Everybody forest here. We'll try to change title here ripping 20 21 22 contenders optic basketball hobby 10 box half case number 33 picker team style.  Good luck Brandon Brian C. Lakers Carson H. Kings Chad W. Pelicans Mavericks Chen M. Sixers Danny C. Warriors Raptors Gregory M. Cabs Harper B. Rockets magic Ian L. Hawkes Jonathan Q. Celtics Jose V. Pacers Lucy R. With the Knicks Michael S. Blazers Mike Y. Has the Grizzlies Noe G. Nets Ronald G. Bulls Shingonori with the Wizards Sorin Vichy and Spurs and Jazz Steven W. Nuggets Thomas C. Thunder Timothy F. Bucks and Victor F with the Hornets. Good luck everybody. Here we go Not gonna recap this one picture Not gonna recap this one gonna be super quick break So Green Bay's winning it no matter what happens here That's that's it, right? Okay All right, so it's either Seattle Green Bay. Sorry Lions. I was I was room for the Lions. I wanted them to make it They're fun They're fun because their offense is really good and their defense is really bad. So it's like every game is high-scoring That's what I want to see Has a neutral fan going forward in the playoffs. So I want to see I Also want to see like Bill's Chiefs Bengals. I want to see those three teams duke it out I want to see Cowboys Eagles Niners Or you know, Jags Cinderella on that be cool Jags Eagles Doug Peterson revenge Super Bowl Good luck everybody got to love it last game of the regular season There's a Packers need to win You know the Lions are gonna want to beat the Packers so bad now though They ain't gonna try any less Murray 75th anniversary Cole Anthony nice card magic Black Pandora Time Lord for the Celtics Robert Williams the third About one every ten box case on those Celtics Pelicans are the first auto here Jonas Valentunas perennial contenders auto 57 of 75 that is an on car No, that's a sticker Valentunas. Excuse me sweet shot silver Luka Donchich Mavericks and a red ice legendary tandems will Chamberlain Jerry West Lakers I would have loved to see the Pats lose the bills and still make it in the playoffs to play the bills and then beat him somehow They played him well today though outside of the two kickoff returns Red Basley for the Thunder after what happened Monday with Hamlin There were there were some calls that definitely went Very very head or non-causal and heavily in favor of the bills in the first half of that game I read Basley for the Thunder a silver rookie ticket Primo Spurs And we've got okay see Josh Giddy up and coming rookie auto 25 a 25 orange color match to there you go Giddy Thunder Giddy Big card will get that magged Got a kisspert hoop dreams is that a oh, that's a gold vinyl behind it. That's gonna be big It's gonna be big Kisspert hoop dreams for the Wizards. It could be big. It could be could be So it's a lottery tickets lottery tickets gold vinyl one of one of Jonathan Kominga hers Incredible insert there Danny see Super happy for Danny That looks unbelievable with the gold vinyl Heck yeah, man, you know, it's cool though the magic owned the Warriors It's a fact Sick card joking aside Sick sick hit man. What a break so far that box had the Giddy and We're starting to heat up tonight to some big triple threads heads as well Starting to heat up it's on a no be silver book night for the Hornets a black bandara Davey on Mitchell Kings awesome parallel there Well, take what I said about one every ten boxes and throw that out the window because that's wrong At least for this one Nice hit Kings there again. Those are a non numbered short print parallel and Gold rookie ticket auto pistons Luca Garza to ten four out of ten. Holy cow, man Yeah, I think it's gonna be a great game still. Well, if you know the Lions want to knock the Packers out Plus that's a team that overachieved all year. They're gonna be hype no matter what All-star aspirations Chris Paul Suns silver and blue ice Kyrie Irving sweet shots Nets 72 75 Yeah, it would have been cool to see like a winner take all I agree with that. I agree with that Danny if you're here, there's someone in chat currently on you in YouTube chat that says they are a Kaminga collector Be interested in talking about that card if you're here YouTube chat pool silver rookie Quentin Grimes Nicks having a good year a Blue og on a no be raptors to 99 season ticket Blazers up and coming rookie auto Greg Brown the third 109 of 149 Yeah, it is the same Danny actually they hit that Obi-Wan sketch on the random earlier Vin Sanity and T Mac legendary tandems raptors and red ice Superstar LeBron James Lakers There's Danny right there. Hi. You're welcome Danny Danny, I was just saying there's someone in chat right now Brandon S that Be interested in talking about that Kaminga. I want to let you know got red Zach Levine Board of Bulls blue clay Thompson Warriors number 21 of 99 Lakers rookie auto Austin Reeves. He's had a really good second year as well He's like the one bright spot on that team. I feel like you go Lakers On card also our aspirations Russell Westbrook thunder and a red ice also aspirations sons Chris Paul Yeah, so are the Saturday games determined like ahead of time that it'll for sure be on Saturday like way ahead of time But was it like always on like a Saturday Red Kawhi Leonard Clippers Silver Silver Time Lord Celtics Jericho Sims is 75 rookie auto for the Knicks up and coming All star aspirations Mavs look at Donchich and blue ice uniform any Jimmy Butler heats 68 of 75 Yeah, I mean they still had a prime-time game. It was 8 p.m. On a Saturday with no college football. So I I Get it though This one's still a win it in yeah, the last the last week and they always do Ends on Sunday, but also I mean that's to get teams like the right amount of rest and also because So it's like the right time to do it Time Lord we got silver Shengyun Rockets rookie. We got a silver bones rookie for the Nuggets Red rookie out of Kings Davion Mitchell nice hit 12 out of 99 See more gold coming. This is getting crazy, man. There's a wild case lottery ticket service. I Williams Grizzlies and Gold Joellen bead uniformity Awesome card Philly 10 of 10 That's awesome, man. Sixers is Chen M. We'll get that bag for you guys pretty good at basketball You think he's MVP right now Luca or Yolkich? He's a Lucas fourth. I probably look over Tatum right now Probably over Giannis right now Really Red Jalen Brown Celtics. Yeah, it's gonna come down to like the last couple weeks in season Jimmy Butler site of fifth anniversary heat and who stays healthy really like who plays the most games essentially Window Carter jr. Magic to 99 on the perennial contenders auto yeah, Katie as well It's just the competition is getting pretty crazy this year Like guys are doing ridiculous things and they're like he's like third Sweet shots bull Zach Levine Red ice uniform and he John Marant Grizzlies That's awesome Brandon. That's that's awesome Brandon. That's how I feel. I love seeing things get hit I love cards going to you know people that want to collect especially at a PYT because that means like you bought the team for a reason I Mean sometimes people just fill but most of time I think people buy the teams You know hoping to hit you know having a certain card in mind It's just really cool to see that happen Thunder will ask him in the next one Silver Lonzo for the bulls red John Marant Grizzlies Grizzlies Zaire Williams red rookie ticket auto At least a 75 You'll see it a lot man Silver Lamello also our aspirations Hornets and blue ice shy Gildus Alexander Thunder uniformity He's having a hell of a season to 47 a 75 All right y'all last one again not gonna recap this one actually let me see though the Maggots cards only because there were some big There were some big ones in this I'll show the Maggots again Yeah, Danny, thanks for filling up the Star Wars breaks earlier too, man Appreciate you. Oh We got a redemption that could be big It's probably a vet honestly most of the rookies are live a silver Durant nets red Lamello for the Hornets We'll see the red for the end. I don't know what it is silver numbers game Trey young Hawks red ice pick and roll Kyrie and KD And it is a Kate Cunningham. I was wrong big hit pistons now up-and-coming autograph blue Detroit Congrats Attila that was a heck of a case Up-and-coming blue it's a rookie the valentunis earlier was what's a 75 that's 75 or less I'm gonna say the rookie one might be lower Prano contenders somewhat up-and-coming it's it's it's it's lower numbered the inserts lower There we go. Congrats on that one pistons Thanks again everybody make sure the other Maggots cards really quickly we had that gold uniformity and bead for the Sixers The orange out up-and-coming rookie Otto Josh Giddy thunder to 25 and Gold vinyl lottery tickets Jonathan coming Love to see that good-looking card, too That's what's up, man. Thanks again everybody another contenders out to go next


You are eligible for weight loss surgery if you have a body mass index which is a quick calculation between your height and your weight which you can get on an application of 35 and above with a comorbid condition such as diabetes high blood pressure sleep apnea or if your BMI of 40 and above without any medical problems.


