All right, traders, this is Falstow. Let me do a quick audio check. If you guys could hear me loud and clear, give me a chat back where you guys can hear me okay. All right, good, good, good. All right, everyone, just give me a second. We'll be getting started about a minute, okay? All right, ladies and gentlemen, let's get started. Let me just do a quick audio check one more time. Just share a couple of screens over here. Just, there you go. Okay, thank you very much. All right, good, good, good. All right, everybody, welcome. My name is Falstow Pugliese from Cybertree University. I'd like to thank you all for being here on this lovely Wednesday afternoon. We're gonna have some fun today. We're gonna talk about mastering the real-time order book and analyzing for stock trading. So the whole idea about being here today is to learn how to play the game of trading. Now, my goal today is I'm not here to entertain you. I'm not here to talk about the winners and how great we are and pound our chest why we've been around for so long. I'm here to teach you how to stop losing money. And the real reason why people lose money in trading is they just don't know exactly what they're looking at. They don't realize that you have the access to see where buyers and sellers are, all that good stuff. So we're gonna cover that. We're gonna go out there. I'm gonna show you a couple of things how to trade today's markets. But before we do that, just really quick. Just wanna touch upon a couple of things here. First of all, this is strictly for educational purposes. So just let you know that I'm not making any guarantees or promises or anything that stuff. So please be very careful that there's a big risk when it comes to trading.  Now, everybody here, if you have your phone, right there is a QR code, okay? And if you turn on your camera, you'll be able to have access to a one week trial. Now I will be posting this QR code and the address again at the end of the presentation. So if you wanna cut the line now and have first priority, here's your chance. But everything I'm gonna talk about, I'm going to invite every one of you to come see this live in the market for one great, wonderful week. So listen, I can only make it very drooling in your mouth in the next 45 minutes.  So go out there and register, all right? So let me change the slide here and tell you.  I've been investing, it's been a couple of weeks, couple of years, how long you guys been doing it for? Just give me a little chat back, three months, okay, Don? All right, good, good, good. And another question I'd like to launch while you guys are filling that out, what kind of trader are you? Are you a stock trader, an options trader, futures, forex? Let's see what kind of traders everybody is. But once again, if you're in watching on social media, just put it in the chat room, you got a chat over there on the right, just let me know. All right, so let me end this poll and try to share it, everybody. So you could see right here, we've got about 75% of you stock traders, a lot of options traders, wow, futures and forex. Okay, so let me explain something to you. The way I look at this for, the way I look at this is that if you've been doing this more than 60 to 90 days and you're not making money, you're pretty much wasting your time, the way you were trained or what kind of market you're trading. I'm an actual day trader. And what exactly is a day trader? I'm in and out the same day. The reason why I love day trading is I'm in cash every day. Okay, I still swing trade, I do do some options, but you can't be a good swing trader unless you know what's happening over the course of the day. You can't be a good options trader if you don't know what's happening with the stock over the swing. And a lot of you guys are doing things backwards and once again, I'm not here to knock anyone's style, but this is an easy way to answer it. If you have anyone here on the business or you work somewhere, if you're training someone for, after doing all that research and doing all the diligence and all that vetting and someone's finally hired someone and they're working for you for 60 to 90 days and they're not getting it, guess what? They gotta go. You're no different, okay? But there is something you could fix because maybe that's just not your style. So let's get on with it right now. Now, before we get to further and start getting to trade, let's get started and tell you where we will begin and how I started. Cause some of you probably wondering, Paostal, I never heard of you before, okay? Even though you've been around for 30 years, okay? Me and Doris, brand new brokerage firm. Let me tell you how I got started because my story is no different than some of you, okay? I'm 52 years old, been doing this for over 30 years. I was one of the original day traders that started. When I started, it was about maybe 1,000 traders. Now there are millions of you. Right on the left-hand side as a bunch, basically I was one of those kids, a bunch of kids tormenting Wall Street from Page on Orbs. You could see we used to make money on a 15-inch monitor. Some people came and make money on four. I'm also a number one best seller of how to beat Market Make as their own game published by Wiley, big, very well-known publishing company. Now that book right there, you could buy it on Amazon for $47, but if you stick around and you click on that QR code, I'm going to give you my book for free, okay? Very fast