. This can be done by ellipsometer or, you know, X-ray nutrient effectometer coupled to a humidity channel. So what we performed or what we did here is to compare the theoretical prediction versus the experimental data. What I show here on the left and right are the results, computed results on thick, thin and thick cellulose. On thin cellulose, when we added up the van der Waas, black, hair free, green, and hydration repulsion blue, we get the net force showing in red. You see a clear crossing of the force zero. So corresponding to the equilibrium distance, which is about 140 angstroms. And if we compare this value to the values we obtained from experiments, they are in pretty much good agreement. In contrast, when you look at the data on thick cellulose, first you note that the position of zero force is much less prominent. For instance, when you look at the y-axis skating of left and right, it's different by a factor of 30. So although it looks similar, it's 30 times shallower than it appears when you put it here. So this means that on thick cellulose, hydration repulsion is more dominant so that the point of zero force is very vaguely confined compared to the syndrome. So as a result, we demonstrated that the counterplay of attractive van der Waas interaction shown in this massive black curve and hydration repulsion, which is an exponential repulsive force, confines indeed the membrane position. But in this case, the role of the sample and relation of membrane is less common. So along this line, we also made a small breakthrough where we succeeded in controlling the density and orientation of protein by spreading of native cell membranes directly onto banana supports. What we did first was the use of human red blood cells. So we took the blood of Stefan Kaufmann in my lab and also my blood. 20 years ago, it was allowed. Nowadays, it's no longer allowed. But in that time, we just took our blood and prepared the ghost cells simply by removing the cytosomes from the cell. We just throw them onto banana substrate with and without polymer and see the orientation of cells by putting antibodies, recognizing inside or outside of the cell. What we first observed was on growth and blood substrates, we saw nothing. No adhesion, no rupture, which is a kind of understandable because the surface of adult animal cells are covered with negatively charged sugars called cyanic acid. But when we did the same procedure on 10 nanometer six cells and applied the antibody recognizing the cytoplasmic domains of band three proteins, we obtained such a picture. It's a very frequently asked question, so I want to tell you that it's not the paint, so it's not a paint in a photo shop, but it's an immunofluorescence picture of my blood cells stained from inside. You see uniform and continuous coverage. On the other hand, when you apply the antibody going to the outside site extracellular domains, or glycopherin for instance, we saw nothing. So this means that now we have the scenario of complete waiting of cell membranes simply by having 10 nanometer thick polymers. The cell first adheres on the surface pulled by tension and rupture. Expose their inside and fuse with each other to form a continuous film, which is exactly as I mentioned in the introductory part about the scenario of complete waiting. This was indeed the first example of two-dimensional cell membranes, where the three D cells were transformed into quadruped two-dimensional films with a perfectly defined membrane orientation. Also we scratched the membrane from the surface and threw them into SDS page and found that the protein compositions were also kept very well. This enabled us actually to go to X-ray neutron because we now can cover macroscopically large substrates with native cell membranes spread in 2D. You can imagine that's a very nice tool to look into the structure of real cell membranes. Here I show you on the left the high-magic X-ray reflectivity data for healthy human red blood cell membranes and on the right the neutron reflectivity data. So the right was shown in Flanelplot just to show you a bit better features. We actually demonstrated that the same principle works for other native membranes such as microsome, sarcoplasmic reticulum, and plasma membrane extras. So now we are going to a very realistic biological same-surface remix and this is very intuitive that the wetting concept or wetting physics concept is very important. Like the bare glass no adhesion no wetting on soft polymer or on cellulose films no complete wetting. So this kind of wetting contrast between solid substrate and polymers supports brought me to another step to utilize wetting contrast to make a micro pattern of cell membranes. So now I have been talking about the macroscopically large substrate. For neutron reflectivity we cover the substrate with the size of five by eight centimeters 40 square centimeters substrate. Now I want to make them like a micro pattern. To do so we micro pattern cellulose support either by UV lithography or by micro contact screen the BSA, so bobbin serum algorithm which avoids the adhesion and spreading of cell membranes. Both worked out beautifully. So the top one is the same immunofluorescence picture of human red plus a membrane grid say printed onto the substrate. So only these grid parts are showing immunofluorescence positive signal and these are all backgrounds. The bottom picture shows the sarcoplasmic reticulum extracted from rabbit muscles and the green is showing the BSA you know stained with FITC and the right shows the grid of sarcoplasmic reticulum membrane which was stained by the antibodies