The Jack Benny program presented by Lucky Strike! Ego Lucky, get better taste today! Friends, smoking enjoyment depends on taste and taste alone. Yes, in a cigarette, it's the taste that makes the difference. And you can taste the difference in a Lucky Strike. So mild, so smooth, so firm and fresh with better taste in every puff. Yes, Lucky's taste better, and here's why. First, LS MFT. Lucky Strike means fine tobacco. Fine mild tobacco that tastes better. There's no substitute for fine tobacco and don't let anybody tell you different. Second, Lucky's are made to taste better. In fact, they're the best made of all five principle brands. Yes, that's a fact. Established by tests, measuring those important factors of workmanship that affect the taste of cigarettes. Tests made in the research laboratory of the American Tobacco Company and verified by leading independent laboratory consultants. So remember, your smoking enjoyment depends on taste and taste alone. And you will find Lucky's taste better. Always so mild, so smooth, so firm and fresh with better taste in every puff. Make your next carton, Lucky Strike. Be happy, go Lucky, go Lucky Strike today. The Lucky Strike program starring Jack Benny with Mary Livingston, Phil Harris, Rochester, Dennis Day, and yours truly, Mrs. Don Wilson. And now, ladies and gentlemen, I bring you the star of the Lucky Strike program, Jack Benny. Hello again, this is Jack Benny talking and Mrs. Wilson. I must say you read that introduction beautifully. Thank you. Now, if Don were introducing me, he would have tried. Hey, Jackson, what's going on? Why isn't Wilson here? Because last Sunday on my television show, he made one little mistake. Just one little mistake and he had a nervous breakdown. No. Yes, at the close of my television show, Don was supposed to say, be happy, go Lucky. But somehow he got mixed up and said, be Lucky, go Happy. It was nothing. Well, you say it's nothing because you don't understand the complexities of emotional reactions. Complexities of emotional reactions? And I know what I'm talking about. I once made a mistake. And for months, I couldn't look people in the eye. I was shunned, a social outcast. My friends wouldn't talk to me. Phil, for heaven's sake, what did you do? I put a cherry and a martini. No. A cherry and a martini? Why, Phil, I don't blame your friends for shunning you. I didn't mind that, but they tied me to a post and gave me 20 lices with a swizzle stick. Phil, go sit down and stop making up jokes. Now, Mrs. Wilson, is Don really so upset about that mistake he made that he couldn't come to work? Oh, yes, Mr. Benny. Last Sunday, when he came home right after the television show, I had to coax him to the dinner table. He just sipped at the consomme and nibbled at the salad. But I knew something was wrong when he didn't eat the T-bone. Don didn't eat the steak? No, the bone. He ate the steak. Wait a minute, Mrs. Wilson. You mean to say when Don eats the steak, eats the bones too? That's why we had to get rid of our dogs. Such fights. Oh, I can imagine, but Mrs. Wilson, if Don is as broken up as you say he is, I better call and reassure him that his little mistake was nothing. After all, anybody could have said be lucky go happy instead of be happy go lucky. Such a serious thing. Hello, Don Wilson's residence. Jack Benny calling. Is this the butler? No, this is the doctor. Doctor? Oh, my goodness. How long have you been there? Oh, I've been taking care of Mr. Wilson for the past week. Past week? He also has a nurse. A nurse, too? Oh, my goodness. He must be a nervous wreck. Well, tell me, doctor, when do you think Mr. Wilson will be ready to go back to work? Well, when is your next television show? Five weeks from now, March 9th. Oh, good, good. By then, I'm sure he will have calmed down enough to shave. Shave? Yes, in his present condition. I wouldn't dare let him have anything sharp. But this is ridiculous. Just because he made a little mistake and said be lucky go happy? Doctor, let me talk to him. I'm afraid he won't talk to anybody.  Well, you tell him it's Jack Benny calling. No, yes, sir. Hold on. Oh, Mr. Wilson. It's be happy go lucky, be happy go lucky, be happy go lucky. It isn't be lucky go happy. It's be happy go lucky, be happy go lucky. So simple. How did I ever mix it up? I never mixed up that other one. Strawberry, raspberry, cherry, orange, lemon and lime. Why couldn't I have said be happy go lucky, be happy go lucky. Mr. Wilson. Be happy go lucky. Mr. Benny. Yes, doctor. What did Mr. Wilson say? Be happy go lucky. Well, at least he's getting it right. Goodbye, doctor. Goodbye. Did you talk to Don, Mr. Benny? No, Mr. Wilson. He shut himself up in the closet and the doctor can't get him out. Oh dear, I hope he isn't stuck again. Again? Yes. The last time he made a mistake, he forced himself into a closet. We had to break down the wall to get him out. The last time he made a mistake? Yes, don't you remember? Two years ago, in one of your programs, he was supposed to say, I saw it in Drew Pearson's column. But instead of saying Drew Pearson, he said Dreer Pooson. Oh, yes. Well, don't worry about it, Mrs. Wilson. I'm sure Don will be all right pretty soon. Now kids, let's get on with the show. Hello, Mr. Benny. Well, Dennis, it's about time you got here. I hope you have a good excuse for being late. Oh, I have. You see, I was walking down the street and I passed a gas station. There was a car standing there getting gas. And you had to stop and watch the car getting gasoline? Oh, it wasn't that. There was a dog in the back seat of the car that attracted my attention. A white French poodle. Oh, well that is a rare species. Yeah. The man told me the dog was worth over $2,000. Gee. And while I was standing there, the attendant happened to accidentally spill some gasoline on the ground. And before the man could stop him, the dog jumped out of the car and lapped up all the gasoline. Gosh. And then he made a crazy dash down the street and when he got about two blocks away, he suddenly stopped and flopped right over on his side. Dead? No, he ran out of gas. Dennis. Dennis. Hey, Phil, look how red he's getting. Yeah.  Phil, did you give Dennis that story? Yeah, Jackson Loosen up laughily. That's a funny story. It's funny. It's funny. Dennis, how much did Phil charge you for that story? Oh, he didn't charge me anything. It was an exchange. An exchange? Yeah, he told me the story and I told him all about the complexities of emotional reactions. I wondered where he got it. Now I'm wondering where you got it. The doctor wrote it on my birth certificate. That I can believe. Now look, it is time for your song, so let's have it. Okay. Oh, hold it a minute, kid. Hello? Hello, Mr. Benny. This is Mr. Wilson's doctor. Yes, yes. We just x-rayed Mr. Wilson and found some broken bones. Broken bones? Where? In his stomach. Tell Mrs. Wilson he finished his dinner. I will, I will. Same Dennis. It's some talk. Cloud that cried sung by Dennis Day and accompanied by Phil Harris and his Gruen wristwatch orchestra. And now folks. Well, that's a new one. Jackson, why did you call my band the Gruen wristwatch orchestra? Phil, if I've got to listen to them, I might as well get something for it. Now, wait a minute, Jackson. You have just cast dispersions on a musical group that only last week played at the formal opening of the Pismo Beach grunion festival. Very funny, Phil, but I happen to know that last week those little fish called grunion weren't running. That's why the committee hired my band. What? They put my boys on a barge, towed them three miles out. They played, that's what I like about the south, and the grunion hit the beach like it was D-Day. Really drove the fish out of the water, huh? Well, I don't want to brag, but it was the first time they had halibut dancing in the streets at Oxenard. No, no, Phil, that's not bragging. If you did it, you did it. However, tomorrow I'm taking a rumble lesson at Arthur Murray's, and if my partner turns out to be a flounder, I'm going to punch you right in the nose. So Phil, the next time I say anything about your boys, just let it go. Oh, Mr. Benny. Yes, Dennis? How come Mary isn't here? Huh? Oh, oh, Mary's in Palm Springs. See, next week I have to go to New York. Mary's going with me, so I thought I'd let her take a little vacation. You never give me a vacation. Well, Dennis, when I give Mary a week off, we can fill in with more dialogue, but it's difficult to have a program without a song, so I can't do without a singer. Excuses, excuses. Huh? You gave Kenny Baker a vacation. What? He's been gone 12 years. Dennis. When's he coming back? I'm getting tired. Dennis, I didn't give Kenny Baker a vacation. He left because of another job that paid more money. Gee, didn't that upset you? No, I was his agent. Anyway, Dennis, that must be Don Wilson's doctor with another report. Hello? I have a long distance call from Mr. Jack Benny from Palm Springs. Oh, this is Jack Benny. One moment, please. Palm Springs, Biltmore Hotel. I have Ms. Livingston's party. I'm sorry, but Ms. Livingston went out to play golf about 10 minutes ago. Mr. Benny, Ms. Livingston's not in now. Gee, and I was so anxious to talk to her. If you like, you can talk to me. To you? Yes, I have a message for you. Oh, what is it? Will you go over to Ms. Livingston's house and leave a note for the milkman? Certainly. What shall I say? Just say sorry. I couldn't meet you last night. Ms. Livingston wants me to leave that note for the milkman? No, I do. I get to town once in a while, kid. OK, I'll do that for you. Now, when Ms. Livingston comes back, I'll tell her, AHHHH! Ms., Ms., what frightened you? Two mackerel just came through the lobby and they were dancing. Gee, Phil's orchestra really drove them inland. What did you say? Nothing, nothing. Goodbye. Now, Phil, since Don didn't prepare a commercial for the