read, great read. And let me tell you, nothing has changed 30 years ago until today other than technology got better. You didn't have to get licensed. You don't have to come live in New York. And that basically is the QR code. Actually, what we'll do is John, I see Alex, I see you guys in here. Could you put the link in there for Tammy so that you could just register on for it? Just give it that link in there. All right, there you go. You're more than welcome. Now over the years, I've competed against every school in the industry and I beat every school in the industry, okay? Actually, some people even show up. But you know what I've learned? It's not about how good of a trader I am personally am. It's am I a good teacher, okay? And what I'm about to show you is exactly the way I was trained. Now there's a lot of people out there will teach you and they'll talk about indicators and they'll talk about all these shortcuts and will tell you about, oh, let me give you a watch list, like a hot list and scanners. It's not about that. Autopilot in a car is pretty cool, but do you really trust it? You got to drive the car. You need to know what's wrong with it. You need to know how to change the tire. And that's what happens when it comes to trading. So I've been doing this over many years. I've trained people all different walks of life from engineers, first responders, grandmas and grandpas, professors, scientists. I mean, some of the smartest people that you would even imagine. And some people not the most smartest but the most greatest entrepreneurs. And I know a lot of you here maybe look like a lot of the people that you see right here that are students at CTO. So my goal is to have some of you to be some of them. Now to do that, I need to show you exactly what trading's about. Now, before I go into talk about the trading, just to let everybody know, tomorrow I will be live at NASDAQ at 1 PM tomorrow. Now NASDAQ just to let you know, we're one of the probably the only educational schools out there that they want you to learn how to trade the market. So for everyone that registered or once again, we'll post that link up there. You'll get to see me a little bit more about it. And why is so important that you need to know how to trade these markets because it's not that I'm telling you so, it's even the exchange telling you so. Now, question I want to ask everyone regarding about a strategy. Okay, so let's start getting into the fun of the trading part of it. What is your strategy buying a stock? Is it charts, is it indicators, is it news? Just want to see what everybody's strategy is behind trading. So just put in the chat out there if you can. Charts, chart indicators, okay. Indicators and news Donald, okay. David says gurus. Well, David, remember you got to hit the button the other day, all above 10, candlesticks, charts. By the way, the buyers are Brian, okay charts. Listen, this is how majority of traders fail. Everything that you said, Brian, Robert, Kessner, Ira, you're all wrong. I hate to say, I know that you might be looking and be like, what, what did this guy just say? Yes, I'll tell you why 90% of the people fail in trading because they go out there and they're not using the right tools that they need. Okay, remember, you want to know how to read the future not read the past. What is the past? It's an indicator that is delayed. It's news that already came out. You ever heard of something buy on rumors, sell at news? Well, why are you buying at news Samuel? You know what I mean? But what you do need is the right tools they're going to show you. Now, how do I know this? Let me remind everybody, I was a market maker. What is a market maker? I was a trader for a brokerage firm. What I've learned as a trader, I realized that everybody's trading backwards because it's not about what you think is what the street thinks. You need to know where the orders are and that's what I'm about to show you. So let's talk about some of the scanners and let's talk about some of the catastrophes that we have been. Let's talk about the horrible, horrible effects that happened in Hawaii, okay? And first of all, let's hopefully, let's pray for those people out there that are alive. And once again, a heart's to go over the people that lost their loved ones there. It is a huge major catastrophe. And you know what? We deal with this all the time. How do we deal with it? What about COVID? What about 9-11? What about Hurricane Sandy that affected me here? I mean, there's always these catastrophes that you see, but we are traders. And unfortunately, these catastrophes do make opportunities for us traders. Now there was a stock, now does everybody know who's being held accountable for the fires? Anybody here know what's going on? Like who's being held accountable for it? Just out of curiosity. Donald, yeah. Okay, well, that's only one person.  