Wow, what's going on this engraving here with another video and in this video sorry that I'm breathing heavy But I was just walking fast because This is crazy. That's I'm walking fast like I'm getting ready to go walk out and sign a contract for that much money But anyway team keep it clean. What's going on this engraving? The contract's so big it made me forget my name Let's try again YouTube team keep it clean. What's going on this engraving here with another video and in this video? We are here to talk about them San Francisco 49 us They gave Jimmy Garoppolo. What did he go like 5 and 0 or 6 and 0 for him? I forgot, but they gave Jimmy Garoppolo The biggest deal in NFL history The biggest deal in NFL history five-year deal 137.5 mil 137.5 million dollars to Jimmy Garoppolo Who they traded from the the New England Patriots last year 137.5 mil The biggest deal and not not not even the not even the biggest deal right now for quarterback But just the biggest deal in NFL history period period period Jimmy Garoppolo Now really quick before we get into this video. Let me introduce myself me. I'm engraving this This is my YouTube channel engraving visit I'm a Ravens fan and even though I'm a Ravens fan on this YouTube channel We talk Ravens, but then we talk football period too So if you're Ravens fan or if you like Jimmy Garoppolo on the 49ers or if you like the Patriots If you upset that the Patriots got rid of Jimmy G you can subscribe if you like that The Patriots got rid of Jimmy G you can subscribe or if you anybody in between and you don't even care You can still subscribe turn your notifications on and once you do that make sure you leave a like on the video I'll click that thumbs up button, but anyway Jimmy Garoppolo highest paid player in the NFL and NFL history now and everybody that's coming through I appreciate y'all man really quick when I first when I first saw this come across my phone. I Was thinking Like you really just hate Jimmy Garoppolo all this money. I know he came out there last year and killed it for you I know he did his thing last last year for you and he looked good doing it But you really just paid him all that money to become not not just a high-paid quarterback Not just starting salary, but the highest paid ever ever ever That was my initial thoughts, but then when I think when I thought about it I say wait a minute. Well 49ers they got a lot of cap room one and Jimmy G from what he showed them last year I guess that was enough for them and he obviously showed them some stuff when he played those games for the Patriots too But what they saw out of him last year led them to believe that hey We know that Bill Belichick gets rid of a lot of guys and a lot of the guys that come from Bill Belichick maybe system quarterbacks or whatever, but Jimmy G obviously proved that our system is working for him too and the fact that Jimmy G did what he did with The lack of weapons that he had there in San Francisco His receivers sucked His receivers were almost on Raven's level. Who did he have Pierre Garcon? He's better than all the Ravens receivers though. I think good win I don't even know who else But the fact that Jimmy G did it with the lack of weapons shows a lot and it obviously showed the 49ers like Hey, this guy may just be real deal and again when I thought about it some more I was like, okay, you know what? This guy came out last year those last five six games and he didn't do anything But when he also and when some people may think about it They may be they may be like, oh, but he didn't really play anybody this dude played the starting Jaguars defense And hung like 40 like 40 something points on them starting Jaguars defense Jaguars was one of the best They were one of the best Defenses in the NFL Them and the Vikings and this dude put like 40 some points up on the Jaguars Jaguars And their defense is real their defense is fast their defense is strong their defense is real deal And he put up 40 some points on him So Jimmy G showed them last year that he knows how to win and knows how to win against real teams, too But like I said when I first heard about the damn like man, you will really give Jimmy G all that money but then Yeah, that's the same thing that I thought to dream on I thought it was kind of early at first two But then when I thought about I say, you know what the one they got the cap room to If I love how aggressive they are with Jimmy Garoppolo I love I love how aggressive they are because it shows that they believe that Jimmy Garoppolo is their guy and I love Shout out to rhodo rhodo said garoppolo is the opposite of me beautiful and rich But oh, yeah, now you're excited about d-way going back to the heat Um, who knows how much you got left in them legs though. But anyway, um, it shows I love how aggressive they were to get their guy. They got their guy They got that guy and they don't want any drama with them. They don't want him to be playing on a rookie deal They don't want any of that. They got that guy and they paid that guy So their guy is their guy and now with 49ers I know they they wanted the teams that got some of the most cap room in the nfl. So now You take him and again, he was with a team last year that really didn't have much Of a team at all um But he was winning with them Uh, Nate carlin just brought up some numbers. He said seven touchdowns and five interceptions Smells like Brock Oswiler. Hey, Nate. We'll see we'll see But the the number that means the most out of those how many touchdowns he threw versus interceptions The number that wins that that means the most out of those are wins Are wins you can throw 50,000 Touchdowns and throw 60,000 interceptions but if you're winning Doesn't even matter if you can find a way to win you can lead your team to winning None of that stuff means anything Doesn't mean anything means absolutely nothing Um, has he lost as a starter? No, they said the last time he lost was like was it in college or high school? I forgot which one it was um But he just keeps on winning one with the Patriots He won with san francisco Um, he he's been doing his thing for a while. Um, so hey shout out to him anyway man jimmy g jimmy g I didn't think anything like this was happening. Um And again, not even not only the highest paid player now Not only the highest paid player Uh, at not only the highest played paid quarterback right now, but the highest The highest paid player in nfl history nfl history nfl history It's crazy George said he only gonna be the highest paid for a couple of weeks We'll see either way whether he is the highest paid for a couple of weeks or if it's throughout the entire season doesn't even matter because that dude He cashed in man He cashed in and cashed out Hmm Jimmy g anyway um wow That is crazy. Anyway, I appreciate y'all. Thank you for tuning in um Nate said kirk is getting more no question We'll see we'll see Anyway, appreciate y'all make sure you subscribe to the youtube channel if you haven't already Turn your notifications on so when we go live later on you will be here And you don't miss a video and click the thumbs up button Leave a like on the video and you can follow on twitter instagram and snapchat all three are engraving vids I appreciate y'all. I love y'all. I see you later on and i'm out


The MAGN platform is a novel approach to delivering therapeutic agents to cancerous tumors. It involves engineering macrophage cells to carry biologically responsive nanospunges, which are loaded with drugs and then delivered to the tumor site. Once there, the nanospunges release the drugs when exposed to the acidic environment found in tumors. This allows for more effective treatment of advanced metastatic cancers than traditional methods. This article was authored by Xueliao, Guidong Gong, Meng Yuan Dai, and others.


Salt Support Tactics 3 is a non-combatant evacuation operation, a NEO exercise put on by Marine Aviation Weapons and Tactics Squadron 1, based out of Yuma, Arizona. It's executed on training day 40 of their twice a year exercise, and it tests the perspective of weapons and tactics instructors, the PWTI's ability to conduct a non-combatant evacuation operation of American citizens while concurrently conducting a foreign humanitarian assistance operation to remove refugees in a permissive or uncertain urban environment. The ground teams involved in this operation were primarily the security force that were dispatched from the aircraft to secure the landing zones. The fourth of the major evolutions that we do at Weapons and Tactics Instructor course, and what it consists of, is a dual-site night aerosol into two separate objectives. So one objective being here in range 210 with a infantry company to clear the range of 210, and then the other objective is over in the Delta T, and that is another company establishing a blocking position that will then sustain notionally for upwards of 48 hours. We have all elements of Marine Corps aviation, we have all elements of the Marine Air Command and Control Squadron, and then we have the GCE that's all integrated, so we have every element of the MAG TAF integrating into this single evolution.