sportsman, you will have to- Just a minute, Mr. Benny. Yes, Mrs. Wilson. I have a commercial for the sportsman to do. In fact, we rehearsed it this morning, didn't we, boys? Well, Mrs. Wilson, I think it's wonderful that you- Oh, excuse me. Hello? Hello, Mr. Benny, this is Rochester. Rochester, what did you call for? I want to ask you, boys, how long will you be in New York? About a week. You want me to run the usual ad in the paper? What ad? About running your house while you're gone. No, Rochester, not for just one week, you know. Boss, you sure are a change man. What do you mean? You once ran your house when you went to the movie. It was a Jane Russell picture, and I didn't know when I'd get back. Now, Rochester, hang up and get back to your work. Boss, I have been working. I wash the dishes, polish the silver, vacuum the rubs, wax the floors, mop the kitchen, and after I finish listening to your program, I'm going to clean the woodwork and wash the windows. Wait a minute. You're taking time out to listen to my program? What do you mean, time out? That's word two. Oh, well, Rochester, I'll be home right after the show. Goodbye. Goodbye. Oh, say, boss. Now what? Mr. Wolfie Gilbert was here to see you. Wolfie Gilbert? The songwriter? Yeah, I told him you were at the studio, and he's on his way down to see you right now. Oh, that's wonderful. Rochester, did you tell him about the song I wrote? No, boss. Why not? That and how you look in the morning are my guarded secrets. Well, I'll tell him myself when he gets here. Goodbye. Hey, kids. Wolfie Gilbert, the songwriter's coming over to see me. What a songwriter. You know, he wrote Waiting for the Robert E. Lee, Down Yonder, The Peanut Vendor, Lilac Time, and oh, a great bunch of songs. She'll be glad to see him. Now, Mrs. Wilson, before he gets here, we better do the commercial. What's the thing you've prepared with the boys? Well, Mr. Benny, since Don is so upset over the mistake he made on your television show, I thought it would be nice if the boys sang something to cheer him up a little. Well, good. Don will probably be listening to it. Take it, boys. Be lucky and go happy. That is what Don Wilson said.  But don't you worry, Don. Oh boy, you'll still collect your pay. If in the future you make sure that this is what you say. Be happy. Go lucky. Be happy. Get better. Taste me happy. Go lucky. Go lucky. Strike today. Get out of bed and take a walk. The air will do you good. Don't try to hide to save your pride. Your fluff was understood. Why any one of us could make a similar mistake. But don't feel bad. Just watch it. Dad, be right for goodness sake. Be happy. Go lucky. Be happy. Get better. Taste me happy. Go lucky. Go lucky. Strike today. Be happy and go lucky is a slogan you know well. So say it right on Sunday night or I will get bonzelle. Be happy. Taste me happy. Go lucky. Go lucky. Strike today. Now that was wonderful Mrs. Wilson.  Now kids, as soon as we, hey, that must be Wolfie Gilbert now. Come in. Hello Jack. Wolfie. Wolfie, it certainly is a pleasure to see you. Well, I stopped over at your house and Rochester sent me to the studio. Yes, I know, I know. And Jack, I wish you'd tell Mary. I'm sorry that I only had a dime. What are you talking about? Mary's in Palm Springs. She is. Yes. Well, who's that girl who checked my hat? Oh, that's Barbara Stanwyck. Barbara Stanwyck checking hats for you? Wasn't she on your television show last Sunday? Yes, and she didn't read the fine print in her content. Now Wolfie, I'd like you to meet my orchestra leader, O'Fell. This is Mr. Gilbert, the famous songwriter. Glad to know you, Gilbert. We're Sullivan. This isn't Gilbert and Sullivan. This is Wolfie Gilbert. He had nothing to do with Sullivan. Now Dennis, come here. Now Dennis, don't you want to shake hands with Mr. Gilbert? Sure, not. He wouldn't be after having anything to do with the Sullivan and Truth we got in bad tests to him, too. Dennis, behave yourself. Now Wolfie, how he got mixed up with an Irish line. A straight line, yes, but an Irish line. This I will never figure out. Now Wolfie, was there something special you wanted to see me about? Yes, Jack. I really had a trip to make over here. I wanted to talk to you about the song you wrote. I heard that you've been having a little bad luck, I mean about publishing. Well, trouble with it. Yes, and frankly, I can't understand it, Wolfie. I wrote my song three months ago, and it still isn't on the hip parade. I haven't even been able to get it published. Well, Jack, believe me, it's nothing to worry about. You mustn't become discouraged. You know it isn't easy to get a song to be ahead. But Wolfie, you didn't have any trouble. Look at Robert E. Lee, Lilac Time, the peanut vendor. And how about your latest hit, Down Yonder? Everybody is singing that. Yes, I know, Jack. I'm grateful. But that's what I wanted to tell you. It's true, Down Yonder is a hit today. When I wrote that song 30 years ago, it was a flop. Down Yonder was a flop? Jack today is a big hit. I know, I know. Well, so you see, Jack, 30 years from now, your song may be a success too. I mean, but who can wait that long? I'm 39 now. In 30 years, I'll be 45. By the way, how old are you? 65.  Quiet, Bill. By the way, why do they call you Wolfie? Well, I didn't spend all my time waiting for the Robert E. Lee. You know, that waiting for the Robert E. Lee was a great song too. Now, Wolfie, as one composer to another, how do you... Wolfie, how do you go about writing your songs? Jack, it is really inspiration, mostly inspiration. Now, you take Robert E. Lee. I happened to be in New Orleans one morning. I was sitting on the levee looking over the broad Mississippi. It was a beautiful day and I felt wonderful. As I watched this solitary riverboat loom out of the morning haze and majestically drift by an idea, some words and lyric came to my mind and I just had to put it down on paper. Now tell me, Jack, what inspired you to write your song? Well, it's an amazing coincidence, Wolfie. One day I came home after a broadcast. It was a dismal, rainy day. I had a headache. My stomach was upset. My feet hurt. And as I walked into the house, I tripped and broke my glasses and split my lip. Then I sat down and wrote, when you say I beg your pardon, then I'll come back to you. And yet, from the lyrics and music of my song, you'd never guess how much I was suffering, would you? Who's got enough money in the bank to answer that? Well, be quiet. Well, anyway, Wolfie, I do want to thank you for your advice and it made me feel a whole lot better. But as long as you're here, how about you and I doing one of the numbers you wrote? Robert E. Lee, how about it, folks? Now, Wolfie, I'll get my violin and you start the verse and we'll each take a part of it, the quartet too. Wait till I get my violin. Okay, boys, let's have it. We're down on the levee in Old Alabama. The daddy and mammy leave from in Sammy on a more life. Night you can find them all while they're waiting.  What's that they're saying? What's that they're saying while they keep playing? I'm humming and swaying in the good ship. Robert E. Lee, that's come to carry the cotton away. Watch them shuffling along. See them shuffling along. Oh, take your belly down. You'll really have. Go down to the levee. I said the levee and just join that shuffle and throng. Hear that music and song. It's simply great, mate. Waiting on the levee. Waiting on the levee. Here I come, folks. Thank you, Wolfie. Thank you very much. You're an inspiration to young songwriters. Thanks, Jack. I hope I inspired you, too. You did. You did. So long, Wolfie. Goodbye, Jack. Ladies and gentlemen, your armed forces are short of 300,000 pints of blood a month, a shortage that may cost us thousands of American lives. We know you are going to give blood. We ask that you give it now. While you're Red Cross today, this is an urgent request. Remember, a gift of blood is a gift from the heart. Thank you. Mr. Benny, we'll be back in just a moment. But first... Friends, whenever you want the smooth, mellow, completely enjoyable taste of truly fine tobacco, reach for a lucky. For the difference between just smoking and really enjoying your smoke is the taste of a cigarette. And luckies taste better for two important reasons. First, LSMFT. Lucky strike means fine tobacco. Fine mild tobacco that tastes better. There's no substitute for fine tobacco and don't let anybody tell you different. Second, luckies are made to taste better. In fact, they're the best made of all five principal brands. Yes, you'll be happy when you go lucky because luckies taste better. So mild, so smooth, so firm and fresh with better taste in every puff. So next time you buy cigarettes, try a carton of luckies. You'll find luckies taste better. Be happy, go lucky, go luckies like today. Well, folks, this ends another show. We'll be with you again next Sunday night at the very same... Excuse me, man. Hello? Hello, Mr. Benny. This is Don Wilson's doctor. Oh, yes, doctor. Is Don feeling better? Yes, I'm happy to report that he came out of the closet. I took his pulse and it's normal. His heart beat is strong. His blood pressure is fine. I took his temperature and it's 46. 46? Isn't that a little low? Not for a man who's hiding in the deep freeze. Well, when he thaws out, tell him that I hope he'll be better soon. Goodbye. Thank you by Lucky Strike, product of the American Tobacco Company. America's leading manufacturer of cigarettes. This is Mrs. Don Wilson reminding you to listen to your hit parades with Guy Lombardo every Thursday night presented by Lucky Strike. Consult your newspaper for time and station. Exactly, any program is being selected as one of the programs to be heard by our armed forces overseas to the facilities of the Armed Forces Radio Service. Stay tuned for the end of the show which follows immediately. This is the CBS Radio Network.