I can't go after climate change. But a lot of it is being blamed on an electrical fire, okay? And you're right. And it's this company right here, H-E, Hawaiian electrics, okay? Now, listen, I'm not here to debate you about telling me, oh, you got it wrong, this and that. Let me explain something to you. Why did this stock go down from $35 down to 10 in three days, in four days? Now, as much as you might look at it, listen, I was involved in 9-11, okay? I remember sitting and doing a coaching class with one of my students. He was in Las Vegas, and he was a, actually, I don't forget, he was a blackjack dealer. Even he wanted to learn how to trade. All of a sudden, we're watching on the news in a plane crash into 9-11, and that's right, right at the block from me. And what do I do? You think I run out? First thing I do is I hit the short button on everything, because if it really was true, right down on a heart of Wall Street, I knew it was gonna be closed forever. Well, what do you think happened with HE, with the fires? Boom, look at that stock from 35 down to 10. Now, you think about it, if you had 100 shares, not even 1,000, let's say 100 shares, 100 shares, and you knew that that was happening when the fires started kicking in, and you could start seeing it's going down, down, down, down, and you shorted it at 35. And then you buy it back at 10. You made 25 points, 25 points on 1,000 shares. 25 points is $2,500. Who doesn't want that job? Okay, because it all started with a catastrophe. So not only that, that even though you could have shorted it on the long-term or in options, but the only way you knew it was gonna go down is because being a day trader, you could see it, the stock kept trending lower and lower and lower and lower, okay? And that's what trading is all about. Let's talk about Hurricane Sandy really quick, okay? So big catastrophe, huge catastrophe here in New York. Do you know who benefited from a hurricane? Would everyone realize? Generac, Home Depot, those stocks took off because what happened? Everyone realized they lost power. We lost power for two weeks here. People like, well, I'm not having that happen again. You heard what happened with the gas. People were fighting online because you're gonna get gas. There was no backup generators. Generac made a fortune, okay? So COVID, I just did an event on that. Who killed it on COVID? Moderna, you could have bought the stock at $24. I didn't even know what Moderna did. I didn't know they had the COVID vaccine. I actually, you could see a video on our website when I was at NASDAQ talking about it. Where'd it go to $500? Why? Because some of you probably didn't know this, but how much you take a course for a COVID shot? How much you think they were getting paid? Didn't cost you anything. We know what of course what they were getting paid. $2,000 a month for one shot. Not $10 copay, not $500, $2,000. Nice way to be in the business, right? So anyway, there are always stocks that will benefit and there will also stocks that will bounce back from it. So the goal is what made that stock go down? What made the stock go up? Well, that's the beauty about trading. And let me just tell you one more catastrophe. Remember PCG back in 2008, the fires in California, who were they being blamed for? Another electrical company. And look what happened to that stock. We're from $50 all the way back down to almost $5. And then, oh, no, no, no, no, no. Wasn't their fault. Wasn't their fault. And the fire hit. And you could see the stock hovered down November to, you know, for about two months, hovered around 10, 20. And all of a sudden, oh, no, it was their problem.  It was someone lit up a fire in the woods. And all of a sudden it happened again. And we all said, what ended up happening? One of those big transformers blew up. Okay.   You can't take this personal, okay? Because as a trader, those catastrophes make opportunities and help pay you bills. Because if you can't and accept it, you should not be trading. Now, the question everyone's always asking is, how do you find these stocks? How do you know that that one was moving? I don't have a crystal ball here, but what I do have is what you all have is a big percentage gainers and losers. Those big percentage gainers and losers will drive the stock, will help us find those stocks that are moving. Like today, there was a whole bunch of them that moved pretty cool today. And we'll talk about them. Now, so for some of you here, you're saying, oh, I subscribed to somebody's newsletter, this and that, you're not subscribed to anything. You know what you need to subscribe to? Our trading room, that link that I gave you. And you'll see that we'll show you how to find these stocks and how to pick them out. Knowing which one is going to give you the least amount of risk with the high amount of reward. Now, even though we find these stocks, question is, how do we know which one's gonna continue to go higher? How do we know the one's gonna continue to go lower? Who's gonna, you know, what's making going up? What's making going down? So what I wanna talk about is something called level three and level four. So let me just do a poll here, if I can. My question is this, do you have NASDAQ book viewer? So let me do a quick poll. And once again, I know if you're on social media, you can't do this unless you're in our trading room. But if you guys are willing to, if you're watching on social media, just go in the chat and just write it in that if you have it, yes or no? Yes, no, don't have no idea what I'm talking about. So let me end the poll here. And you can see a lot of you don't have it, almost 80%. All right, now, listen, I need, we have over, you know, 100 people in this room, okay? I need your 110% attention right now because what you're about to learn and what you're about to see is going to be the most disturbing thing you've ever, were ever told. Because you're gonna realize that everything that you were trained was the total opposite. And I can almost guarantee that. Now watch very careful, I'm about to show you. You heard of something called level one quotes, you maybe heard of something level two, but I don't know, you spent a lot of you never heard of level three, and that means that none of you probably even heard of level four. So once again, I am going to be on NASDAQ tomorrow talking about NASDAQ book viewer. And you're all welcome to join, okay?  