How a beginner can be successful at email marketing, okay? So you've heard about this email marketing thing and you either want to get started or you want to get better at it This is the video for you. I'm going to walk you through all the steps What you need to do the resources you need to have so that you can make money at email marketing now I got to tell you in the beginning. I did not collect email addresses. I didn't realize the importance of it now Fast-forward to today and I have a lot of email addresses and I can send out an email and make money within minutes So I want you to be able to do the same thing before we get going Don't forget to subscribe hit that subscribe button down there turn it from red to gray. Don't forget to ring the bell Turn all bell notifications to your notified each and every single time we go live or if I upload a video So in this video, I want to walk you through all these steps now I'm going to assume That you need help in this area or you wouldn't be watching this video So I can guarantee you that when we're done You are going to have the tools you need the skills you need and exactly what to do to get better at email marketing Now one of the oldest forms of online communication is email and even though it's one of the oldest forms It's still one of the most successful keep a mind that out of seven billion people in the world Four billion people actually use email. So that's a pretty good chunk of the population of the world This is something you have to have in your arsenal on top of that It said that thirty eight dollars get returned in sales from one dollar of email marketing So that's a pretty good return. I don't think most social media ads could actually do that So that's why it's so important that you have this in your arsenal. I'm not saying don't do the other things I'm saying you have to do this one thing if you do anything Now take a look at this chart right here and it shows the growth of Email users worldwide and the cool thing about Email is that you know, you don't have to worry about some social algorithm Changing and wiping out all of your traffic because once you own those email addresses You own the traffic and you can talk to it on your own terms now I've heard people say well email marketing doesn't work J. R. I've tried it. It just doesn't work No, that's not the truth The truth is you aren't doing something right and that's what we want to correct in this video is to show you how to do it Right because obviously it works if there's people out there making money If you're not making money, then you're just doing something wrong and that can be corrected Now keep in mind when you send an email, especially business emails, you are an interruption Okay, you're interrupting their inbox of their email. So it's got to be good and it's got to be useful So let's take step number one is you got to have an email list It's not just accumulating names and sending out emails We've got to know why they opted in how they opted in what their interests are in other words We got to put tags on these people so that we can segregate our audience now The best way to do that that I found is through Cartra now Cartra is fairly due to the market maybe four or five years old and when I started out I use constant contact. I used infusion soft, which is now keep I use mail champ I used a web or I used them all and I got to tell you Cartra is the best one out there now I will put a link below in the description where you can actually watch a video on Cartra I highly suggest you watching this video. You don't have to opt in you have to give your email or anything to watch The video but at the end of the video you can actually get Cartra for a trial of only one dollar So you definitely are going to want to check that out Now the next thing I'll tell you is once you've built this list you have to define what your goals are. What's the point? What's the point? Okay, are you trying to drive them to a website? Are you trying to build your list even larger? Are you trying to get them to opt in are you trying to get them to attend a webinar? Are you trying to get them to buy a product or service? Make sure those goals are Extremely clear when you send out that email so that you can judge if it was successful or not I've had people say well You know I sent out an email and it was not successful and I say what do you mean it wasn't successful? They say well a lot of people open the email and a lot of people clicked on it, but nobody bought anything Well, that's not the fault of the email That's the fault of wherever you sent them to the page the landing page the website or whatever That's where the breakdown is so you've got to understand all the different components of email to see if it's really working or not Now before you get started an email marketing campaigns You've got to understand the rules. There are rules out there that can spam act the GDPR All these things are super important You cannot just randomly send an email to somebody because you have their email address They have to have opted in to get on your list or you can get in a lot of trouble So make sure that you're getting people to opt in properly and that once again That's where a cartridge is going to help you because they filter all that they show you what to do They give people opportunity to have a Double opt-in on their email. So get cartridge. It's going to help you out a lot Okay, so let's look at a few opt-ins right now So you can kind of see what people are doing and how they're getting opt-ins Now the first one we're going to look at here is tionic tionic And their subject line is getting low on wellness and then it has a question mark on it And then it shows their product here And it says friends don't let Friends run out of tionic order a refill. So this is how they're marketing here, which is pretty cool And what do they get right in this? They got a great subject line They leverage the color psychology the bright warm colors trigger feelings of health and happiness It's well time the email is targeted at people who have purchased the product and is sent when the customer supply is about to run out So that's a really good one Let's take a look at blue host here real quick. Okay, that's the blue host one And their subject line is blue host new website builder makes building simple So they're hitting on a big high point here, which is Making websites is difficult And then they give you all the reasons why you should use blue host and you can click the button that says get started So this sells the benefits not the features focusing on the benefits makes the copy more compelling They have a really clean copy design the simple design makes the email Aesthetically pleasing and easy to read and it has excellent targeting blue host knows who their audience is mainly made up of small businesses owners Without technical expertise. So they want to make it look easy as possible Now when you're writing emails, it's super important that you understand all the different components of it Your first component is going to be the subject line Your subject line cannot give up everything that's in the email because if your subject line says, you know 20% off sweaters for men today Then they know everything that's in the email. There's no point in clicking on that subject line But if it says huge sale on most popular products Click to reveal then they've got to click to reveal because a subject line is just there to get them to click That's it. That's all a subject line is supposed to get them to click And when they actually open the email itself It should give them some information That's going to make them want to click again Because you want them to go from that email to your landing page or to your webinar or to your opt-in page So understand you've got two different components here subject line Shouldn't give up all the information should not do that. Okay It should just create enough curiosity where they're click and they're going to actually read the email The email should give them enough information to where they're going to want to click and move forward Those are the two main components of an email and if you get those right, you're going to do great And if you don't you won't but the other thing I would add to this right now is make this easy to read Okay, I think a lot of times people think they need to write in some type of business jargon And they don't the average american if you're marketing in america, which I know some of you are that are watching this I know I have a huge audience in india too, and I appreciate you guys out there. I really do Thank you so much for watching. But if you're writing to america, they actually read on about a fifth grade level That's what the average american reads on. Yes. I know they go through high school and college and all that but they Currently and comfortably read on a fifth grade level So if you're writing on a fifth grade level, you're good And I would tell you you want to leverage storytelling storytelling is really good I use that a lot in a lot of my ad copy, but what's the story behind your business? What's the story behind you? What's the story about what you're trying to accomplish? If you interweave that into your emails, you're going to have a lot more people reading your emails and clicking through Also, you want to use the psychology in your emails psychology would be fear of missing out a fomo Okay, fear of missing out could be that maybe your offer ends in 72 hours Or maybe you have limited inventory Or maybe you're just offering this to the first 20 people to click or 50 or 100 Make sure you have some type of fear of missing out in your email copy Now I like to offer bonuses. So, you know, we own a survival food company survival cave food You can see it right there and when somebody buys. Let's say a case of our meat. Maybe we offer them a free 10 steps to protecting your home or seven steps to survive in the wilderness And it's valued at $29 or $59 or whatever it is, but we give that extra bonus And we will put in our emails get our free $29 report when you click here when they click there when they buy the product They're going to get that free report or maybe they can just opt in and get the report for free and we can keep emailing them Now when they first opt into your email list, don't automatically try to sell them something You want to welcome them to your list. You want to let them know what your list is all about Here's an example here from Spotify. It says welcome to the band. You signed up for regular doses of Spotify designed straight to your inbox We're so pleased to have you on board. Please know that you can unsubscribe at any time by clicking unsubscribe From this list. It's right here in the footer So right up front, they're telling them what they're going to get when they're going to get it And if you follow this practice, people will appreciate the stuff you do send to them because they've been told up front What they're going to get Spotify says, hey, we're going to be emailing you on a regular basis So they let them know that if you don't let them know these things, you're going to have people get mad You're going to have people report you with spam and it'll just be a big mess Now to really email properly you need email automation. What is email automation? Well, I've mentioned cartra a couple times here and once again You can get cartra by looking in the description of this video But cartra will schedule emails. So let's say somebody gets on your email list And they opt in to get a free report Cartra will send out another email to them thanking them and welcoming them and then maybe three days later It'll send another email with some content another three days later another email with some content And then three days after that maybe it'll try to sell them something and what it does is it automatically sends out all these emails As people get added to your list So you could have some people getting the welcome email on a monday another person getting on a sunday Another person getting it on a wednesday because of when they entered your ecosystem And this can all be done automatically through cartra. So I highly recommend if you're going to do email sequences If you're going to try to build your email marketing, which that's why you watch this video You're going to want to get cartra once again There's a link in the description where you can watch a video You can see all the things it does and it doesn't just do email marketing It does you know all kinds of product funnels. It does courses. It does Memberships it does all kinds of stuff for less than what some of these email providers are charging And on top of that at the time of this recording They don't charge you more based on how many emails you send out and that's huge Especially if your list starts to grow guys. I want to thank you so much for watching this video I hope you got some useful information out of it. I hope you understand what email marketing is all about now And if you have some more questions, which you very well may it's hard to get across Tons of information in a short video like this put your questions below I'll be happy to answer them one on one If you just want to say hello do that too put it in the comment section below guys Thank you so much for watching this video if you haven't subscribed yet Please do so hit that subscribe button turn it from red to gray and Turn on all bell notifications so that you are notified each and every time I go live Thank you so much for watching this video and I'll see you in the next one Hey, thanks for watching my video. Don't forget to subscribe to my channel and click that little bell right there So you can be notified every time I do a new video Also, click on one of those videos there. Keep watching on my channel