to causing ATPs. So as you saw these papers have been published already quite some time ago and actually afterwards quite some followers came up but up to my knowledge maybe I'm wrong but you know we are the first one in the only one team that can replicate both microscopically large as well as micro patterned two-dimensional cell membranes. So now I come to the last part of my lecture where I want to show you how one can model the interactions between neighboring cell membranes mediated by a membrane-bound carb hydrate which is nothing but glycocomics you know. So as a model system we designed the stacks of synthetic and natural glycolipines which are shown on the left with structures and when we deposited thousands of these membranes you know membrane stacks and solid supports I call them as multiple supported membranes or polymer supported membranes. So as I show you know indicate with red characters or the red text so I was very lucky to get an outstanding synthetic chemist as well as microbiologist who can offer me very unique and well-defined sugar lipids. So either mimicking the lipids in animal and plant systems or in bacterial systems. As an experimental technique we utilize off-specular neutral scattering you know where we detect the scattered beam you know impinging to the plantar stocks of membrane now using 2D detectors. And what I show on the right here is the say raw detector readout you know presented in the angular coordinates gamma and omega. This experiment can be done in ILL D16 beamline you know and this was strongly supported by the local scientist Bruno de Me. So the advantage of this system is that owing to the planar geometry of the sample one can discriminate the momentum transfer perpendicular and parallel to the membrane plane. Simply from the geometric consideration so you see only gamma and omega are in this equation so this means that the specular signals reflects the vertical structure information and inter-membrane potential while the off-specular intensity reflects the lateral structure ordering and membrane mechanics. So after you measure the signals using nice 2D detector you have to work a little bit. So here we have to compute the scattering signals. To do so we have to revisit we had to revisit the basic framework of discrete symmetric Hamiltonian which was theoretically written by Leibniz and Lipposti in the 80s. Here the Hamiltonian contains two key parameters. First is the compression model B so that's a vertical compression model B and the bending rigidity copper. On the other hand when you see the data from the analytic viewpoint the scattering function from stratified rough interfaces within the first bond approximation is written in that form. Now this was written by Sina in the mid 90s and although this looks very complicated most important bits here is this G as a function of R called displacement correlation function and this displacement correlation function is determined by two key mechanical parameters lambda and eta. Lambda is named the jump parameter and no eta carrier parameter and what's notable is both lambda and eta are the products of copper and B. So this means from the analysis if you can find the best combination of lambda and eta you can calculate the copper and B which are corresponding one by one. What I want to emphasize here is there is an important the number we need to introduce called R that's a cut-off radius which represents the finite size effect. If you do not limit this lower limit the displacement correlation function becomes dispersive so you know the roughness becomes infinitely large when you make the separation between two points larger and larger. This size can be determined by the size of the membrane patches which you can determine by optical microscopy or atomic force microscopy. So plotting these numbers you know we actually computed the scattering signals. What I show in this slide is the direct comparison of the second black sheet from experiments and simulations. As you can agree probably yeah this is a very nice agreement and what I want to emphasize here is the introduction of the cut-off radius indeed allows a full calculation of scattering functions with no floating parameters. So this means if you plug in instrument resolution function and also the cut-off radius you can be fully quantitative. This is an advantage compared to the previous approach where you have to approximate something to fit the signal. So this technique was applied to shed light on the influence of the carb-hydrate confirmation on the mechanical properties. We started from a simple glycolipids possessing glucose and galactose unit on the head. One is gentioburus which is mimicking the lipotheic acid in gram-positive bacteria and the other one is lactose lipid and the difference only difference between two lipids are the glycosylic junction either 1.6 or 1.4. By comparing two modally you find that simply by bending the cylindrical lipid head groups through the bent one the bending rigidity decreases by a factor of 4. On the other hand when we look at and extend this idea to a bit more complex lipids you know we performed experiments with Daniel Veratz. He's an organic chemist in Göttingen and now in Branschweich. He designed the GB3 lipids which are known to be the glycolipids or galactoside accumulating in the eyes of patients suffering from fabric disease. So here you have the lactose unit plus one more branch here. So by adding just one branch you see that the bending rigidity decreased by a factor of 2 and the bending rigidity decreased by a factor of 2 and compression modulus as well. This was extended