So level two only tells you the buyers and sellers out there, but it doesn't tell you all the buyers and all the sellers. So let me explain to you what we're looking at. I think we all could agree, is that stocks go up because there are buyers out there and stocks go down because there are sellers out there. Now think about this, how much smarter and better trading decisions would you make that you could make if you're able to see those buyers and sellers out there? Okay, so what we're looking at NASDAQ book viewer, let me get my crayons out here so you guys can see a little bit better. Hold on, let's say we draw something here. Here are your buyers and here are your sellers. Greg wants the QR code. Could you post the link up there again? I think we have the link there for you. Here we go, here. You can just click on this link, Alex put it in there. There's the link right there, Greg. All right, so you got buyers and sellers and you got two columns.  That's what you got right here. Buyers, shares and the big price. Now here you're seeing a stock, it's going down, right? Now for some reason it decides it wants to stop right around this price around 1565, which is right around here. And then it hits it, it hits it, it hits it, it hits it. The volume goes back up. You notice how it never wanted to go lower than 1565? Why is that? Well, the correct answer is there was a 84,000 share buyer that there were 15 orders that made up the 84,000 shares at 1565. Now there are buyers at every single price level, okay? You got 100 shares, 10 shares, 25 shares, but you're talking 85,000. That's not, you know, chump change. That's a lot of shares when you compare to all the other buyers and sellers out there. So buyers equal support. Now everyone's doing the opposite. They want to know where to support resistance. But you know what? You cannot have a support at resistance levels without those buyers and sellers out there. So there's your confirmation of buyers that are out there. Now let me change the slide and show you another one. Here's the resistance levels. Here we're looking at a stock Mara, okay? It started right around 9.30. Hit this price over here right around, it was at 10.20 or something like that. Hit all we went to a price, hit a resistance levels around 10.70 and it came right back down. Why did it 10.70 and came back down? Well, because there were sellers out there and when you look over here on the right, you'll notice that there are a lot of sellers out there right at 10.70 for 92,000 shares. Now there are 4,000 shares, 10,000, 3,000, 8,000. You're talking 92,000. You're talking between 10 and 20 times more of the average than at any single price than 10.75. Let me repeat it. How do stocks go up and go down? Buyers and sellers. What are you seeing right here? Buyers and sellers. So when you wanna wonder how a stock goes up and goes down and wondering how it got there, well, that's the reason why because that big seller out there. So how much smarter and better trade decisions which Vue made if you were able to see those orders? Michael, it's not only visible to me, it's visible to you. The Mike's question is, when was that 92,000 share visible to you? When all those 20 people put it out there. It could have been aggregated and the attitude. You're gonna see this live in my market. I'm not holding any punches. Listen, I'm not a fan of PowerPoints because anyone could cherry pick, but when you start seeing this moving, you're gonna see a total different perspective of how things move. Now, let's go over a few stocks that we traded this week. So let's talk about CVS. Now, my question to everyone is this, what is the direction of CVS? Is this going up or down or going down? See if you guys can answer this question. Up or down? Kay, Donald, you're right. Michael, you're right. David, right. Tammy, Xavier. Everybody should be able to answer this question. Now, let me say it, and please don't take this the wrong way. I'm a little brash sometimes, but if you can't answer this question, like Brian, I didn't hear from you. Charlie, I didn't hear from you. Alberto, I didn't hear from you. You should not be trading. Every bunch should answer this question. What is the direction? Please, don't make me waste my time. I'm here to stop teaching you from losing money. Why? What is the direction of this stock? I'm gonna ask it one more time. Is it going up or going down? Thank you. Okay, good. Thanks, Juan. Thanks, Brian. Thanks, everyone. Okay. Now, this is important to answer this question because this is gonna lead to you from stop losing money because if I had a stock at 69, I only had 65. I'm crapping in my pants right now. Do I sell it here? What do I do? Well, I'm gonna ask you a little question. What's gonna make the stock from stop going lower? What is gonna stop this from happening? You need buyers.  Is it that easy? You know what? Yes, it was. And the issue is this. I don't see any buyers on a chart, okay? So, let's go check over here on the CVS and NASDAQ book viewer. And now you'll notice that. Now, I'm gonna look at the buy section. Right when we get around 64.75, there are 35 orders out there that are looking to buy the stock at $180, 108,000 shares, okay? That make up that 35,000. Now, there are 20, there's 200 shares, 1,000 shares, 300 shares, 100 shares. You're talking 108,000?  