All right welcome everybody. It is my great pleasure today to introduce Barry Honig to various somebody that I met very well I I met him through his papers almost immediately When I entered this field and met him shortly thereafter He's made enormous contributions, especially to the field of protein electrostatics and implicit solvent models for proteins and macromolecules Today, he'll be talking to you about some newer work so Barry is a Investigator of the how are you Howard Hughes Medical Institute and a professor at Columbia University in Molecular biophysics Systems Biology and Medicine. He's the director of the computational biology and bioinformatics Institute there And he has numerous awards. I almost feel like I should save half of this for tomorrow because there's such a long list here, but he has the Hollander award in Hollander award in biophysics from the National Academy of Sciences and is also a member of the National Academy of Sciences and The American Academy of Arts and Sciences He has the Founders Award from the Biophysical Society the Delano Award for computational bio Computational biosciences and the Anfanson Award from the Protein Society and honestly I can't think of any other possible award that well maybe just one but you know any other possible award that somebody could win in computational structural biology and And very also I asked him for a couple of hobbies And he let me know that he is an avid scuba diver and also wanted me to let you know that he played a year of football at the University of Florida before he went to Went to Brooklyn to get his his degree in chemistry and then on to the Weizmann Institute for PhD So if you'll please help me in welcoming Barry Honig as our Steenbach lecture Thank you, Julie I Heard for many years about the Steenbach lectures one of these things you don't actually think you'll ever be one I'm really excited and delighted by the honor and thank you for attending. I this is the title of this talk and It's it represents an area. I began working on about 15 years ago and I say right here with Larry Shapiro because when I became I've done computational work all of my life, but When I became a user investigator, I was able to expand my interest to do experimental work as well and Nobody would ever fund me to do experimental work And but Larry's a accomplished crystallographer and together we set up the joint lab And I think it's perhaps a way that some modern science can be can be done We have a single experimental lab and actually share computational students as well. We do crystallography Everything that goes along with that cell assays biophysical measurements and the people working in the lab Aren't even sure who pays their salary. It's it's really a joint effort. So All of my work on adhesion is done with Larry. We publish everything together and I Just want to start with that. I also wanted in part to advertise Tomorrow's lecture in part because I sent the wrong title There was a typo that I never caught but there's a fundamental difference in what I'll be talking about today and tomorrow Today is I'd say more traditional biophysics. There's some cell biology in it It's really understanding how molecules work in a biological context Tomorrow is I'm going to be talking about the use of three-dimensional structure to Basically to predict what proteins interact as it says on a genome-wide scale It's sort of low-resolution structure biology today will be high resolution structural biology and the differences are are quite dramatic So the sorts of problems we're interested in Are summarized here and I'll be getting to some of them But the basic question is can we use the information about protein structure and protein protein interactions to understand how cells Organize how they interact and this is a picture or diagram of motor neuron pools there are different motor neurons that connect to muscle and They separate into into various pools as you see here dependent on the proteins that appear in them that are expressed in them And these are the proteins that I'll be saying a lot more about that These are proteins called nectons I'll be discussing that or play a role in organizing the inner ear and in many other roles and in both cases I'll be trying to try to convince you that there's a clear connection between molecular and cellular properties And then the second half of my talk will be devoted to some very new work Which involves the how protein protein interactions play a role in a specific problem in the design of the new in the wiring of the Nervous system particular how neurons know when to form synapses and when to avoid each other So that will be the second part of the talk, but they'll all be based on common families of proteins So the proteins that we're interested in and these are generally how adhesion proteins are organized This is supposed to represent a cell membrane. They're multi-domain proteins I'll be talking first about what are called classical cat herons. They have five immunoglobulin like domains and a Trans-membrane region and a site of plasmid region. I'll be talking about proto cat herons nectons They all look like this though multi-domain proteins With some connection to the cytoplasm This shows in the case of classical cat herons what that connection sort of looks like there's a Unstructured region the outside the extracellular domain is structured the cytoplasmic domain is not but it forms complexes with beta-catenins alpha-catenin and ultimately links to the actin site of skeleton Almost there in fact everything. I'll be talking about today involves Recognition which takes place outside the cell. I won't be talking about the various Cosmic processes that are initiated by recognition. So the basic question is how does protein recognition? translate into cellular recognition so what got me into this field was 15 years ago or so when Larry actually who's in our department Gave a talk in my group meeting trying to get my lab interested in the following sort of problem. This is a Image of the developing chick embryo after six days. This is the ectoderm and This shows that the cells here express a protein called ecad here an e for epithelial And you can see that these cells are separate from this inner set of cells Which form the neural tube and these proteins express these cells express N ket here and N for neuronal and It has been they were discovered by Takaichi quite a few years ago And the basic thought then was that this is very simple ecad here and doesn't stick to N ket here And so these are the thought was that they were homophilic proteins So that when cells start expressing it get here and those cells would break off from the cells creating ecad here and giving rise to this sort of early developmental process so We had at the time models, but now we have crystal structures of E and N ket here And you can imagine the membrane over here and here so these coming out from the membrane they sort of form a banana shape and Only bind at the external domain the ec1 domain and the there's a lot to say about this But I won't today the interaction is involves what's called a strand swapping You see there's a tryptophan from one protein from one monomer that's Inserted into a pocket in the other and vice versa. So they they sort of exchange the beta strand That's how they bind and the question that actually posed a graduate student at the time was Why how are they different? How is the in N ket here and different? They're about 70 percent identical and it was The fellow working on it was an empty PhD student and they all want to get their PhDs within three years So I told him that if he could tell me why they were different he would get his PhD and it could go on to do other stuff so He he basically we had structures or models of this is just the membrane distal interacting domain of e ket here and N ket here and he built a model of E N and all he had to do was tell me What was wrong with this model? why he doesn't bind to N and How these two guys are different and after a year or two He couldn't figure it out and the models looked as good as the heterophilic models looked as good as the homophilic ones And at that point I became an experimentalist So we actually used an analytical ultra centrifuge and SPR surface plasm on resonance to Measure the binding affinities of these proteins and we found a few things number one that N ket here and sticks to N Quite more strongly than e to e. This is a hundred micro molar. This is a 20 micro molar binder and E N actually bound better than e so these molecules actually did bind heterophilically and This came as some somewhat of a surprise But it sort of set us on our path after these experiments were done He did Peter Chen his name was did get his PhD. So it all worked out for him but this Gave us a different picture of these proteins in that they were heterophilic and then the question was well, why do the cells that express e and n ket here and why do they separate from each other and The answer to that we believe I'll just set up the answer I'll just point out that there are proteins I'll be talking less about that are called type 2 ket herons that are very similar, but they have a larger interface They have insert to trip defense with strand swapping and they don't bind to type 1 ket herons And the reason I tell you that it's because we did the following set of experiments So these are cells are labeled with red and green dyes And if they both express both sets of cells express n and n ket heron as you might expect they mix together because n binds to n When they express e ket heron again They mix together and this is a control with ket heron 6 of type 2 ket heron and again You see the same of the same result now if you have some cells expressing n and some cells expressing ket here and 6 They form separate separate aggregates because n doesn't stick to 6 if you have cells expressing e and Cut here and 6 again. They form separate average aggregates But when you have cells expressing e and n together they actually form separate aggregates, but they stick to each other and in a way that's Very crudely reminiscent of what one sees here that n aggregates in the middle any around it And why is that true? Because n sticks to n has a higher affinity for n than e to e So the concepts that might work at a molecular level work at a cellular level the most strongly aggregating Cells will maximize their interactions and therefore form a central aggregate The weaker ones will aggregate around the stronger ones interact with each other and with the central core so in very very crude, I know I'm going through this very sort of Superficially, but very quickly the concepts that simple thermodynamic concepts are working here at a cellular level So that's what I want to say for type about type 1 ket herons Now another family of proteins that are cell-cell recognition proteins called Nectons and These are these actually have three domains. They're immunoglobulin domains and we solve the structures of a bunch of them and they all look alike They form an interface again at the membrane distal region and when we Measured the binding affinities of Nectons that we found that their behavior is very different than classical ket herons. These are Homophilic binding affinities and they range you can see from 0.4 to what 200 or so micro molar actually a point I want to make Membrane binding proteins tend to have much weaker affinities than Soluble proteins than than cytoplasmic proteins. These numbers are actually quite quite typical Anyway, you see these are the range of affinities. You see that Necton 1 Binds to itself say more strongly than strongly than Necton 3 binds to itself But now when you do an experiment with say a Biocore apparatus where you flow Necton 3 or other Nectons On a chipped containing Necton 1 you see that 3 binds to 1 more strongly than 1 binds to 1 So these proteins are heterophilic binding proteins primarily. They're also Homophilic and what they doing all the measurements you get some kind of a matrix and binds in 1 2 and 3 and 4 Etc. And how this works is is ah So these are experiments done in our collaborators lab Sergei Troinovsky and what's nice is I'll explain this in a minute You see these molecular properties manifest at a cellular level So these red cells express and Necton 1 and the green cells express Necton 1 and you can see At the interface between these between red and red Red and green and green and green you see a build-up of proteins those proteins are diffusing to the cell-cell Interface because they stick to each other and you see what are called will call junctions from now on at all interfaces Now if you express Necton 1 with Necton 2 Necton 1 doesn't bind to Necton 2. So you only see junctions Within a single color within a single type of protein. You don't see anything in between them But now when you express Necton 1 with Necton 3 all the sudden you see the strongest junctions between the red and the green Because Necton 1 Necton 3 is the strongest binding interaction. So these proteins. It's not surprising They're diffusing they're finding their partners where they can gain the most interaction sort of free energy That's where they're going to show up. So again the properties of the individual proteins are being Reflected by their behavior on cell surfaces, maybe not surprising, but it's it's nice to see it By the way, I