Hi everyone, Basel Chapman for the Tiger Financial News Network at 11 o'clock market update and we're looking at the Dow up 580,000 to 35,000, 880,000. That was a fantastic candle as it accelerated yesterday, it did pull back just a little bit from the high, but following through with a gap like this, because it is actually an island reversal potential here that is telling us something that is really very important and it's saying that the to overcome all the negativity of the past to reach just like that in a couple of sessions says that if I would normally expect some kind of a dogey small candle like a plus sign tomorrow and then a pullback the following day or tomorrow there's a pullback as long as the 35,000 805 as long as the 35,500 to 35,400 area holes of support in the short-term that's very good action I am kind of impressed I must say the S&P at this particular point is up also sharply it's up a hundred points at 46.91 all-time high 47.43 wow it's almost there this is a really good new unless it's just a sudden bad news thing who knows what it could be anything but whatever it is if the market doesn't react to news negatively but dismisses news and moves up or cheats every bit of news is positive I will not fight that that means of the market the buoyancy is there it's wanting to move up and even the QQQ is now up 2.8% a break in the resistance is at 397 no it's lagging definitely lagging that's a rotation correction we've been calling for and that's unfolded until December 1st and we're looking at the IWM store very very it's strong today but store week gold is really struggling it's up $5 a 1784 it needs to get up into the 1800 and 8 area or higher to be able to say I've changed course at this point is just sideways if you're looking at the TLT I didn't go through that very much during the show I'm calling this an alternate count with a 152.99 higher three days ago I'm watching this closer because you know the rectangle formation you come back in so let's just see if yields are going to go higher or lower right now they are going just a fraction higher and Crudo has had a very nice move to the upside it's up 3.22 it's 72.71 so far the VIX index is my key and I spoke about this yesterday during the show my target technicians I said if this starts to pull back sharply that's the sign we'd be looking for because look at this every time it's hit the 28th or higher level for the past eight months or so it's pulled back the following week very sharp have a wonderful rest of the day say to you for Larry and all the rest of the show I'll be back with Tom a little later on should be great program yet this is a great day to do here for them back then have a great day


Personal question, Tammy says, why aren't there more men like you in the world? Tammy says, why aren't there more men like you in this world? Folks, my backstory goes like this. I was raised by my mom and dad from a foreign country. Their teaching was basically this. After high school, go to college. After college, get a good job. After college, or get a good job, meet a good woman. After meeting a woman, get married, buy a house, start a family. That was my blueprint, okay? I wasn't prepared to how to be in a relationship, and I operated from a very selfish perspective for a very long time because I had childhood wounds and traumas.  Imagine those people that had horrific parents.   And so when my world imploded where I got divorced, lost my quarter million dollar-year job, I got wiped out in the market crash, I got completely humbled, and in that humbling moment, I almost wanted to commit suicide. I went to bed wishing I didn't wake up. I went to bed wishing I didn't wake up. And interestingly enough, my drug of choice, my drug of choice was online dating. I mean, I was addicted to online dating. I was addicted to talking to women incessantly, listening to them share their stories. And then what happened was along the way, you know, I had a Tony Robbins CD, I started to read Abraham Hicks, I started to read Wayne Dyer, and I realized after one year of dating, the common denominator was me. I was the problem.  I kept thinking it was the women, it was the women, it was the women.  And that's when I began the beginning of my journey, roughly about 2010. And then in 2011, I met a really fantastic woman who happened to be a therapist, and she helped reparent me on some level. Our relationship helped reparent me. And then I did a deep dive with the Hoffman process. I did a deep dive into insight seminars. I read the book, The Course in Miracles. This took a lot of work. I was in the tunnel, as Allison Armstrong talks about, for over 15 years. So I've done heroic work, especially in the last four years after Connor passed away. And it inspired me to write my book, What the Heck Is Self Love Anyway? What the Heck Is Self Love Anyway? It's a journey of personal development, self-help, and spiritual work. It's actually a bit of my story. And I invite everybody to do the inner work so they can find a level of inner peace. Inner peace. Just, you know, look it, I'm not in the fully committed relationship, and that's okay.  And that's my invitation for everyone. So how did I get there? It took a lot of humbling events.  And most people, when they have humbling events, they go down the rabbit hole and they never get, they come out.  I was a fighter inside of me. That's why when some women say I'm feminine, I'm like, fuck, I started my own business, I make, you know, I make hundreds and hundreds of thousands of dollars a year. I'm a fighter. I lean into the most bravest thing anybody can do is to be vulnerable, to be authentic and transparent. And yet, sadly, that's called feminine traits for men.  Anyway, that's my judgment over that. Coming back to your question, why aren't there more? Because sadly, people use drugs and alcohol to suppress their pain instead of doing the inner work. And this is true for men and for you women as well. You're just as bad at this as men. Men and women are equally bad at this. So my invitation, do the work, find that inner peace. And you might be surprised on what comes around the corner. So I want to thank you so much for that question. Lynn says, sounds like men are more feminine than women.  Men, okay. By the way, I've been characterized as a feminine because I have a capacity to communicate my feelings in an emotionally healthy way. Now, to me, that just means I have a good balance, not of my masculine and feminine energy that men are more feminine. It's just that I have a capacity to navigate life from an emotionally secure place. This comes back to my book.   So to suggest men are more feminine. Well, then I can make the argument that women are way more masculine than ever before. I mean, they're riddled with masculinity. And all this does is create divide amongst men and women. And that's why I'm not a big proponent of labeling men or women this way. Because you can complain about men being feminine and we men could complain about you as being masculine. And all that do is complaining for unhealthy behavior, most likely in someone's life. And rather than criticize them, how can we uplift people? That's my invitation rather than judging and criticizing. What can we do going forward to uplift people? That's my invitation for all of you. Or at least that's my invitation for myself. So Lynn, thank you for sharing that allowed me to chime in and point out how I feel about it.


Do you know we have the maximum species diversity near the equator and as we move further from the equator either towards north or towards south, this diversity decreases. Why is that? Let's discuss all about it in this video. And we will begin by talking about the different zones we can divide our planet into. These are the climatic zones. So the area near the equator, we call it the tropics. Then adjacent to tropics, we have the temperate areas and then the poles. And as we move from tropics to poles, the species diversity decrease. That means the diversity of species is not evenly distributed. Well, are there any particular species or all the species on this planet follow this pattern? Well, with few exception, almost all the life forms that we know of follow this pattern. The plants and animals that we see on a regular basis, or if we talk about the world under water or about the world which we can see through a microscope, all these different life forms show a huge variety near the tropics and not in the poles. So what is so special about the tropics? What do you think? Well, to some, it might seem obvious or even like a silly question because it is quite prominent even from the picture that the tropic has a very huge area. It occupies almost 40% of the total area of our planet. So more the area means more number of species, right? Simple as that. Well, let's check if that's true. For that, let's take an example. Let's take an example of a frog species. Let's just call it the green frog. Well, this is not the one which you can kiss and it will turn into a prince charming. This is just an ordinary neighborhood frog, okay? Now let's assume that they leave near the equator or in the tropics and if they have more area and more resources, they will reproduce more and their population will increase. But here we are not talking about the abundance or the population of a particular species. Instead, we are talking about the diversity of species. Why are there more different kinds of frog in the tropics? And more land area alone is not an explanation for more number of species, right? So the more you think about it, the weirder it gets, right? Because life has shown that it can grow pretty much everywhere from the poles to the tropics. Yet, it's most biodiverse places are in the tropics. Interesting, right? Now let me bring to you another map that shows species diversity. Here as you can see, as the color moves from red to blue, the diversity decreases. And from the color you can tell that it depicts exactly what we just discussed, right? But let me give you certain data. So let's say Colombia, which is somewhere here, it has 1400 species of birds, 1400 different kinds of birds are seen in Colombia. Now moving a little more towards poles from Colombia, we have our own country in India here. So India has 1200 species of birds. Let me write India here. We know major portion of India is in the tropics. So we also have a good diversity. We have 1200 different species of birds. Now moving a little more towards the poles, we have the famous New York here and New York has just 105 species of birds. And moving further towards poles, let's talk about Greenland. We have Greenland here and Greenland, poor Greenland only has 56 different species of birds. So you just saw, we moved from tropics to poles and the species diversity decreased. And since we see this decrease and this gradient as we move along the latitude, we call it the latitudinal gradient in diversity. Alright, now let's get back to the prime question of this video. Why the tropics? Why are tropics this chosen area for more biodiversity? Well, a lot of hypothesis has been proposed explaining as to why biodiversity is so rich in the tropics. And in this video, we will discuss about three most important hypothesis or the hypothesis that is widely accepted. And talking about the first one, the first one says that the tropics are older than the temperate