Well, guess what? That loser that you saw was going down, all of a sudden became a winner. Because when that buyer showed up, you had a few minutes to see that happening. And that went from that price of 64.75 and shot all the way back to $66. How about that? Here you're losing $5 going down. Now, if you were patient and knew where the buyer was, you could have made $2. Did that seem hard? Did I lose anybody yet? Anyone get lost? Anybody on YouTube? Because I didn't hear anybody from there. Good. Simple enough, right? You found a long one. That's what I want to hear. All right. What is the direction? Why is Apple going up? Why would Apple go up? What's the theory behind it? Oh, very good, Don.  Buyers, right? Does everybody agree? There must be buyers. It's the only reason why it's going up. Not because it came out with a new iPhone, whatever it is. At this moment in time, there is buyers. So, is it going to continue to go higher? And being a good trader, if I owned it at 184, my goal is where am I going to take a profit? Well, where do you see the big orders on Apple? Can anybody tell me? Let's see if you guys are going to be able to figure this one out right now. Brian says there's a big order on PLTR for 300.  I'm going to bring that up. If nobody knows the answer to this question, just give me a question, Mark. Oh yeah, that's a good one. Thanks a lot for sharing that. Well, the stock's going up. We're looking for a seller, right? So we have a seller here, the sellers. We look down the seller column. Oh, right here. Is this what you're talking about? Just sell it right here, the 50,000 shares. I don't know if you think it's a lot, but for me, when I see people looking only sell 400, 600,000, 1,000, yeah, 50,000, I'll take that very seriously, all right? So, clear this out and change the slide, see how that worked out for us. Well, doesn't show that in the chart. It's going to go down, I do start to see, it's starting to consolidate. And guess what? It hit it, came back down. Hit it, came back down, okay? Not once, but twice.  I don't care about that stuff. Professional traders don't use indicators. We follow orders. You want to know where the institutions are, you want to see where all the algorithms are, you want to see where the dark pools are, that's where they're at, when you're looking at the NASDAQ market. Now, where can we get NASDAQ book where you're all going to ask? This is what I'm going to do. Here's my email address. So if you've been paying attention and you're interested in book viewer and you want to know where to get it, I'm going to tell you something pretty cool about it. When I started, it cost me $1,000 a month for this data. Okay? Now, is that a lot of money? I don't think so.  There's nothing less than 60, 70,000. You go to community college, there's still 5,000, 10,000, okay? So if I was looking to buy a six-figure salary and it cost me $1,000 a month at 22 years old, I'll pay for it. Guess what? It doesn't cost 1,000. It doesn't even cost 500. It doesn't even cost 100. It costs $15, 15 bucks. You're telling me that you're going to get the opportunity to see 50% of the volume in the market have a seat on the exchange and I pay the $1,000 a month and you don't want to pay $15 a month for it. You don't think that's going to help you make smarter and better trading decisions than trading is not for you. And that's why I'm going to be at NASDAQ tomorrow talking about it. And that's why NASDAQ wants me to talk about it to you because there's just too many people losing money in the market.  Why are you making that mistake? Let's take care and go to the next level. Now, what does 50% of the market mean? It means following the money. Michael, that's what it means. All right, let's upgrade to something called level four. All right, so everybody got my email address so you put it in there one more time. Thalstowe P at TPU corp.com. All right, all right, good. Now let's talk about level four. Now, level four is more of a heat map, okay? Where you're going to see orders how long they've been out there for. It's going to aggregate other exchanges. You could use this towards futures, options, cryptocurrency, you know, you could see, think about it, it's just an amazing tool. Now, let's look at some stocks and let's see what support levels look like, okay? So here's a stock that's trending down, okay? And you'll notice that there's a little red line right here, right around 105 for about 35,000 shares. Now there are sellers at every price level. The COB is current order book. Look at that as an Aztec book viewer. But now you're seeing time in sales, you're seeing trends, seeing all that. And the thing is what's cool about this is when you get to 105, you notice that there's a 35,000 share buyer out there. When it hit that buyer out there, it went up. It hit it, it went up.  Maybe I want to buy it. And he's probably the one that ran the stock up a dollar. Look at the stock, AMD. That's a stock that, that's basically what happened. It even works on brand name stocks. Let's see how stock reverses at a resistance levels. Here's a stock going up, all right? And then all of a sudden here comes a seller around 955 for about 32,000 shares. See that little red line? What do you think's gonna happen when you get a stock that goes from 1340 and now within about, I don't know, 30 minutes, you're coming up to a big seller that decides he wants to sell it. Exactly what we thought it was gonna do. It's gonna come back down.  