should say I'm happy if anybody wants to interrupt at any stage. Please please do The way these proteins are designed is very clever and very simple If you want to design a heterophilic protein put a charge there and on the protein it binds to most strongly have an opposite charge So if these two proteins dimerize you'll have this sort of red negative region interacting with another Glutamate there'll be some repulsion, but in the case of Necton 3 there's a lysine There'll be an attraction. There's a very nice way of designing Homo heterophilic proteins and there's the way that the Nectons work What's very nice at a cellular level this these are cells Which Contain either Necton 1 and Necton 3 and the and this is these cells are in the inner ear. I Admit I don't remember of what organism, but I assume it's mouse But you see they form a checkerboard pattern and when you knock out Necton 3 the checkerboard pattern basically goes away And this is exactly what you expect from heterophilic proteins if you want to max Maximize the favorable interactions, then you're going to have their heterophilic at the molecular level They're going to be heterophilic at the cellular level and that the way to optimize this is through a checkerboard so again the properties of the individual proteins are being reflected by No by the by the behavior of the cells that contain them and This is sort of I guess An example of someplace. We're going. It's just something I I like showing because I never in my life when I started As a computational chemist dreamed I'd be showing a picture of a stained kidney But this is what this is These the kidneys being stained for different types of cat here and this is called a nephron, which is a filtering unit in a kidney and Some regions of it contain ecat here and which is a type one Cat here in six, which is a type two. I already told you that Type ones and type twos don't bind to each other So what happens in between? There's a region that contains both type ones and type two cat here And so we combine in both directions so Again what at least very qualitatively, and I know I'm hand-waving a lot We can understand some of the simpler aspects of this development of a nephron based on the adhesion properties of the individual proteins We are hoping to now using CRISPR technology to design artificial kidneys By changing we can change affinities of these proteins almost that will and that's something that we're hoping to do and this Work is being done with rosemary symponia a nephrologist and a PhD at Columbia So that's a Sort of almost an introduction. There's the properties of proteins on cell surfaces are reflected in the properties of the Behavior of the cells now. What happens at the interfaces between cells? This is it's not enough to say recognition. There's complicated things going on and this is a Picture I took from a textbook central cell biology and what you see these are cells stained with ecadherin and See the ecadherin is distributed all over the surface of the cell and as soon as these cells come in contact You see basically the proteins diffusing to the interfacial region and you could see the increase in fluorescence intensity So that's that's what's going on well It turns out that in the case of classical cadherins these interfacial regions called a form eventually fixed junctions called adherence junctions and We're interested in the properties of those junctions and what that means for signaling So we when we solve the structures of E and N cadherin And also another cadherin C cadherin We found that they formed they all formed a very distinct crystalline lat looking lattice Crystal lattice in the case of the structures Where this is the trans interaction trans in the language I'll be using in this talk now means interactions of proteins in different cells sister interactions of proteins in the same cell So this is the interaction. I already showed you but in addition. There's a second interaction. You can see going This way. This is trans. This is cis You can see the lattice It's a it's a two-dimensional lattice seen in a crystal and we Suggested that maybe this lattice is what forms between cells, but before I get to that What's very interesting is when we I already told you we can measure the binding affinities of catherins And they're depending on which one 10 to 100 micromolar or so We couldn't measure the this interaction which we thought might be a crystal contact Because we could we when we just tried to measure this in solution. We couldn't measure it. So it's very weak less than one millimolar but when we Connect these Cadherins to liposomes first you see that the liposomes form a there's a there's a clearly ordered structure between the liposomes, but when we make mutants when of this Once we have the cis interaction which has some hydrophobic groups We simply replace one or two hydrophobic groups with aspartic acids. We disrupt this interaction and you lose structure So we're disrupting an interaction. That's too weak to measure and Yet we're seeing a strong effect on structure, but more interestingly this is a image of Adjunction formed in live cells Transfected with wild type e-ket here and when we mutate again. We the system interaction We no longer see aggregation or there's very little aggregation in the cell cell Contact region so again disrupting an interaction. That's too weak to measure has very significant effects on what happens in cells So how is that possible? I'm going to go through this quickly. There's a whole theoretical story, but I couldn't resist Telling you about it quickly what you have of this is I'm going to show you a simulation a minute but these are supposed to be cadherins moving on cell surfaces and If they formed a cis interaction on their own as I'll just tell you that would signal to the cell that something has happened But so they can't be allowed to form a cis interaction until they've seen another cell So there has to be a mechanism whereby this interaction becomes strengthened by interactions with other With proteins from another cell and the way we think this happens and caught myself here You have two proteins that are Connected to the surface. I'm not telling you how I do these simulations, but Have linkers that are fairly flexible between them and they move around quite a bit if they're connected to Another protein from another cell surface then they have much less conformational freedom because they're linked at both sides to a membrane in the middle to one another and See I'm not good at this And the point is these guys don't move so much once they form a connection with a protein on another cell and We believe that it's this trans interaction that strengthens the cis interaction and this is This sort of leads to the next step. I'm about to show you is The question the question we're asking is can these proteins form a two-dimensional assembly on their own of this of the sort I showed you on the cell surface and This leads to sort of all kinds of theoretical issues, but the basic point is that when we calculate or think or measure binding affinities in solution these proteins are moving in three dimensions and When proteins interact on cell surfaces, they move in two dimensions Sort of two dimension quasi two dimensions so that the affinities We measure in three dimensions if we have somehow relate them to affinities in two dimensions And we have a whole theoretical framework that we use to Calculate though to make the transformation But I just want to show you a another simulation before I get to the second part of the talk So here's here a cat here ends Green and red means different cell surfaces here When they in contact we draw them blue so there's three colors here green red and blue You don't see the blue yet and the only difference between this lattice on the left and the lattice on the right is we've made the cis interaction a little stronger Consistent with a theory. I haven't told you about and this light yellow region is a is a contact region and I just want to show you the two sets. So we're moving these proteins around whenever they form a contact This is a Monte Carlo simulation. You see something come up in blue So here on the left the cis interaction is weak the proteins diffuse to the interface, but There aren't they move out because there's nothing keeping them there here. There's some sort of a phase transition Where the stronger cis interaction the trans interactions the same in both cases? But the cis interaction is strong enough to lead to some ordered structure Which is at least consistent with a self-assembly process we think is going on on Cell surfaces so in general and this is a Point I want to take for the second part of this talk which begins now is that these proteins have the capability of forming ordered structures on cell surfaces We know that once these clusters form they they involved in signaling to the side of plasm and ultimately connect to the Act inside of skeleton We don't know if the ordered structure is important is if there's a connection between order outside and order inside Or whether order is just a way to assemble lots of proteins in one place We don't know that yet, but we are what we do know that ordered structures form that can be disrupted Experimentally by disrupting the cis interaction. So that's sort of an overview of What we've worked for many years And now I'm going to discuss a totally different problem which will relate closely nevertheless to what I've been talking about and I mean, this is a presumptuous title. How is the nervous system why I'm not going to tell you how but This tells you how many synapses and how many neurons there are in different organisms and that these Neurons wire in precise ways is certainly absolutely remarkable and it involves different features of neurons and The one I'll be concerned with is a phenomenon of self-avoidance So here's an example of a cell body in Drosophila and these are Neurites extending from the cell body and what you see is that they don't Touch each other they avoid each other And here's an example of neurons in vertebrate in mouse and you see the same self-avoidance phenomena So the question is that I'll be addressing is what is the basis of this self-avoidance? For Drosophila much of our understanding at the genetic level and the neuro biological level comes from work from No, I don't have his name here Larry's a Pereski's lab who Worked with talk and study the properties of a family of proteins called these scams down syndrome. So he's in molecule Flies don't get down syndrome, but these proteins are related to vertebrate proteins and here's what happens. This is a Drosophila neuron when you knock out this gene cluster you see now that the Neurites bind to each other So this establishes the role of these scams, which I'll be talking a little bit more about this is again work from Larry's a Pereski's lab in vertebrates This is Sort of a starburst anocrine cell Again when you knock out a gene cluster You see now the dendrites are sticking to each other and the proteins that do this are Called press clustered proto ket herons, and they're related to the ket herons. I've been talking about And what I'm going to do is briefly tell you At a molecular level how the d-scams work Again, it's a Pereski's work and then get to our own on vertebrate neurons, but there's a very very sort of counterintuitive thing going on So here's a diagram of two neurons Red and blue and they can form synapses with each other But they don't see redness binding to blue But they don't synapse within the same cell and it turns out each neuron has a unique barcode That defines it and tells it that it's different or and some other neuron, but where it where it gets counterintuitive is that the proteins both in Drosophila and Invertebrates are homophilic adhesion proteins And it's adhesion that leads to repulsion and Why and in fact it must be that way because what you want is a specific mechanism whereby two Cells will repel each other and I don't I've never found anyone who could tell me how you could make two proteins repel each other specifically We have there are lots of ways proteins can bind specifically, but how do you make them repel specifically you have positive charge negative charge There's not a lot you can do so specificity begins with adhesion and then adhesion Leads to repulsion Through activation of cytoplasmic phenomena So when you have two neurots from the same neuron they bind and then repel When they're from different neurons, they don't bind therefore they don't repel That's the counterintuitive part of the story But you need to remember that because it can be confused other I get confused all the time so in the case of these scams there are The proteins have three domains which are alternatively spiced IG 2 IG 3 and IG 7 we know that the structures of These proteins from David Eisenberg's lab and basically they IG 2 binds to 2 3 to 3 7 to 7 there's 12 48 and 33 spiced forms if you multiply out all the possibilities you get 19,000 distinct proteins and What Zaperski's lab showed is that these proteins are all strictly homophilic they only bind to each other and That leads to very interesting protein design questions How do you design 19,000 closely related proteins only to bind to each other and? Obviously nature has done it and I can Maybe isn't all that complicated. This is how they this is