areas. How can we call one part of the planet older than the other? Well, by this they meant that the tropics remained comparatively stable than the temperate areas and the temperate areas were exposed to frequent glaciation and it also went through the last ice age where most species went extinct and the newly found species there are believed to have newly colonized those areas. So compared to the biodiversity there, the tropical biodiversity is older. The species there got more time to evolve, to specialize, right? And time is the main factor for spaciation. We all know that new species take millions of years to form to evolve from its ancestral species, right? And the tropics were fortunate enough to have had that evolutionary time for species diversification and that is why tropics have more biodiversity compared to other parts. Now moving ahead to the next hypothesis which is my personal favorite one is niche specialization. Now niche has a number of definition but in simple words it simply means the conditions inhabited that is required for an organism to survive. Okay, now how is this niche specialization helping in having more diversity in the tropics because that is what we are interested in, right? The tropical environments are less seasonal and relatively more constant and predictable and if situation like these prevail, organism can take the risk to specialize themselves into one particular niche. Let me explain this to you with the help of an example. So tropics have almost equal hours of day and night and it is constant throughout the year with slight changes of course but it is mostly constant and since it has equal hours of day and night, birds which are active during the night get equal time to feed and to be active just like the birds that are active during the day and since these hours of day and night have remained constant over millions of years, birds have slowly evolved into nocturnal and diurnal birds and this is their niche and this niche specialization is possible because the conditions in the tropics are stable. But this is not true when we talk about the poles. There are seasons and months where sun do not rise. Let's take the example of animals at the poles, let's talk about the bears there. So one season there will be all snow-caped mountains with no food. The other season can be the happiest one with food and fruits and trees everywhere enough resources to survive. Now talking about niche specialization, just imagine if this bear specialized itself to stay in conditions where there is abundance of resources, what would happen when the season change? They will probably go extinct right? And again specialization takes millions of years of constant environment and here the environment is never constant. So in the poles or in the temperate areas, rather than specializing the animals there has to spend their time and energy into adapting to new conditions every month or every changing season. So animals there have to be more adaptable and therefore spaciation is much slower. On the other hand, as we discussed earlier, due to constant condition in the tropics niche specialization is possible that finally leads to formation of new species, right? And that again explains why there is more bad diversity in the tropics. Now let's move on to the third and another famous hypothesis which says that the position of the earth and the sun is such that the tropics receive higher solar energy than the other parts. And higher solar energy means higher productivity. Now let's think of it this way. If the productivity in an area is more, the growth of the plants and animals there will be faster. Now how is that helping form new species? You ask? Okay. We will take the example of microorganism spectator. It is very easy to see their generations, right? And a slight change in one particular generation might give rise to new species fast because their growth is faster. They give more generation in less time, right? Same happens in the tropics. For us it may seem slow but actually compared to rest of the world due to higher solar energy the growth is faster. And if the growth is faster there are more generations in less time. And that helps in speciation and not just that. Solar energy indirectly affects all other hypothesis that we discussed about. So higher solar energy, higher productivity in the tropics have helped in formation of new species over millions of years. So these are the three famous hypothesis that tells us why there is more biodiversity in the tropics. But again you can go look up for more hypothesis that explains the biodiversity through a different perspective. So this was all about the diversity of species as we move along the latitude.


Hi, I'm Dr. Gene Preuss. This lecture is going to look at Christopher Columbus's legacy in what we call the New World. We really mean North America. And we're going to look at the native people of North America. We are going to look at why the Spanish wanted to cross the Atlantic and at Columbus's legacy. The early Native Americans, the Paleo-Indians as they're sometimes called, began migrating to North America across land that was then above water because of the ice age ice caps. So the water table was much lower in the seas. And so you have this bridge up at the top part of this photograph in Asia. And it's connecting over across what's today the Bering Sea down into across the Atlantic and into North America beginning at Alaska and coming on down. And the migration began about 70,000 years ago, we think. And it lasted until about 10,000 years ago. So it was land that was available for a long time for settlers to cross for people to migrate over. They usually were hunter-gatherers moving in small bands. But about 12,000 years ago, the climate began changing and it began getting warmer. And so large game that many of these people hunted became scarce and the land changed. So people could now domesticate plants and smaller animals. For example, you have about 10,000 years ago, the development of what we would today call corn. The ability to domesticate agriculture and have plants and animals allowed for a secure food supply or a more secure food supply than that of hunters and gatherers. And so you do have population growth because people are able to reproduce more. And if you have population growth, you're also going to have permanent settlements. And you're also going to have a diversification not only of the food, but of people's roles in society. And you have the development of religion, the development of trade, gender roles, become more fixed leadership roles. Now not all cultures are going to adapt to this change at the same rate across the world, but many did. And you have people who are hunter-gatherers, fishers, and then you also have people who are developing agriculture as well. In North America, the people began developing civilization. About 3,000 years ago, you have the development of more modern cultures in North America. And some of these are the Adina Hopewell, about 800 BCE. These were mound builders. They lived among the Ohio River Valley and made large mounds, often representing people or animals. Then later on, moving further south along the Mississippi River, you have what's known as the Mississippian culture. And this develops about 25, 2600 years ago. And the largest city that they possessed was one in what's today East St. Louis. And it's called Cahokia. It's a World Heritage site. And you can visit it today. They've excavated two large temple mounds there. Further south, in the Four Corners region, the New Mexico, Colorado, Utah, Nevada region, and that area, you have Pueblo cultures or Pueblo-Holcom cultures, which still exist today. Mesa Verde is also another place where there were large Pueblos built. And then further south in that, you have in what is today called Tenochtitlan, actually what is today called Mexico City, it was called Tenochtitlan, the development of the Aztec Empire. And they were around about 1200 to about 1500 when the Spanish conquered them. Now let's look at why Europeans came across the Atlantic in the first place. Now we think that sometimes Christopher Columbus was the first person and that certainly is not true. We have evidence, although it was largely a myth, a legend before, that about the year 1000 Vikings came and settled in north of New England in what's today Newfoundland and Canada. Those villages have been unearthed, excavated, and we now know the Vikings did actually settle here. Probably fishing, but they didn't return. There were some myths that had developed in the Iberian Peninsula, what's now Spain and Portugal, about a land of youth across the Atlantic. There was also a myth of seven cities of gold and these were golden treasures that Spanish bishops had taken when the Muslims come up from North Africa and invaded the Iberian Peninsula. And when islands off the coast of Africa, the Azores were discovered about in the 1420s, people began putting a little bit more faith into these myths and legends. One of the other things that the Europeans had, and this is a common myth that we have today, is that Columbus and the people at that time thought the world was flat. Most people and most educated people knew the world was round. Most sailors knew the world was round as they had sailed and seen the curvature of the earth. So the thing that they didn't know, however, was how big the world was.  And so they did not realize there was a large continent between Europe and Asia. And what was their mindset? Well Europeans, we have to remember, when they came over wanted to recreate Europe. They wanted to recreate what they knew at the time. And that's not unlike many people today. When we travel abroad or into other countries, we want to take with us what is familiar. One writer has said that they, what they wanted to do was recreate the new world in their own image. European society at that time, it was a religious society, of course.  And they had been fighting for over 700 years to retake control of the Iberian Peninsula from the Ottoman Turks. And so this was a religious war that ebbed and flowed over the centuries and it was very much built into their mindset. The Encomienda system. This was a system of enslaving people and putting up large haciendas so that they could, landowners could enslave the people that lived on the land. It's very much rooted in European nobility's tradition that peasants worked for the landowner and essentially were owned by the landowner. It was a hierarchical society. And by that I mean, we think of hierarchy today in the military where you have commanders and you have people underneath them. But life was like that in many respects for Europeans. The church was hierarchically organized. Government was hierarchically organized. Society was hierarchically organized. And so everything moved from the people who were the best people at the top to the average person at the bottom. And it was also a mercantilistic society. Now what does that mean? The economy was mercantilistic. It meant that in their eyes, colonies and other lands were there to serve the benefit and purposes of the homeland. And so colonies were simply to be exploited. And so were the people on there by the way. So that was their mindset. They were an acquisitive people. They wanted to gather things. The only way of getting gold was to take it from somebody else or to find it. And so they wanted wealth. In Europe, they needed wealth. You had a whole bunch of rulers who were trying to establish kingdoms at this time. And trade between the east and west was equally important because they wanted the goods and services from the east in Europe. At the time, there were also technological advances. And what is today Portugal? Prince Henry, the so-called navigator, established a school or a think tank for people from the Arab world, from the Christian world, from the Jewish world to come together, engineers and scientists to discover new ways of advancing navigation on the seas. And this was beginning to have influences on the way boats were built, ships were built, and in the way people sailed. And so they can now sail outside of the Mediterranean into the Atlantic. Before that, it was simply too dangerous. There was also religious zeal. I mentioned this idea of taking back the land from the Ottoman Turks. And you certainly influence, you see this influence in the Crusades of the 1100s when they wanted to retake the Holy Land, the Middle East. And the expansion of the Ottoman Turks from Africa and into the Middle East on up into Europe, this led to the Reconquista, the Spanish, for