to more complex glycolipids by taking the bacterial lipopolysaccharides. That's what I collaborated with Klaus Brandenburg from Research Center of Bostel. That's a northern part of northern edge of Germany where he could actually extract the high impunity lipopolysaccharides from various bacterial mutants by chopping the sugar chains at the position of interest like I indicated with black lines. One can test a very short one called lipid A up to the rough mutants with the inner and outer membrane cores as well as the wild type lipopolysaccharides including polysaccharides chains. So what we found, so Rafael my fellow postdoc found was the bending rigidity and compression modulus is motivated by the genetic mutation of sugar chains. For example if you compare the shortest lipid A and the longest mutant APSRA which has I think 8 or 10 additional sugars, we see that the compression modulus decreased by a factor of 4 because these sugars are softer so that they are say you know the compression modulus gets smaller. So we showed that the off-specular nutrient scattering is a good tool or powerful tool that enables the influence of chemical genetic and epigenetic modification on membrane mechanics. To conclude so I hope that I could give you a flavor that cell membranes are just not a boundary but doing more you know. So they act as biological reaction hubs regulating many critical functions and the unique mechanical properties of cell membrane shown by red blood cells are tightly coupled to biological functions you know which can be described within the framework of smectically crystals. Then the recent development as I showed in the last part of both biochemical and instruments enables to design more precise biological relevant models of cell membranes. I started from very simple supported membrane developed 35 years ago and now we are dealing with much more realistic and complex mimics of cell surfaces. The combination of neutron and x-ray reflectivity is also a very good tool to probe the detailed structure of complex and buried interfaces under water enabling the interplace of interfacial forces regulating the wetting contents. And finally the specular scattering though is a good tool that you know quantitatively unravels the how chemical genetic and epigenetic changes in membrane boron carb hydrates would affect the mechanics of membranes. So since this is a lecture so I do not want to give a acknowledgement to the funding sources but you know I want to convince you that the near future development of instruments will enable us to tackle more challenging problems using neutron. So instead of talking to the money I want to thank my students postdocs and colleagues now many of them got the permanent faculty positions outside Germany or inside Germany as well and you know the supports by the large facilities like INL and ASF were instrumental and as you see the interdisciplinary collaboration with chemist medical scientists as well as microbiologists made us very unique and strong and I thank for your attention. Tommy I'm done. Okay thank you very much and I think there is time for a lot of questions. I hope there are some. Yeah you have some questions here waiting waiting waiting. So I think it's very interesting that you actually can look at real cell membrane. I think we always are struggling we always are doing the UPC with 5% the UPS or whatever so I think it's very useful to be able to look at the real real membrane and so on and sometimes one can argue it's complex but complexity is sometimes necessary I think. Yeah because you have quite a sense of biology. Yeah. So how much? Who has the question? Who is the question? Yes thank you very much for a great presentation and I just have a general question maybe to hear your point of view like what do you think are the main challenges right now where the research is going in the this like interface lipid kind of systems because it feels like you are also a pioneer in this in this field right now so maybe some comments. Yes so one direction that I think is interesting is to going through the influence of disease and aging on the membrane you know because you know many biochemical studies suggested that you know the structure as well as the composition of lipid membranes are modulated by aging and diseases and indeed you know what I plan to gather with this form is to spread the red blood cells infected by malaria parasites on polymer supports and look at how the malaria parasites modulate the site skeletons as well as membrane proteins and for the mechanics using microfluidics and other methods I really demonstrated that the disease actually modulated the mechanics of the membranes a lot so now I want to shed light on the structure in another direction that I would be very keen on testing or going myself you know until the end of my career so I'm not too old yeah so is you know to go into the dynamics so now I'm looking more or less now on the equilibrium systems not because of the time window that we can assess but maybe you heard that lecture of Vicky yeah about the inelastic neutral scattering and spin echo so back in the days you know using ILM I mean if we want to look at the mechanical properties of cell membranes you know using spin echo I mean I needed two weeks or a month not in the reality and we need to have tons of samples but no not if we imagine the situation of using the spreadation source I think that's something that I'm definitely interested in doing myself there as soon as the instrument is ready and that's actually the one of the reasons why you know I am coming in close contact you know so I