Now let's check the live markets. Now there was one student, one trader in here who just found stuff. I want you to go check out PLTR, okay? So PLTR, this is a pretty cool one. And let me bring up PLTR. So PLTR, everybody, let me go get this chart. PLTR is doing what? Is it going up or going down? This is the live market. Don said it's going up, right? Up, up, up, up. All right, so the question is, what's your game plan? You had the stock here at the open at $14, $14.50. Now we're at $15.30, okay? Is the stock gonna continue to go higher?  What do I, I mean, is it gonna continue to go higher? Is it gonna go down? Well, what's our game plan? Let me see how you got, how would you analyze this? Mike, I think it's gonna go higher.  How did you know that, Dan? Davis has taken a profit. Dave, why are you gonna take a profit? What's giving you the reason? Well, let's look at this. Let's go to, let's go here to the heat map, okay? Now, you notice here's red line right here.  Guess what? He got executed. Once he got executed, the stock went higher. There was two sellers out here and they all came out at 9.30 in the morning. That's called program trading. This is what you call an iceberg order. Here's a seller right now for 300. Let me zoom in here a little bit closer so you can see it. There's a seller right now, right here if you notice. How many shares is that guy looking to sell, everyone? How many shares is that? 350,000 shares, okay? 350,000. What is, let's bring up our calculator. 350,000 shares, right? Hold on. Times that by $15.30. It's about $5 million worth of stock, okay? That's trying to sell out there. Now, are you telling me that as much as the news could have been great, let's say earnings, I don't know what's making the stock drive up, but you're telling me no matter how great the news is right now that you want to still hold on to the stock and want to think it's going to go higher, because in theory, the stock did do pretty well. I mean, you're looking at this right here and you could notice that, wow, the stock was at like 20, took a big hit, it's on the uptrend. Oh yeah, definitely, the stock is going higher. The stock resistance here on my indicators after talking about the moving average, my sarcastic, my RSI, my bolager bands, what else you guys use over here? MACD, I heard that's pretty cool. That tells you a lot, right? Let's just see, how about that? Does that answer your question? After all that education that you spent in, does that tell you what's going up? Yeah, that's right, you better laugh out loud, Brian.  But if there's anything I'm watching, it's I'm watching the big orders. That's what controls the market. Is this guy right here? Now, one last thing before we go, before I can get you guys registered again, some people ask me, well, how would I use this towards swing trading? How could I use it towards long-term trading?  And looking at the chart, technically, the resistance levels was at 16. I'll show it to you right again. But for me to do that, I gotta take off a lot of this junk because it's driving me nuts. I'm getting confused right here. Let me take off all these indicators. So if I drew a trend line at 16, right here, you could see clear as day that, yeah, 16, support levels there, resistance, support, resistance. So why is this resistance? Why isn't that resistance? Well, because when we look at this, you gotta deal with that seller first before we can deal with this seller second. That's all I'm saying. Now, NVIDIA. Tomorrow, I'm gonna talk about NVIDIA and NASDAQ. Now, what's going on with NVIDIA? Anyone know? What's going on with NVIDIA? Exactly, earnings are coming out. NVIDIA, you know, really big move here on NVIDIA. And bring this up. NVDA. So everybody knows the story of NVIDIA and NVIDIA had a really nice move today. You could see a ramp from like 452 to 460. You could see it right here. It's got resistance levels. Now, your question's asking like, well, is it gonna continue to go higher? Well, I can tell you this right now. You got a big seller here at 470. You got another biggest seller right here at 475. If I zoom out, you could see another big seller more majority at 480 and then 490. And we look here in the chart, you could see here, and I'll zoom in over here and make a little bit better for you. Here's your chart at 470, 480 and 490. So you could see clear as day. Oh yeah, I see the resistance levels here at 470. Oh, I see resistance here at 490, but is it really resistance? Well, there's no way to find out. Look at the orders. They're out there. They're out there and they've been out there. All right, so no, it's not. No, it's not. So there's no better time to learn than now, okay? I just showed you for $15, you can get this data, okay? Well, how about this? How about I told you it's gonna even be cheaper than that? How long, how about that? So, but for me to do that, I wanna prove it to you. I wanna bring you to the real markets. I wanna introduce you to all my traders and not judges by our winners, but judges on our losers because 80% of the people, 90% of people lose money in trading and that's really what we gotta fix. And then you'll get to meet guys like John G, how he traded AI and he killed it