example, the Reconquista. How do we take it back? And we must take it back. And you have a militaristic society that develops around this. Now there were some early successes at sailing. You had Bartolome Diaz and Vasco de Gama who sailed around Africa and were trying to establish trade with India. And de Gama eventually made his ships, made it to India. So there was this idea of needing to explore and acquire and having the technology to do it. Now when Columbus set sail and took advantage of some of this new technology, he left Spain in August of 1492. And it wasn't until October that they sided land. In the meantime, the governments of Portugal and the governments of Spain divided the world. These are the two countries that had used Henry the Navigator's techniques and his discoveries in the field of navigation in order to sail around the globe. And because Portugal had pretty much sailed east around Africa and on into India, it was a disagree that they would get the eastern part of the world while the Spanish would get the western part of the world. And this was divided, actually, this is why the Portuguese get Brazil because it's on the eastern side of that line and they were there first. Now, what was Columbus's significance? Well, he sparked European interest in the New World, in North America, a place that they hadn't known before. And so because of the technology that they could repeat that adventure, Europeans began going regularly to the New World. Now, a big surprise were all the people and animals living there in the New World, people and animals they had never seen or heard of before. And so for many, they thought this may be a new creation, a creation that wasn't in the Bible that they didn't know about. And it confused many of the Europeans as to who the Native Americans were and what those new animals were and were they the same as everybody else? And I want to talk also about another part of the legacy of Columbus. And that was the Columbian exchange. We talk about this quite a bit. This Columbian exchange is, as one historian has described it, the greatest biological revolution in the Americas since human beings came over. This meeting of the old world and the New World, Europe and Africa and North America, exchanging animals, exchanging people, exchanging food, exchanging disease. That is the Columbian exchange. And when we look at this and look at the differences that Columbus made, this is why it's named after him, is because his ability to come to the New World and the people who followed him brought about this rapid exchange. And so let's look at some of the animals that were new to Europeans. They had never seen before. Now, some people may not like rattlesnakes and think, well, it would be better if we never knew them. But there's other animals, too, that they didn't know what they were and they wanted to find out more about them. They were foods that Europeans had never seen or tried before. Potatoes, beans, pineapples, chili peppers, pumpkins, cotton, many things that were new to Europeans. But there were also things that Europeans brought with them that people in the New World had never seen before. Onions, radishes, grapes to make wine, olives to make olive oil and to eat, just as importantly, were the animals like horses, cattle, pig, sheep and goats and rats. Many of these traveled with the explorers, with the conquistadors as they came to the New World and were allowed to run wild once they came in because there was no fencing. They were just rounded up later, but they also bred and they multiplied in their numbers and took over large parts of the New World and thus helped to spread some disease. So you see what happened as a result of Columbus's landing. Who were the native people of North America? They were descended from prehistoric migratory people who came from Asia largely. There probably may have been other routes and there may have been some people who were in boats who traveled over further south, but largely they came from Asia. And by the year 1500, they had developed many long established settlements and empires in the New World.  He was actually coming in and finding civilizations. Why did the Spanish want to cross the Atlantic? Well, they simply wanted to look for a new way of continuing the trade they had already established with Asia. And now they had the technology because of new sailing techniques, because of new ways of building ships and sails that they were able to repeat this time and again. And what was Columbus's legacy? His miscalculation, this accident on the location of Asia, sparked interest of all of Europe in the people and places of North America. And they now had the ability to continue to do that. And the effects of that repeated contact were tremendous. Thank you very much for listening. We'll see you soon.


now let's talk about how to talk about pregnancy after you receive a job offer or during your in-person interview especially if you are in your store trimester there's no way you can hide it's very challenging if it's a virtual interview you receive the job offer and I will tell HR regarding the pregnancy by saying the following saying that I am actually pregnant my due date is abcd date which is several months from now on what I can do is I am happy to work until now or before my pregnancy and take three months off and during the three months off I am okay to not get paid because I'm new to the company or you can directly discuss and negotiate with the HR based on when your due date is


I can start from the second lecture of Dr. David Baker, who is a professor of chemistry at University of Washington and an investigator of our youth's medical institute. Yesterday he presented a protein prediction problem and today he's talking about protein design. It's a very important application, so we're going to do what we can teach us about more about proteins and also we can think about what we can do with protein design. One of the things we're going to learn about is how to design enzymes. It's an extremely difficult task, because the proportion of protein design is not the complexity that is required for optimizing a transition state. And so we learn our strategy that includes how to create a combined conformational search that can be successful. I would like to start with this. Thank you, everyone. It's a very simple question. What does it mean to Europeans to be able to walk on the lake and all of you who managed to scare off everybody yesterday, I guess. So, as I said yesterday, I spoke about all the sequences that we have in all the wonderful biological organisms that exist in trying to be kind of compute from those with help from experimental data and where it's available to the structures and functions and interactions of the proteins that basically carry out all of biology. But today I'm going to talk instead about the inverse problem of trying to make new things that don't exist in nature and generally what I'll talk about today is some new function that doesn't exist. How do we work backwards to come up with an amino acid sequence or an RNA sequence that will pull it up to give that new function? I showed this slide yesterday, too. Before we were going, we were using this model of the interactive, the energetics of interactions to give the amino acid sequence, for example, find the lowest energy state. Now we're going to use the same model. And now we're going to, rather than starting with the sequence, we're going to start with a function or a structure that we like and find the sequence that has very low energy when it folds up to a structure that carries out that function. So we'll be working backwards from various types of functions to structures using the same energy function. I think that's really important that I really need to emphasize is that most of the time when you try to predict a structure, it doesn't work. You get information there that's very valuable about how your model is wrong, likewise with design. So by doing it, it's very important, I think, when you're doing computations for the computation, or that it's possible to complicate the new problem. That's really where you learn that model, if your model isn't right. And I think one thing that we do know for certain is that complication of biology is that current models are not very good. So it's very important. I feel like there's too much emphasis on trying to show how good models are. I think really important points to show how bad models are is that's the way to make them better. And fortunately, traditional design are very good ways of showing how bad models are because you fail very frequently. And every time we fail, we go in and we improve the model a little bit. So I think that's sort of how we look forward. So this is basically the workflow for all the things I'm going to be talking about in this talk. We started off with computer calculation of a design protein. And in some cases, and I'll talk about this at the end, for fun, we're trying to see if we can get some human input into this. But basically, you have a calculation on a computer where you're trying to design an infold or a new end sign. Once you have done this calculation, you have your protein model. You can read off the amino acid sequence from your computer model. And then, of course, we know the codes and we can backfence that into a DNA sequence. And then it's a very difficult step. You have to fill out a gene order form, which you then email to a gene synthesis company.  But basically, this part is what makes protein design very much easier than it used to be. And then, if you assemble all of those, you have your gene in a week. If you are lazy and you want any weights for the company, you do it. It comes in two to three weeks. And you get the gene back and we always make out these things in, usually, the coli expression vectors with his tags on them. So you put them in the coli. You break the cells.  And then you assay. And then you find out how weird your design was. And I wasn't really sure where to include this, but this we just talked about a few days ago. And you can see that... I would say the opinion is not unanimous about whether protein design is a worthwhile endeavor. So here's how the public calculation works. One starts with a protein backbone, in this case. And now instead of... Now we need to search through all possible sequences or a sequence which is very low in energy when it's on this protein backbone. This is for the problem of designing a new structure. And basically, what's happening is that each step we're going through and putting in a new confirmation of a new amino acid or an alternative combination of an alternative amino acid. And we can do this very rapidly. Again, this moving, this batch of calculation doesn't take much longer than what you're seeing here. So we can very quickly come up with amino acid sequences that pack very well in any arbitrary structure. You can see here that the blue things are nonpolar so we end up with a completely nonpolar solution. Now in practice, when we want to make a new protein with a new pool, we can't just draw a backbone on the back of the envelope and then use this method to find an amino acid sequence which attacks the core well because there's no guarantee that there is any amino acid sequence which will really fold up to this structure. So because of that, we actually need to iterate after we do this first calculation to get the sequence. Then we go into structure prediction of what I described yesterday in that pie resolution movie and we let the backbone and then we repeat the sequence optimization and we let this go into we have a sequence and structure combination that really fit well together. So we used this a number of years ago now to see if we could make a protein with a topology that didn't occur at nature and this is before the days of gene synthesis so we've sampled all of those and when the protein was purified it was found to be extremely stable and we were able to solve the crystal structure and it was very, very close to the structure that we were trying to make. So we had the computer law model which is shown in blue and the x-ray structure in red and they are very, very close to each other. So this showed that we could make from scratch from scratch we could design amino acid sequences that fold up to new topologies and we're very, very stable when they fold it up. Now there was a big problem going actually before I get through them so since then what we've done is we've been trying to make new folds and the reason why we're trying to make more very stable scaffolds the