how they work and this leads to sort of an issue which is talked about but It's essential that we think about there aren't clear answers for these scams you have 19,000 distinct proteins and 10 to 50 isoforms per cell so what that means is through stochastic splicing Each neuron say neuron a will select 10 to 50 of the possible 19,000 choices it has Let's say in this case. They're only six To simplify the figure so this shows that there are six different isoforms chosen There may be a thousand copies of each but six different isoforms chosen Here's another near a neuron with six isoforms chosen of which two are the same The yellow and the blue so let's say they were all the same then you'd have Homophilic interaction the cells would be the same But by having this opportunity to choose six in this case from 19,000 you get differences, but then you get to a question. Well, you don't want these cells to stick to each other What if five out of six were the same? You know, what if 19 out of 20 were the same you'd expect them to bind You know the one that doesn't bind would get out of the way so to speak and one doesn't really know What the number is and the people working on drosophila haven't worked this out but it raises sort of a Term the tolerance for common isoforms how many isoforms can be different and the cells still don't bind to each other They they assume 10 20 percent But putting that aside It's clear that given a choice of 19th given a choice of 19,000 isoforms It's not that hard Intuitively to imagine Selecting a few so that the probability that any two neurons would have the same set is very small So drosophila solve the problem with 19,000 proteins Okay, without now. This is what's known today now in the case of vertebrates There's actually Gene clusters of protocadherans. There's there's an alternative Splicing going on here, but I'll turn stochastic promoter choice And there are different clusters But the point is that in the case of vertebrates and sake of mouse for example, they're only 58 proteins not 19,000 So mice accomplish Being having more complicated brains than flies mice accomplish this problem, you know solve this problem with 58 proteins So the mechanism working in drosophila can't be working in vertebrates But again, this is to remind you that if you knock out a gene cluster You lose self-avoidance. So We want to figure out I'm going to try to tell you how I think mice accomplish the same task so working with Tom maniata's lab with postdoc in his lab a to chan we first study the binding properties of these proteins and These are cells expressing Different protocadherans red, you know, and they're red and green Fluorescing cells and you can see this when if we look at the diagonal you see that All the cells aggregate together Which means that these are homophilic proteins beta 4 to beta 4 you see there's mixed aggregates Any other combination they don't bind to each other You see the set they form separate aggregates So we're really using cell aggregation as a probe of binding and what you see again as is the true for drosophila. These are Completely homophilic proteins They don't bind heterophilically But the really interesting and surprising observation we made in studying these cells is that Well, you know, let me just take you through it You have to start on there on the right the way this is organized These are cells that express three different protocadherans. Don't worry about their names and in the figure on the right All three in the red and green cells are the same and they form nice aggregates But if two are the same and one is different the cells don't don't bind to each other So one incorrect protein is getting in the way of two correct proteins Is that clear I hope I hope it isn't so there's what we call an interference mechanism You might think intuitively if you have three different classes of proteins on the cell surface What I said a few minutes ago if one of them doesn't bind It'll just diffuse away and the other two guys can bind to each other But they don't one incorrect protocadheran is enough to disrupt cell-cell contact and we've done experiments with up to five Expressed in the same cell one incorrect one interferes with binding and We believe that it's this mechanism that lies at the heart of how protocadherans work So we solve the structure I I Say we solve the structure. It's I couldn't I don't know how to solve a structure say But Larry does and we have this common lab So I always feel a little comfortable saying we we solve but I'll say it we solve the structure of protocadherans. I see this figure is not so clean we solve Structure with few of them. This is what it looks like as opposed to classical cadherans These guys form anti-parallel contacts these very similar proteins to the cadherans. I've been showing you yet They interact in a completely new way instead of forming a banana shape, they're very straight and You can see they form this anti-parallel sort of head-to-tail structure Where ec1 the membrane distal region connects to ec4? ec1423 this is how this is the interface they form But that doesn't tell us anything about interference I guess we've mapped we we understand how they bind we understand which residues lead to specificity I Won't be discussing more of that this slide is crucial So I want to take you through it We made constructs with different numbers of domains So and then assay they're binding both in a biophysical assay basically Analytical ultra centrifuge and in cell assays and it's this combination of information that taught us something very different So if we have a construct of only three domains It's a monomer in solution. It doesn't form a dimer because you need all four and it doesn't affect cell aggregation If you have a construct of four domains they form a dimer, which is what we see in the crystal structure if we Transfect cells with four domain constructs the cells aggregate nicely So that shows you that the four domains that we see in the crystal structure are enough to aggregate cells And we do that with large larger constructs as well The critical observation is this when we cut out. Well, we move EC one so we have a construct with two to six it forms a dimer, but it doesn't aggregate cells and From this and other evidence we know that this forms a sys dimer So sys dimer on its own. We've killed the trans interaction. We've only left the sys interaction and See it doesn't aggregate cells it forms a dimer when we have full constructs in solution We already know what aggregate cells and we know it forms a tetramer So this series of experiments tells us that the recognition unit of a protocad heron is a sys dimer which then interacts homophilically with proteins on the other cell surface and There's two ways this sys dimer could interact could Could form a tetramer and solution it certainly this is this is what happens in solution But there's also a possibility of some sort of a zipper Which I'll show you in a second where it forms an extended assembly Which I'll get to in a second, but the basic notion then is that this is what happens This cartoon is what happens on cell surfaces. We know that the sys interaction I didn't tell you this is is promiscuous any protocad here and can interact with any protocad here and in sys So if you have different isoforms shown here in different colors, you're going to get a combination You're going to get some statistical distribution of dimers But the way to maximize interactions is see some of these dimers are heterophilic like this one here It's red and blue the sys is the same, but they're different trans so this this If you start with red to reds it can bind to a red and a blue which can find another blue and in this way You can get maximum number of interactions between cells and This is at least consistent with the notions. I've been talking about a system trans interactions and lattice assembly So what's nice is that this gives us a mechanism for self-avoidance. So let's pretend That we start with I'm just going to show you how lattice might grow red and blue Red blue green If all the isoforms on both cells are the same any protein can always find a partner of another color Because it's going to be there some in some dimer and therefore these aggregates can grow at will But now let's say we have a single mismatch When you start trying to grow chain you quickly get to a situation where this green has no partner on the other cell and the Assembly stops sort of a chain termination mechanism So if we if you actually calculate which we did here The size of it the average size of an assembly. This is a Monte Carlo simulation Let's say there are a thousand copies per cell shown here in black The assembly if if there are no this is the number of mismatches if there are no mismatches The assembly comes up to be this basically related to the size to the number of proteins if there's even a single mismatch then the size of the assembly goes down to What is this 50 or so and then the decrease is very sharply after that so The argument we're making the suggestion we're making is that this effective poisoning of this assembly is The recognition mechanisms how cells tell Self from non-self it it translates into the size of the assembly they form which we would then Suggest effects what happens in the cytoplasm That the model is consistent with the with all the data at least it explains this Remarkable ability of 58 proteins to do the job of 19,000 proteins As shown here these scams have 19,000 they basically function by Having this great molecular diversity We think at least we're suggesting that protocol adherence function with an interference mechanism Which allows them to generate the even greater diversity by the way than protocol adherence Do we're obviously in the process of testing these ideas and there are number of ways to do it But the critical connection then between binding and repulsion remember ultimately binding leads to repulsion is Is is we're claiming in the size of the assembly? We'll see if that's true or not This is just to sort of want to summarize that From point of view of protein design it's sort of remarkable that these proteins there are other Conforms so many different interfaces and it's just one of the wonders of nature that this that this can happen I'm not going to be discussing them So let me just finish by mentioning sort of key people I say we have a a large lab and lots of Wonderful collaborators. We have people doing solution biophysics Computer simulations the protocad here and work the people in red worked on the protocad here and wrote them Rubenstein Carrie Goodman did the crystallography large protein production group the Simulations were done together without being no one been shown in the Hebrew University in Jerusalem and the protocad here And work is done in collaboration with the money out of slab and I briefly showed you some Kidney stains from Rosemary Symponia's lab. So thank you very much for your attention Mechanism So it may well be we don't yet know how many isoforms are expressed we know it's on the order of 10 to in Amicron cells is I think it's 15, but we don't really know the actual number. There may be more isoforms in Invert in invertebrates. We're not sure yet Well, so you have this is where there's there's a random selection of different proteins that takes place at the RNA level or at the DNA level so That's the mechanism for choosing these different proteins is is these different isoforms is unclear, but The point is in the same cell there's no problem because they're all the same They're dendrites coming out from the same cell body, but the dendrites come from the same cell. They have the same Protein, so they'll always Stick to each other and then repel each other so the trick for any other cell is Not to stick and what we're suggesting is that even a single mistake is Enough to make sure you don't stick and that happens randomly. It's just the probability that the same cells will be Produced the same proteins will produce in different cells is very small so Yes The monomer we would argue that the monomer isn't there there may be small clusters formed So but but once you're starting us, maybe I don't understand your question Implicit implicit in this is that diffusion on a surface is fast enough that things will happen out at the proper time There's there's so many many assumptions in this Model, but implicit is yes is that is that if something is there it will find its Place in a short enough time to let the chain to grow Yeah, I mean there's a there's a there's a lot of work to be done I say it's consistent with everything we know about these proteins, but maybe there's something we haven't thought of that's always possible Well, you did notice I had a simulation of the molecules moving yes, so the the cat herons The classical cat herons all the cat herons have three calcium's between each domain that make them semi-rigid Nevertheless, they're fairly flexible. So if I do if I in that simulation I showed you If I fixed the membrane here and just asked what's the region that the External domain moves it's about 2030 angstroms so but you have five flexible regions So they're quite flexible the protocol here is that bind this way are far less flexible because they have a much larger interface So they're far less flexible. So they are You could so one question is does what effect does flexibility have on their properties? We actually think that Flexibility interdomain flexibility affects binding affinities. We've shown that There are alternate Conformations of binding that we think there's an entropic driving force for some of these proteins, but I don't I Think I think beyond affecting binding affinities. I'm not sure what else but again, we're in the earliest stages Unless unless as I say unless this size Unless the size of the cluster is what's responsible for signaling in Which case no it wouldn't make any difference in the case of the classical cadherans what one of the experiments we're Doing now is I said that you need cis and trans to get this assembly So what we're doing now is we're making the trans interaction much stronger in Which case perhaps we can assemble as many proteins in the interface as we do with weaker Trans and stronger cis and the question we're asking is is the order important So if we if we make strong trans interactions, there'll be no order, but there'll be enough the same number of Proteins, so that's the kind of game. We're playing so we we don't really know the answer to that yet But yeah, I mean that's the kind of experiment thinking