bring some strange systems with a bit of idea of physics and you know the experts like Tommy and you know Vicky and no these people can support us to realize this idea yeah and in synchrotron it's also interesting look at the dynamics using x-ray photocollation spectroscopy and other methods and this was also very much limited in the past because of the coherency of the x-ray as well as the intensity and I was luckily involved in the upgrade committee of ESRF and we used as influence the panel to strengthen the coherence so in two years now or five years now I think they should have 30 times higher coherence which enables us to really do xpcs for also biological memory so you know to conclude you know my interest was to aging diseases so as well as to dynamics I think these two are what I think to be an exciting field in the future thank you very much and I'm also thinking that this new like methods with time time resolved experiments and free electron lasers are also like enabling like another level of this dynamic and structural motion analysis yes yeah so if FEL is a nasty method you know because you just burn the stuff with an intensive pulse and observing the shot but on the other hand I mean this is a very you know cute methods which I didn't try myself yet but I think this opens up also the future yeah thank you so much you're welcome and any more comments oh there is a hand Ying I have a basic question about the interplace of interfacial forces regulating the wetting contact and I remember you mentioned many forces including the hydrogen force and can you give a comment on what is hydrogen force is it only and persists between the bilayer membranes and what is it originated from yeah so hydration repulsion is a generic force you know and I think in Lund Hogan Venastro is one of the pioneers and another one in the US is no adrian precision and I think I just mentioned about adrian which I felt a bit dangerous here yeah okay so this is how the hydration project decays so D is a distance and lambda is a characteristic decay length and this is a long set so I didn't get your question so this is not a special thing for lipid bilayer or anything you know for instance you know when you say measure the hydration of polymers in controlled humidity what you see is nothing but the hydration repulsion you know so it exists between and hydrophobic hydrophobic surfaces hydrophilic hydrophilic surface because we are looking at water or hydrophilic parts and so it is not necessary to be surface it can also be like hydrophobic on some parts yeah but it is decaying over distance from the surface you see it's not the bulk osmotic pressure so of course now in bulk you know we can describe the chemical potential of water simply by osmotic pressure well when you go back to high school not high school but the first class physical chemistry you know you find found Hof's law you know the osmotic pressure is given by her kt divided by molar volume of water times natural lock of reactive humidity well done yes that's how i remember from the textbook yeah so there you know you can tune the osmotic pressure namely the chemical potential water in the system by changing the humidity what i'm talking here is how the repulsive force now decays in the presence of water yeah i hope you get it yes and so so what you find here is that in combination of all these forces that's why you can get the blood cell rupture in the surface no here i'm talking how the distance between the membranes are kept constant because now you know when the force is balanced there is no motion newton's first law yeah yeah so in order to have an stable equilibrium distance you need to achieve a balance of different forces if this is destroyed then you have continuous sinning which results in the collapse or your membrane is just separated more and more and they are gone and that's what i showed by computation here as i said so you know on thick cell loss you see the scaling of y so this is a pressure is very shallow you know so the deep here is compared no negatively small compared to this one yeah but still you know one can see how this osmotic repulsion is pushing away this the system from the deep well or fundaverse attraction yeah this is what i showed yes so what is the magic that's rupture that make this break the cell and onto the surface yeah so in order to write you know you need to achieve the gain of the free energy so this is described by surface tension and second is this no no force balance uh where it is so what is at the very beginning i explained this balance oh wait a moment wait a moment i think you probably are not so much familiar with physics so yeah i mean okay so this is the first point this is from the viewpoint of surface free energy but if just look at this now you can imagine that every hydrophilic surface should do the job but here well okay so if you have quartz or glass substrates the interaction is just purely repulsive so those cells do not adhere on the surface okay so they are just repelled when you have some polymers no you have the good balance of attraction and repulsion they sit on the surface and since they are hydrophilic they wet yeah so this is very similar well not similar so the first part is very similar if you consider the spreading of lipids on hydrophilic substrate and hydrophobic substrate well if you have hydrophilic substrate you have the spreading of bilayer but if you have a hydrophobic surface you know you have the spreading of lipid monolayer because the chain of hydrophobic tears that loves to spread on the surface by this no balance of the spreading coefficient