thing about proteins that we're designing if you notice them they look much more ideal than most native proteins do they don't have long loops the secondary structures are regular and you might think of them as sort of being like a platonic ideal this is a very common protein called fold called a paradoxical fold design protein is very much similar so again we use the same protocol this protein is very much more stable than naturally occurring proteins again these proteins typically are penetrating in five or six molar quantities so they're very, very stable and then there's another one a Roslin fold again another very, very common fold and you can see why we're interested in these because of course Roslin folds use these loops to do things so we can make sequences that fold up to new structures either new typologies or similar versions of existing ones and we can do so with quite high accuracy and they can be very, very stable but they don't do anything at all and so where we were a few years ago we knew we could design new protein structures but they weren't very useful unless you wanted to rock because they really didn't do anything so what we've really been focusing on since then is trying to design proteins which actually do things and the problems I'm going to talk about today are designing new DNA cutting enzymes and designing new enzymes new enzymes generally I thought that would be my talk we have been working on designing HIV vaccines and there the idea is to pick epitopes from GP120 which are recognized by neutralizing antibodies but for some reason our bodies don't frequently make those type of antibodies because the epitopes are probably obscured on the virus anyways design small proteins sort of like the ones I showed you which present the epitopes in a very upfront and center sort of way to hopefully elicit a response and their hope is that those would then be neutralizing so this has worked out with my colleague Bill Sheath in Seattle and the result so far is we've been able to design proteins which interact by strongly to be neutralizing antibodies with crystal structures so the epitopes look right they elicit and new responses that bind to these epitopes even bind to the virus but they're not neutralizing so I guess if they were I would probably only talk about that so this is a mystery I think part of the big mystery of immunology of HIV infection okay so now to talk about DNA I think so what are the types of things you might want to do well long term we're working with class of endonucleases which recognize very extended recognition sequence 20 base pairs and if we could redesign these enzymes because any arbitrary 20 base pair sequence then there are a lot of interesting applications the most obvious one is in gene parity so if we go in and design an enzyme that would leave near the site of a mutation and then the idea is we would introduce that enzyme along with the wild pet copy and double strength rate then hopefully it'd be corrected by by copying on the wild pet template there's also possibilities for some antipathogen applications and being able to design a very specific entity basis so in this case now we don't just have a protein we have a protein here on top of DNA but it's basically the same calculation that I showed in that earlier where we made the change in the DNA so we could change the sequence of the DNA here and optimize the protein to recognize that new sequence and so here's an example here's a here's one, this is an extended site I'm just showing you a glow up part of that site here's the wild type amino acids at that position in the new base recognizing the wild type base pair and you can see that if you change this from a GC to a CG the wild type amino acids no longer can make good interactions but if you redesign using that movie I just showed you can get a very nice non-cooler interaction here and a nice hydrogen bonding interaction here and in fact when you make this protein it specifically leaves this sequence this CG whereas the wild type doesn't believe it the sort of data one generates this just shows a number of different designs but this is that new sequence the wild type enzyme the designed enzyme in green cuts this in a little concentration whereas the the wild type enzyme doesn't cut the site and we've gone on and now made multiple simultaneous changes and so I think we can we'll start working our way with a couple different enzymes towards more general recognition but I wanted to tell you about you know in the course of doing all this design work sometimes one comes across really unusual biochemistry which I think is going to be very interesting on its own right and I just wanted to illustrate that here so this is this is what we had to do another enzyme from this family which they had these two domains and they recognized this again here quite extended sequence and it had been known for some time that mutations throughout this site could make space changes to be throughout this extended site you knock out cleavage so this is just a this is just a cat or a ham the effect of the cleavage for all what you're seeing here is substitutions up to each of the four bases at each position in the site from minus 10 to plus 10 and you can see that throughout the site there are mutations which knock out binding and so there's some down on this side which is the internal side of the of the complex and there's others down here so it's very specific throughout so we got our first inkling that something funny was going on when we looked at the effect of these mutations on binding we found that mutations on this side substitutions in this site on this side had an effect on binding which seemed to affect binding at all so to go on on summer time we decided to carry out the K. Lisbon kinetics based on all of these different sites which was a endeavor and what she found was really quite remarkable so this shows for each substitution change in K. Am be the substrate in terms of substrate binding for the K. Lisbon complex and again here's the minus side here, this is the minus side you see that all these substitutions are affecting K. Am on the minus side and this is a very symmetric interface you can see that there's pretty much a two fold axis here but in spite of that there's a real asymmetry of how the interactions are used but not here seem to be used in binding the substrate whereas the mutations here again have very little effect on K. Am but you look at where the effects on K. Kat are, substitutions down here in fact, on this side actually in some cases even make the reactions faster but mutations down on this side almost universally slow the reaction so there's this really strong partitioning of what the binding energy is being used for on this side it's being used to bind the substrate and here it's being used to utilize the transition state and the simplest way to think about what will attract a picture is that this end time needs to scan through a genome and bind this right condition site it binds in the transition state complex which probably resembles the crystal structure to this bent confirmation which is probably not sampled very often in a cell so instead what it can do is it can bind to B form just through this side, it binds in that way and then this interaction, this stabilization of this bent state basically is used to stabilize fluctuations of the DNA that then put into an confirmation that's heavily confident but we can now take advantage of this and we can make specific designs then that either effect K. Kat over 4 K. Am so these are just designs that are designed on the minus side, designed on the plus side so this is one that's on the minus side and again the color coding is, the different colors represent the different bases so in this design see so the wild type shown in dotted lines and the the design is the solid line so this design increases the K. Am makes it worse for this face, this face this face but makes it better rocks the K. Am for this one so it achieves, this design is changing the specificity of wild type by modeling K. Am for the different bases but it has pretty much no effect on the K. Kat for these different substitutions. On the other hand we have this design on the plus side which is not having much effect on the K. Am's for the different possibilities of that position but it's drastically reducing the K. Kat's for the wild type pretty much has a uniform K. Kat's are very similar but this design they're lowered for three of the four base pairs leaving only the blue one highly active so this shows that we can now sort of go one level beyond sort of this great design specificity we can actually go in and now modulate the parameters independently which you can imagine could be very useful and depending on what your bio-athletal application is okay so now I'm going to switch gears and talk not about redesigning in type specificity but about the no going side design and so if you can come to me and say design reactant enzyme which capitalizes this chemical reaction the first step is to compute what the intermediate and transition states in that chemical reaction are then the next step and this is sort of the fun part is to design a disembodied active site around say the transition state for the reaction so you might say well I have a hydroponic group here a positive charge here, a negative charge here but these are completely disembodied and as Alessandro mentioned we can use on-mechanics calculations in collaboration with K. Kat's group to sort of disturb the optimal positions of these you can also use chemical intuition about where where would be optimal how would that best upload and let out flow in a bond rate in a bond making reaction this is really just hypothesis this is like what you see in a biochemistry textbook you know they first show you the whole thing that it's doing on the active site and we just see it as having a triad in the transition state we start with just that low enough view but we don't know what an ideal active site would necessarily look like we just have hypotheses we need to have a number of different hypotheses because we don't know which one is actually going to be able to catalyze the reaction so once we have this ideal active site we design a protein that contains this ideal active site so we have this ideal active site we want to make it now we have to build a scaffold which has that ideal active site and that's why we went back out the top seven to start kind of making bolds these are basically two ways to build a scaffold one is you could construct a noble scaffold that is just perfectly for all the back but it's just perfectly poised to hold the catalytic residues and the other thing you could do is just go through naturally occurring scaffolds and so what I'm going to be telling you now we're going through a naturally occurring scaffold of thermohella proteins that are very very stable so where we've removed large numbers of side chains put in the ones that create this site but this isn't really ideal because these proteins really evolve to do something else so we'd like to be able to do it from scratch so given the description of ideal active site then we need to next up is to well let me illustrate this first part we need to find we need to figure out what scaffold we can use to actually hold that ideal active site so here's a really simple example this is called the chemical elimination reaction where we abstract this proton and then it's going to hopefully lead to the breakage of this bond here so this is the product so the first thing we need to do like I said is to choose a chemical peak this is like a disembodied active site so here's an example here we have a negatively charged group that's pulling off the proton that's forming a space here and we have a positively charged group that's stabilizing the negative charge that's appearing here and here's another scheme where we have a histeme that's pulling off the proton and a proxylate group that's backing up and holding the histeme in place so we have a hydrogen bond in this case that will stay close to the negative charge so you see there are a lot of different options that you can pour a very simple reaction on this so the next thing we do is we take this large set of staples and we ask where can we geometrically we constitute this site so the backbone we need some backbone here, here and here and we need to have room for the substrate so we fill a geometric caching algorithm for that after we found these places where you can support these residues the next step is design all the surrounding of the elastic that's