Okay good evening all. I hope you're all doing well. It is Friday evening but live at five o'clock and I know that many of you this evening will be looking forward to some football and also to the weekend but before you do I need to make an appeal to you. First things first this evening I had well yesterday actually I had arranged to speak to someone who had been subjected to anti-white violence in Batley and just as we were sitting down to record this time yesterday I was told that the person in question had pulled out pulled out for fear of reprisal and we have to understand this and I have nothing but sympathy for this person. You have to understand we all have to understand what it is like for people living in these enclaves. These areas of the country absolutely dominated by Muslim gangs, Muslim politicians and police who protect both and keep both from and oppress that keeps both from real scrutiny or opposition. Violent gangs a police force that looks the other way and politicians that side with openly side with the Muslim contingent in this. The police frankly have openly sided with Muslim gangs as well so people are frightened and they have no support so for example if we had gone ahead with this interview yesterday and there were threats made against the person in question and his family there would be no support. The police would not take it seriously certainly local government wouldn't take it seriously so such people are on their own and this is the core of the problem. This is why we're going to badly in the first place. So because we can't do that interview I want to share with you just a few news stories that I've picked up from that area. Now these are and I know I know I know I've been talking about this book a lot I promise it's the last time but there's just so much in it that is so relevant that I need to share as much of it as possible with you. This is from Danny Lockwood's book and these are news stories just in one year. When I read these to you I'll just pick down a couple I'm not going to keep you for very long this evening just a few that I have picked out. So we have police report that street robberies have risen by 55% over three years and car crime by 20% in 12 months. Admitting this increase is down to drug addicts. Now who controls the drug trade the heroin trade in that area is Pakistani gangs. This is the ripple effect. The police said there's no reason for alarm. A month later 10-year-old Tiffany is sexually assaulted in her school playground when she arrives at 8 30 a.m. An Asian man tried to kiss Tiffany and rub himself against her no arrests were made. Diane Bentham of Raising Thorpe demands action after three this is a month or so later after three Asian boys taunted her 12-year-old daughter Laura and her friends Sarah before stoning them. Laura lost a tooth and had three others broken leaving her needing dentures at the age of 12 but despite police assuring the family they were treating it as a race hate crime and the girls being able to identify their attackers two weeks later no charges. Same month the Labour-led members of the Dewsbury Area Committee vote to donate 1,000 pounds to Ravenstorpe Community Centre in order for them to celebrate Pakistan Independence Day. The grant was part of 30,000 public budget that Labour councillors held sway over among their plans was a grant of 8,776 pound to the Bismillah project a social club for Asian men and 8,800 for a good health program for Asian women. Going on a couple of months later Safeway Supermarket bosses apologise after an in-store promotion for halal food goes wrong they refused to allow white customers to participate. Going on 13-year-old Sinead and her friend 15-year-old Jordan were walking home at 10pm when an Asian man about 35 pulled up and asked if they wanted a taxi he drove them up a lane and locked the doors before pulling out a knife he slashed Sinead's jeans from ankle to thigh she fought back and suffered cuts and scratches to her face before he pushed her out of the car and made off Jordan managed to get the window down and climb out screaming for help while Sinead fought off the man no arrests were made again and again and again and again this is what you see vicious violent crimes against the white population by Muslims no arrests are made at the end of almost every story no arrests were made in between all of this more money for this Muslim community centre more money for that Muslim community centre and while all of this is going on education schemes are set up to teach white people not to be racists to teach white people to be tolerant and accepting and multicultural no such lessons no money is spent on teaching tolerance or acceptance the other way round this is standard standard across the country and this is why people are so afraid the police have taken sides in this local governments have taken sides in this the national government after national government has taken sides in this and they're not ashamed of that there's no apology it's open it's openly taking sides no matter how much violence is inflicted no matter how much crime is committed police do nothing and governments pour more and more money into Muslim community projects and in teaching white people to be more tolerant this is science fiction horror movie levels of injustices is unbelievable it is unbelievable and it's why people are afraid to speak up because nobody's on their side they could call the police the police wouldn't come it's standard it's standard in areas like this and badly is such an area this is happening to people in that town the chap I was to speak to yesterday told me we are in trouble here in badly we are in trouble that's that's those are the words that he used and they are in trouble there's someone at some point someone is going to have to stand up to this someone and if it's not us then who will it be and if it's not now then when will it be when are we going to do this exactly when are we going to stand up and demand justice because that's what this is about it's about justice this is disgraceful reality this is a disgraceful disgraceful reality this is race hate racial violence and it's complicity complicity by the police and by local government in this area so my appeal to you this evening is this the press is not going to print this this is from an alternative press this is Lockwood himself there's a newspaper he started just because no one else would cover these issues the mainstream press is not going to give us a voice we know this we have to accept it and work around it the press in in badly is not going to give for Britain an article to explain ourselves to explain why we're there what we're doing we'll be dismissed the usual way racist hate Islamophobia the usual way all we can do is as many of us as possible get there and get our leaflets through letterboxes now to pool our resources for this weekend we are going out on Sunday rather than stretch people because we are we need more volunteers we're going to get as many of us as possible together on Sunday and do as much as we possibly can of getting our leaflet through doors I told you on my livestream the other day that we have had our leaflet one of our second leaflet accepted now by Royal Mail all of the shipping and arrangements for that have been completed and thank you to Ed and to Mike and to everyone else involved in doing that that is now underway so when people receive their Royal Mail leaflet for this by-election they will receive this one I will show it to you once more this is the front and this is the back this will go out via Royal Mail this weekend we will be putting out a separate leaflet I'll show you that one as well please do join us please get there if you can there is strength in numbers look you must be as disgusted by this as I am you must be this is wrong it is wrong people frightened frightened to speak about the injustices they are subjected to every single day is wrong this is a moral crisis it really is and it's a moral emergency and it's a it's a requirement it's up to us it is incumbent upon us to stand up to this and to give some kind of voice to the people in Batley who are too afraid to speak up because they have no one on their side it is incumbent upon us and we can we can in numbers get out there and make our presence felt but if we don't go out then it'll just continue and we'll have more and more and more of this let's go with each other watching each other's backs that support each other and get out there and fight this campaign for justice that is what it is this is a moral campaign it's a fight for what is right it is a fight for people who have been terribly wronged and who are continuously terribly wronged and if we're in this for anything at all surely this is it surely this is why we are all signed up in the first place please come along on Sunday please come along on the 26th of January for britain.uk slash batley has a registration page for the 26th we will be posting more details so i've said to you before make your way to the leads area that morning the morning of the 26th or a plan to make your way to the leads area that morning and watch out Friday evening on telegram and various different groups to find out more detail of where we would be because obviously we can't be open about this it's sometimes hard to believe the injustices that are being committed under our noses and it's sometimes hard to find whatever it is you need to find to stand up and fight back but whatever it is you need to find find it because this is why we're here we're here to fight for the people who are suffering like this we're here to fight for our country for the truth for a police force that does its job for politicians who represent everyone and not just their own small closed ghettoized communities and we're here to fight the mainstream politicians like Labour and the Tories who have got us into this mess in the first place so enjoy your weekend enjoy the football but join us on sunday to give people like mark who i was going to speak to last night to give him a voice and to give a voice to everyone who has been subjected to the injustice and i've just given you the tiniest tiniest number of examples from one small area of the country this is nationwide and we all know this is nationwide we all know something else as well we're it we're the opposition it's up to us so i'll see you on sunday i'll see you on the 26th of june go to forbritain.uk slash batley it's urgent it's urgent and it couldn't be more important i will be there i will be there as determined if not more as i have ever been to fight back against this this is wrong this treatment of people is wrong let's show these mainstream politicians press and police that we will not take this forever we won't i'll see you on sunday thanks for joining me this evening i'm sorry the interview didn't happen i'm as disappointed as anyone else but i do understand i do sympathise and i do know why we are in this position see as many of you as i can on sunday take care until then thanks


This month will mark 20 years since the United States illegally invaded Iraq. Over a million Iraqis were killed while weapons manufacturers and oil companies got rich. Since then, we've seen the devastation of countless other wars. 37 million people displaced by the U.S. war on terror. The horrible 20-year war in Afghanistan that was all for naught. The U.S. backing of the Saudi war on Yemen. One of the worst humanitarian disasters in the 21st century. The constant, appalling U.S. airstrikes against civilians in Somalia and in Pakistan. The tens of thousands more bombs the United States has dropped on Syria in its continued occupation. Now we're involved in yet another devastating war. The war in Ukraine. Meanwhile, many U.S. officials are preparing for a war with China. War is an epidemic that is killing our people and our planet. For 20 years, CodePink has been fighting fiercely against it. Enough is enough. If you're as frustrated as we are that our government keeps investing in death and destruction instead of funding the people and planet's real needs, join CodePink and 200 other anti-war organizations on March 18th in Washington, D.C., to stop funding endless wars.