over here, making almost, I did an options trade right there or Alex or Phillip did it on the stock trade. You're gonna meet these people and this is the big thing I wanna point out. Look at these screens right here, okay? That screen on the right is me doing a presentation at Charles Schwab. Even Charles Schwab wants you to learn how to use it. On the left, look at the floor of the trading exchange over there. Look at them, how many indicators you see on this wall? None, they're orders. Those are orders. That's what it comes down to it. So this is why Cybertrain University has been partnered and endorsed by more brokerage firms than any school in the industry. And not only that, but if you ever look at any other school, you'll find that we have the highest, most successful rating on Google and on the Better Business Bureau than any school in the industry. Now, why is that even possible? Because we teach you what works, okay? Not teaching you the past, but we're teaching you how to read the future. So this is what I wanna do. I wanna invite all of you once again to come in my trading room. It's online, you can log in whenever you want, but I'm gonna give you one week access. Now, this is what we do. We have live audio broadcasts from 9 a.m. to 10 30 in the morning, and then live commentary from 2 30 to four. And what we're trading all day, but we have those two valuable times where we trade them first hour and the last hour. Now, we'll also have gurus that will come over the week. We have continue education that goes on. Remember the one that educates wins. So the faster you learn, the faster you'll learn how to control your losses, the faster you make this more of a successful career.  That's it. $9, you can click on the link that we just posted up on the screen or you can use that QR code. Let me post the QR code in YouTube also. There's your trial if you want that YouTube trial. And if you're on the chat, there's that link for the chat right there. Okay, so that's all you gotta do is click on that link and for $9, we'll give you one week in our trading room to take everything that we talked about and see it live in the market. Now, this is what you're gonna get. These are all the things that you're gonna get. You actually open up the trading room at 7.45 in the morning because a lot of the trading we do is in the pre-market.  We're gonna give you a Traders Talk Library, okay? We're gonna give you a coaching class. And you know what? To make this not even worth, make it worth your value and time, I'll even give you a money back guarantee. Listen, I don't need your $9, okay? I basically are looking for people that I could train that could be part of my group because eventually I'm gonna teach you to be so good. You're gonna teach other people. So we're always looking to recruit new traders, okay?  It's time, but listen, what's the worst thing that could happen with $9? You learn something, that's the beauty of it. So with that, you got the QR code and just wanna kind of, I see a couple of people are registering right now. Just a couple of shout outs really quick. Samantha, T from Florida just got your registration. Welcome aboard. Brian got your registration. Welcome aboard. Look forward to teaching you. These people actually registered early this morning. Greg got a registration. Vance got your registration. Welcome aboard. Fairmont got your registration. Thank you very much from Chicago. Welcome aboard. Now, if anyone has any questions, please it's the best time to answer them. I'm not clear, we get 15 acts of broken. No, no, listen, Silvia, NASDAQ sells book viewer for $15, okay? You should go buy it. Send me your link, I'll tell you where to get it. But if you want me to teach you how to use it properly, then for only $9, I will show you how it works. So when you do get it, you know how to use it because it might do more harm than good. When could you start? You could start now. If you wanna start on Monday, you could start on Monday, but lock in your promotion rate right now, because like I said, things could change. And once again, when you register, you're gonna have a registration page. And not only that, you're gonna make an appointment with an education advisor. We're not looking to sell you anything. We just want you to have the best experience as possible because I think I do more of justice for people to realize that trading is not for them than actually learning the hard way, okay? Any other questions? Do I need a brokerage firm to start? No, listen, we do not want you to trade. We don't want you to buy anything. We just want you to watch, okay? And trust me, you're gonna be confused.  I told you before, listen, if you don't get it, you shouldn't be in the first place. You know what I mean? You shouldn't be trading. So wouldn't you rather surround yourself with a guru that does it all day long? Cause that's what trading is all about. Now before we go, I'm gonna play the video that I just was on last month with NASDAQ. And so you can sit back and watch and listen to a video of why it's so important to be a very good trader. But you know what, for $9 fellow traders, what are you gonna lose? What are you gonna lose? So anyway, listen, I'm a little insulted. I'm a little disappointed. We have over a hundred people in here and I feel that all hundred you should have been registered already. What are you waiting for? I just told you, not only are we the most reputable company out there in the industry, but now you're also able to see the future and see orders.  