shown here it's a little hard to see and the final step is to select the best designs and make them and this is now an illustration of the two designs using two of the motifs the two motifs I have on the proceeding slide so if you remember there was one with the carboxylate here and there was the one then we had see you can't see the lysine I think coming in in this case you see the substrate which is in yellow this is being in a nice hydrophobic pocket that's been designed that's part of the second step of the optimization now we have an aromatic residue on top that's sort of planting the substrate in place now the other option to call was having an isthene which is now pulling out the proton out here and this is backed up by a proboscillate group in this case an aromatic group that's planting it in so the the neat thing is that these designs they're very simple but they do have enzymatic activity this is just an out of product form versus time and when you mutate the residues which are like this is the glutamate this is the one that was on me you make the glutamate into a glutamine you completely lose activity and then use the isthene in the sparking if you would move the isthene you are down to here and if you would move the sparking you're down here so it's not as absolute a requirement so that's a very simple reaction I'll just take you through a couple of other reactions and then I'll make some more general comments about where we are with this and so here's another reaction and this one is interesting because it's not catalyzed by naturally occurring enzymes or at least there are no well documented cases of enzyme catalyzed reactions it's called the yield-solver reaction so here we have this group of blue, this group of green and they're going to come together to form this ring here so we're forming two carbon-carbon bonds in order to catalyze this reaction one has to bring these two groups next to each other and that's shown here and also we're pleased to perturb the orbital energies of this guy so that they overlap more to increase the rate of reaction and we do that by introducing hydrogen body groups which an electron-domaining group which will raise the energy levels on this side and an electron-crawling group here which will lower the lumo energy here to increase the overlap again this is basically the same thing I showed before so this shows an example of such a design here we have the small substrate and the big substrate we have a hydroponic group here and a hydroponic group here those are what are doing the orbital derivations and again this is the case where we go through a big gene and this shows a product versus time here notice that this is not a very good enzyme if this were an apple, they aren't actually bringing in enzymes to catalyze this reaction but the word this would probably be second but that's just where things are right now so we have a crystal structure of this design as we did we do also of the preceding ones and in general the crystal structures are quite close to what we to the goal we've been trying to make the interesting thing about this reaction is that these substrates can come together in several different ways and so this is the substrate this is the other and so there are various anti-mersite that can be produced and you can separate these as shown here and what's neat is we only form one of them and it's the one that we were actually trying to make this one here so there's some specificity here so alright so to summarize where we are so far then I showed you an example of how that uses general acid-based catalysis pulling on and on putting one on on the other side we can't eliminate it I showed you a bi-molecular reaction that deals all the reaction and they talked a little bit about the elbow reaction which involves covalent catalysis where the enzyme becomes covalently bound to the protein and knock and knock on ester hydrolysis so we've been able to use these are pretty much the strategies that native enzymes use to catalyze reactions but there's a big difference which is that our designs are much less effective catalysts than native enzymes now I'll come back to that just to show you what the diversity of chemical reactions are here's the camp I showed you I talked about this one already here is the reaction I'll talk about in just a moment forming this carbon bond between these two substrates there's ester hydrolysis so these are quite different and this just shows different types of what the designs look like okay so now these really aren't very good catalysts but we've been collaborating with several groups who are expert at laboratory evolution including the antics group here and the designs I showed you those pictures of are called K78 K59 but also the numbers are important because it gives you an idea of how many times we had to try we don't have all the numbers because some of them work solid some of them we decided not to make but we're trying quite a few designs so it's like a one out of ten sort of thing that we get one that works so that's an important caveat to what I'm telling you but we always keep the numbers on so you can see in his lab and as a student although constantly very talented a student took them and started evolving them just making mutations screening for once that have more activity even though this is a fairly simple reaction there isn't anything in an equal act which catalyzes it so you can just rake over the cells and do the assay and she's now up to about here I don't have her by slide but the best designs now the best evolved version of this K59 design that's the one that uses the the glutamate is now about 5 times 10 to 5 which is starting to get into the range of naturally occurring enzymes so the maximum diffusion control will be 10 to the 9 so dark so it's starting to get respectful and the rain investment is on the order of 10 to the 8 a little bit about that so these are still not as those naturally occurring enzymes but they're getting better of course we'd like to be able to compute what these these are the sequence changes which are spread out throughout the protein and it's been possible to get crystal structures of a number of these evolved variants this is actually for the first design we had activity I haven't shown you a picture of that's the one through which we have the most structures now it's called A7 and we were really really silly when we made the design it had a glutamate that was attracted to the proton and this lysine if you remember it had a negative charge but if you put an awesome next to a negative then they can decide to do other things and that's what you see in this crystal structure here now once we saw this crystal structure of course we got rid of the lysine and that increased the activity considerably but evolution itself follows in unusual ways sometimes so during the direct evolution what happened is a negative chart, a negative cluster from evolved down here which distracted the lysine down and thought it out of the way and what you see happening by the final round of evolution is that this catalytic residue is really held quite a bit into place, really better positioned to lock that proton up so this is this is a the reaction I mentioned before this is the retroalval reaction in this direction we're breaking this carbon-carbon bond here and this reaction is a little bit different as I said we're forming covalent bond between the protein and the substrate and that's shown here this is a complicated reaction these are many many steps you form this enzyme substrate intermediate then you break the bond here and then you need to recycle the the free enzyme well we've been able to do here, well actually you can see so we've had to try as I said we always have to try multiple mechanisms and we tried it in a number of things and the one that worked really well was one which where we used a water molecule that was positioned by two hydrogen bonding groups that we designed did and this water molecule helps facilitate proton transfer between these two oxygens this is the bond that's going to get the lead criteria so what's been exciting recently about this is a couple of things first of all we've been able to using this motif we've been able to design this active sites and catalyzes reactions very reproducibly so we're not at 1 out of 10 anymore we're better than 1 out of 2 so we have a very high frequency of the designs the new designs that we make for this reaction actually work and this just shows the range of protein scaffolds and the ranges of positions of the catalytic lysine that we have now active designs so this we like this one because we actually understand how to reproducibly produce designs that catalyze this reaction and they're quite pretty this is what a design model looks like here's the two residues which are formating the water here's the lysine that's coming in and forming a covalent interaction with the substrate and here's the binding pocket for the substrate here so what's neat is we just have gotten a crystal structure of this and I think I'll just skip to here so in the crystal structure we let's see we've been able to trap the intermediates on the lysine this is the lysine here by using our hydrohypride production we can actually trap both the substrate and product on the enzyme and so we have pictures of what these substrate and product complexes look like one thing that's quite interesting which I'm not showing you on the lysine this one accumulates the substrate there are other designs which accumulate the product so again there are multiple steps in this reaction and it's hard to do them all well it seems like different enzymes did some extent block the different steps what's neat here is these two water molecules which we had designed in we actually see in the crystal structure they're interacting here and they're positioned by all these hydro body groups so this is our we're excited about this because this is our first co-construction of a enzyme and a enzyme with a substrate the substrate lies pretty much in the pocket that we designed in ok so where are we after all of this first of all we can't design active enzymes from scratch I've shown you that now we've done this now before these are low they're going to be increased by directed evolution there are clearly some things are happening that are on their competing reactions the product of the reaction goes back on slows down the activity it's clear that we need more precision positioning and the catalytic groups are a level of control and it's not so high and I think one of the bottom lines is that real enzymes are masters of the art of compromise they have to bind substrate and carry out the chemistry they have to release product they can't get hung up at any of them and if we're producing this it's really not very easy and so from a practical point of view one of the key questions is not really about protein design but about evolution we can make things that catalyze these chemical reactions we can make them from scratch but they're again not very active catalyst and we can also make quite a few of them for the alkali we can make many tens 40 or 50 by now but none of them are very active so the real question is I think we can turn back the clock 3 billion years and look at the ancestors of modern day enzymes how active were they and how many different starting points did they just need to be able to get one that was really really good is naturally occurring in enzymes so I just ordered for that to be better than these so it's not clear whether can we take any one of these designs and do we not by evolving it doing enough rounds of direct resolution to really turn against enzymes or are there many large fraction of dead ends so sort of a question about dead ends and evolution and this is the practical question I don't think it's likely that in the short term we'll be able to design from scratch catalysts that are very very highly active so I think from a practical point of view we'll be making new enzymes we'll be left with sort of making things that have starting activity then optimizing them experimentally one of the areas that we're very interested in here is there are a lot of interesting applications obviously in the energy area because it's, for example, is some you can capture energy using oscillating solar panels very effectively but using that energy to make molecules you don't really have a way to do that but if you could design a design sort of a pathway that could take electrons off an electrode of a solar panel or from electricity or would take a voltage difference that