Welcome to Prague City University. My name is Selena. My name is Skaj. I moved from Mexico to Prague to study my Masters in Fine Arts. And I moved from the Netherlands to practice studying my Masters of Future design. PCU has four schools, School of Business, School of Media and IT, School of Education and School of Art and Design, and two intakes every February and September for most of our programs. The application to PCU was really straightforward. My admissions advisor was there to answer all of my questions and assist me every step of the way. The whole admission process can be completed online. When you are ready to apply, let the admissions team know and they will send you your application to complete. There you will upload all your documents which are a confirmation of completed education such as a high school diploma or a bachelor's degree, an English certificate such as IELTS, so you follow our duelingo, a letter of motivation, a portfolio if you're going to study an arts or design program and a CV and recommendation letters if you're going to study a Masters level. The final step was a final interview with the program leader which was a great opportunity to gain further insight into my program and to meet them before enrolling. It's been great going back into campus. PCU has made me feel safe by introducing multiple measures. Airport fires are in every classroom. Masks are worn through the entire campus and every room it's being disinfected regularly. Staff and students have to present proof of vaccination or of negative test before entering the campus. At reception you can also find masks or tests if you need them and also they have shared with us the measures that the Czech Republic have implemented in a very understandable way. At PCU we are truly an international community so wherever you are from you will feel at home with us.


Wroclaw! Hey everyone, what's going on and welcome back to another video? So we finally arrived here in Wroclaw. I think it's called the Wroclaw. I used to say Wroclaw, but I think that's not correct, which is on the west side of Poland. Our flight went pretty well. It's a bit rainy outside as you can see and the whole week is gonna be like that, but we are super excited to be here. I left most of my camera gear at home and just brought my go-to vlogging setup with me because in today's video I would like to show you how to vlog with an iPhone, particularly with the iPhone 11 Pro Max. It's much more travel-friendly than using something like a DSLR and you really don't need expensive camera gear to get started. While I take you with me on this trip, we're going to have a lot of fun. I'm gonna show you my go-to vlogging setup, the camera setting I use to capture high-quality video, and 10 tips to get started with vlogging. There will be a timestamp below if you want to skip or go back to a particular part of the video. So what are you waiting for? Let's discover Wroclaw, sorry. So before we get back to the vlogging video, let me show you my go-to camera setup for vlogging.  There it went. So this is the whole vlogging setup I used for my trip and starting from the top we have the Rode Video Micro with the dead cat or the windshield on it. I have it attached to the Ulanzi phone mount, which also holds my iPhone 11 Pro Max. The cable I'm using is the SC7. You have to additionally buy this to make it compatible and you have to have this cable. Otherwise, the phone won't detect the microphone. What you also have to additionally buy is this Lightning 2 3.5 millimeter headphone jack, which you then are able to plug in to your iPhone. Now I also have a case for the iPhone 11 Pro Max. It's called the Light Chaser Pro Filmmaking Kit. The reason why I bought this is because I can attach an ND filter to it. This way I can maintain that shutter speed of 1 over 50 to get that natural motion blur. Below I have a mini tripod from Manfrotto. This really helps getting stable footage. Now, the reason why I mounted it this way so that the camera is upside down is because so that I don't have the windshield in the frame. And what I also additionally got, this is called the I Expand Flash Drive from Sandisk. I brought this with me so that I can back up all of my footage in case my storage is full. So this is a really useful feature. I can just plug it in and transfer all of the footage onto this stick. So let's get back to the vlogging video. We're now heading to our next event, which is Axed Pro-E. So let's look at our camera settings. I use Filmic Pro to record all of my videos. Filmic Pro is a professional video application app that allows me to have full control over my camera. So the first thing I do when I open up Filmic Pro is I change my camera from wide angle to ultra wide. This gives me a super wide perspective and is perfect for vlogging since I want myself in the frame and not cut out. The resolution I'm using is 4K, which has more detail. And the encoding rate is Filmic Extreme, which captures more information and therefore gives you a better video quality. As for the frame rate, I'm using 25 frames per second. If I want to shoot b-roll, I switch over to 60 frames per second to get that slow motion effect. As for the ISO, I lock it at its lowest because that way I can avoid noise in my image. So my shutter speed is set double the frame rate, which is 1 over 50. I make sure to lock my shutter speed so that I can just adjust the ISO when I have to.  As for my picture profile, I use flat so that I have more options when color grading. But if you want to be quick and don't want to spend time on color grading, you can also use the dynamic picture profile. The audio I set to my external mic and I make sure that I adjust the levels so that it doesn't peak. All right, so these are my camera settings for getting the best out of my video. No drawing. Lucky I didn't bring one with me. You must keep the green path. There will be junction of the yellow one. Don't go yellow. So enjoy. Thank you. You've got my number. Yes. So 10 fast 1pm the latest time you should show up on the path. Super beautiful out here. Look at this lake. Look at the rocks and the forest. And I think we're the only one here out so far. So it's going to be a nice hiking tour for us. Protein brownies. So while we're on this trip, I would like to give you 10 tips on how to start vlogging with your iPhone. And the first tip is to fully charge your phone, which is quite simple and obvious. And what I like to do is bring my power bank with me, which I carry in this small bag, in case I need to recharge my phone. And that has helped me a lot, even though I'm using the iPhone 11 Pro Max, which lasts me almost the whole day to film, you never really know. So keep that in mind. So the next tip I have for you is to have enough storage in your phone. So make sure to delete unnecessary pictures or videos that you don't need to free up space. But what you also can do is record in a smaller file size. And another thing you could do is use like a stick that you can plug into your phone and then transfer the files to that stick and delete it off your phone. Hey, you smile. Tip number three is to use a tripod or a gimbal. Having this three point contact will allow you to have less shake your footage. So tip number four is to think about lighting. So when it's bright outside, I like using an ND filter to reduce the light hitting the sensor. And this way I can maintain my shutter speed of one over 50 of a second to get that natural motion blur. Also what I do is lock my exposure before I start recording so that I avoid exposure changes during filming. Tip number five is to use a microphone. Now the microphones on the iPhones capture all the ambient noise and you don't want that. You want to use a shotgun mic, which is more directional to have your voice be more present in the video. So I'm currently on top of a church and it's quite windy outside. And the microphone I'm using is the Rode Video Micro, which I plugged into my iPhone. I have a windshield mounted on it and the audio should be pretty good. And this is how it sounds like using just the built-in microphone on the iPhone 11 Pro Max. You'll probably hear more wind noise and the audio. It's probably not as good as using an external microphone. Now filming a vlog is one thing. The other thing would be editing it together to create your story. So I suggest you learn a video editing application. I personally like to use InShot. It's a free app that you can download for iOS or Android. And it's pretty advanced for what you can do. And if you're on the go, that's perfect because you can just edit it on your phone. Now for more complex videos, I use my iMac Pro at home. But for simple edits, like a vlog or so, this could be really useful. I actually have a dedicated video on that, which is called How to Film and Edit with your iPhone, which I will link up here somewhere. So what advice do you have for people that want to start vlogging? Just take it easy, no stress, and talk to the camera like it's a friend. That's actually a pretty good advice. So tip number seven is to film B-roll. This really enhances your story and just makes it more engaging. What I like to do is film in 4k 60 frames per second to get that nice slow motion I can put on top of my A-roll. So tip number nine is also to add time lapses to your video. This is also a great way for B-roll and shows the passage of time. And if you have a tripod, you can just set it up on any flat surface and start recording. On the iPhone, you can just set it to time lapse mode and just press record and it will do the work for you. Oh God, I'm dying. Damn, that was tight. So my last tip is just start. At first it can be intimidating, but the more you do it, the comfortable you get with it. And when I do this, nobody even cares. They look at me, but life goes on. So just do it and think about what you want to say to the camera or to your audience and really just be yourself. And if you mess up, don't worry, you can edit things out. I mess up all the time. And if you struggle with vlogging, maybe start in places where there's nobody around where you can just talk to the camera and be open. And that helped me a lot at first. So yeah, these were my 10 tips. I'm going to head back now to our tour guide. It's probably waiting for us. We have still a long way to go. We're actually going to shoot, not with my camera, but with real weapons. Really easy weapons. It's only noisy. And tell me how was it? Easy or not? Is that easy? Not bad, yeah? Good reason to wear the brown trousers. Try to make hot, okay? I got bad news, but you'll miss everything. No way, seriously?   Oh, my god. You don't need or you don't want it. No, no, I won't. Reload it. No. Reload it. Ach so schreise. It's enough. All right, guys, that was it for today's tutorial. Overall, despite the bad weather, our stay in Vroslav was an awesome experience. One of the great things about vlogging is that you can share your story through video with the world and rewatch it to bring up old memories. I hope this video gave you an idea on how to start vlogging using something simple as an iPhone and a couple of accessories to get the most out of your videos. So let me know what you think of this vlogging setup. Leave a like if you enjoyed this and subscribe to his channel. We're gonna enjoy the rest of the day and I will see you in the next video, guys. Peace. Bye.