generates at least a solar power an electrode, use it to reduce some compound and then get electrons from there into a cell through some sort of assigned pathway so there are a lot of interesting applications that I've designed but we kind of need to know the answer to this to know how many starting points do we need to have and so forth so that's one of the real key questions so just to sort of summarize what I told you in these two talks then today is that for the structure calculation problem the problem was really a search problem that on the lowest energy state per cookie is a very, very good hole for anything but the smallest per cookie structure we didn't really have a problem with accuracy though because once we got close enough to the native structure we really fell into this deep hole and because of that the solution I was advocating was to get experimental data to sort of indicate what the position of the of the native structure was and then do computation to work out all the detail that's for example what we were doing in the structure in the case with NR data we used the NR data to guide to lowest energy search and then we would but we wouldn't use the data in terms of fine detail to the structure because there was a steep, evolved energy gap because that solution had really optimized that sequence to be very low in energy in that native structure now in the case of designing functions it's really quite different we don't have a search problem we can come up with these motifs and then we have these algorithms for rapidly finding places where we can design proteins new backgrounds and cavities the problem is that our accuracy is very high because unfortunately we no longer are dealing with design the problem is that we're dealing with things that we've designed we've come up with so they aren't really as optimized for the structure we want to have that we have for in the structure prediction case but we also don't we're far from having a complete understanding of the requirements for the policies and I think by designing new enzymes or trying to design new enzymes we're learning a lot about what really is important and this policy can be understanding we we do the computation first because we can, again, we don't have the search problem we can just build any site we want in principle but we need experiment to tell us how minimum our designs are and then to optimize them and we need a lot of experimental feedback to tell us how to both improve our current designs and learn in general how to make better enzymes so I'm going to give you a little bit, I want to show you a little bit more about full net just to be in harmony with yesterday but let me first acknowledge people who've done this work so the design of the pieces of scaffold that I showed is the work of two very talented Japanese postdocs the end of the new phase we designed the work to talk about the work of the summer time and no one has ever signed Justin and Alice through the work on the Neal Solverace Eric and Link have done the work on the Retroal Delays and Daniella through the work on the camp and it's been a collaboration with Danny Toffett over at Ronsky and we've got over so let's see now so now what I want to show you is that the Neal and Full Nets set up to not just predict structures but to actually do design and so what I a puffle that's up now, I'm sort of limited by what you can get on the web, is rather than designing new enzymes this is designing approaching much more quiet this is a sort of model problem we're working on and here's why I said here, we give people a starting structure where we use the same sort of matching algorithms to basically build an ideal act of sight sort of copying the strut that hydrogen bonding groups to bind the base of the monotone and you can sort of see that here but then what people can do, in addition to all the things I showed you yesterday they can go in and they can try and make it better and I'll show you how that works here we go so this residue I'm allowed to be taken so I can now go in and I can change that residue to anything I want so maybe I might try and be on a trip to band there for example and now I have a trip to band there but you know I created that little those red and nasty things that doesn't clash, that's not good my score is not much worse we can again compare my score to everybody else let's see yeah so I really have done that thing but I can now I can do the same kind of things as before I can I can move that trip to band there I can move everything around in this case not like this happening so my score is going up and so I'm very excited about this so these problems we're starting to give people now the option too they can change the sequence but what they can also do maybe also one more thing is we can we can add more residues well that wasn't a very good place to do this but people who hold it to those are very good so what they're doing now is we give them starting points and for example for protein protein interactions we can then decide protein stock up against the influenza virus and then they can throw out loops and they can pull the backbone around the sort of ways that I showed you and change the sequence so we're trying to see if we can get all these great approaches that I showed you yesterday for protein protein design but we're actually starting to test some of these in the lab anyway of course most people don't do things like this that's kind of embarrassing but anyway so I think as soon as we can start making things in the lab and people that design all these people around the world and they work and I think that'll be sort of a new point, a new way sort of step and sort of get listening to the general public and sort of biomedical research and also I think really again you get an appreciation for what the molecular nature of a lot of problems are so anyway I'll stop there well yeah I think the most obvious way to do it is to take again using direct evolution if you can truth the activities if you can compare the dynamics of the ball version of the initial design it's very possible because when you're using these thermophilic scalp they're pretty stable so yeah I think that's a really interesting question, we don't know what's missing so I think the sort of two schools of thought one is that these things are too rigid and they don't conclude enough and the other school of thought is that they're actually not rigid they're not binding the substrates like to get a substrate up against catalytic residue will generally be uncapable so you really need to exert force in a very precise positioning to do that and so that may be missing probably both are missing we don't have precision where we need it we don't have dynamics where we need it I think I think I'm pretty excited I think we'll learn from if I can continue to follow up what about the lines that contribute to do the assays to the function of temperature they have pretty much they have thermophilic profiles initially but after you follow them they have more mesophilic lines which is sort of what you'd expect I mean that's the yeah so in the means other reaction that we were trying have you ever thought of putting in I mean the means other transitions state is really high in energy and has got kind of an aromatic headache that's an interesting idea we haven't tried that we've used primarily aromatic groups for satin but that's an example of where this is at there are a lot of different ways of going about trying to solve these problems and you know it could be in some cases we just miss on the optimal idealizer site configuration so yeah yeah so of course what we're doing is we're now going back to the complications we've come up with these substitutions in the first place in fact he was a graduate student here in my lab about a month ago as a postdoc and he's really focusing on that question there are let's see I can't think of a specific case where we know we threw out tonight but it almost must be happening he's only doing the computational design it's going through all possible sequences and if there's something that we find out that it was clearly must have been missed a lot of it is there's a lot of there's a little bit in the companies for example of bigger residues so we sample both ligand confirmations but we can't do that and then we find an example where we find something perfect yeah do you know about designing the loops to make your rise more flexible yeah that's right so that's one of the things we're really focusing on now to go to these and try to design the loops yeah more flexibility maybe to allow the substrate more yeah exactly so we're trying that now you mentioned you need to do the slide changes in the financial state here you clearly can't do that so like the looping and the kind of that looping model that you approach in this case so we have to calculate what that would be yeah well we can also determine it experimentally by looking at the pH and the reaction and actually we kept it since you just started pretty much where you'd expect them to be so the PK's seems like getting a PK shift is not such a big deal but for example you have a very licy that PK dropped so I thought that was a harder problem than it turned out to be we're more limited by other features it seems so you're able to predict I would say that in general designs when we go and look at the PK's there in the range you'd like so the licy tends to have a PK around 6 now for 7 the glue remains around 6 so I'm just saying that now we obviously are able to predict reasonably what the PK's are going to be we're not going to get into turbulence just by burying them when you get them you're shifting what is the number of N? actually it's the address of N what is the N? what's the number? I think the smallest one is about 150 N I don't think you can cut down much because you need to have a pocket at least for if you look at how intense mine substrates if the substrates small generally need to have it looks like that will develop or if you're substrates you can kind of lie on the surface yeah yeah yeah yeah yeah I think that's actually I think that's actually right yeah so I think the water is like you need to get away with prime saver get away with reusing the ends of side chains because they can move around they can easily rotate them that's exactly right well actually what we're trying to do now is replace those with with charged groups because we think that maybe why we have why we're not getting to a certain amount I think it's good for starters but in actually occurring evolution I'll bet that early on there's a lot more use of water and then you gradually replace water with things that you could water's good back in the same piece and it's good and how do you talk about designing the economy for this whole yeah so we are trying now to make design coaching so that we'll be totally solid and organic and so we're trying to basically take the top seven that we've got in version I think I'm trying to figure out how we can make the protein so we can get it to see whether it's actually solid or not then finally the enzyme is in that range they'll come in a common area what about enzymes that can do successive operations so attaching domains, sort of like the polytheta it's interesting yeah well I think that's the end for the future I think we need to understand how to be simple first the retroal law reaction so all the solar reaction pathway we can see even for that simple thing we're getting stuck in different steps so we have to be a lot better than they are and can you in a rational way design allosteric switches something that you could control with light or live in or something yeah well we're trying to do that now actually because it turns out there's a phosphate binding site in this capital core of the back of the company so we're now trying to see if we can put in larger groups of phosphate and see if we can modulate the F2P that way so I think that should be possible finally on the DNA interface aspect of things how frequently do you find a base by base recognition pattern versus multiple bases and affiliated changes coordinated changes of sections that's right so we've now found we have some cases now we've been able to redesign four consecutive base pairs and they clearly are much more more than the individual residue substitution so you need to consider them all at the same time do you see sidechains move in a coordinated way I think that's exactly what happens that you change one and therefore you can change another so we do see exactly that kind of thing going on we do this design