You think you're smarter than them? Unless, Margaret, you're like a member of CTU and that's why you're here. That's why I know a hundred not gonna register. But there should be a hell of a lot more of you should be jumping to your chairs right now and not even thinking twice. All right, now I'm starting to see people coming in register. Michael from Ontario, Canada. Oh, you're from Toronto, Michael? Anyway, I'm gonna be in Toronto on September 9th at the Money Show doing a presentation. So, Mike, we'll send you a link. Make sure you're there. I'd love to see you. So think about this even better for you, Michael. You get to see, you get to do the class and then you get to meet with me live in person and I'll be loved to talk to you. Absolutely, that's great. So lucky for you, because I'm gonna be in your backyard. No, I'm not gonna be at Sawgrass this year, David, but hopefully gonna be the next one. All right, who else we got registered here? Another Michael K from Florida. All right, there you go. Got your registration. Welcome aboard. I start seeing the scenes. I see some of you start to register right now coming in. Go Gators. There you go. And it's not the Sawgrass you're thinking of. It's actually the big golf course that's down there in Sawgrass. Any other questions? How much money you need to get started? That's a great question. $9? That's all you need, $9 to get started. This will be the best $9 you'll ever spend and guess what? After the next, after, if you feel like wasting your time, I'll give you $9 back. And yes, I'm sorry, no. There's not gonna be no hidden charges or I'm gonna hit you with subscription afterwards we don't play that game. Listen, I don't need to scrutiny. I don't need to lock you in. I don't need to jump over hoops. I don't need your money, okay? I am looking for serious traders. And that $9 is separating from people that want free stuff and people that wanna learn from real traders. I can only teach you so much in an hour as far as interest, all right? All right, so with that said traders, let me play this quick video before we all go, all right? And forget about funding account. Don't worry about what you don't only count your funding is at $9 right now, okay? That's the least of your problems. All right, don't worry about all the day trading rule, this, that, that's the least of your problems right now. If you're here, then you're here. You make that investment for $9. Let me play the video. In the meantime, everybody, thanks for watching. Don't go anywhere. Enjoy the video on NASDAQ. I'm gonna play it right now and I look forward to seeing you teach you all you in tomorrow's class or maybe late this afternoon. Thanks for watching, everyone. Happy trading. We're trading university to discuss using NASDAQ BookViewer to trade market trends. And it's so interesting that NASDAQ were almost sector company agnostic. It applies for all different types of companies. All different ones, Jill. And it being earnings week, there's just so much been going on with the market in regard to some of things. But like NASDAQ BookViewer has been such a big help in today's markets. And I wanna start off talking about Rethiom. I mean, this stock in general just took a very, very big beating. It's been on a downtrend. It's been getting hit pretty hard. And sometimes people look at it like, when do I average down here or do I take my losses now? And when you look at the NASDAQ BookViewer, you'll notice that there was a 157,000 share of buyer right around that was at $81.74.  When you look at the BookViewer, look on the bid and offer. There's not that many shares, 10 shares, 50 shares, 100 shares. But when you see 150,000, it's like, why would someone wanna buy at that specific price? But as you work your way down, you'll notice that as soon as it came to that big buyer, the stock just basically bounced. And this is like something people have to learn that when they try to figure out, do I average down? Do I take my losses? Well, listen, the stock's not going to zero. There's always gonna be buyers and someone throws a good trade there. And that's why you have to follow the BookViewer. Right, we can see here that it tested at 81.50, exactly with them. And it bounced about five points from there almost a little bit more than that. So sometimes you can average your cost down, but you gotta know when to do it and when not to do it. All right, let's move along now to our next one, Siri, a name that trades quite frequently, a little bit of news in the market this week with that. So Siri's been on a really good tear lately. Its stock that basically was right around the three, $4 price range, been on a big rally. Now the thing is this, there's an old saying, whatever goes up comes down twice as fast.  Because not only could you use the BookViewer as a day trader, you could use it as a swing trade. So Siri's radio is making a really, really big run. So the goal is going to seven, is going to 10, is going to 20. Well, when you look at the BookViewer, you got a 531,000 share sellers sitting at $7.75, you know. And if you look as we move on to the next slide, you'll notice that it hit that resistance. And listen, there are 10,000, 50,000, but we're talking 500,000, you're not talking chunk change. Hits that resistance a couple of days later, boom, the stock comes right back down, and now you're hovering right around the $4 price range.  Yeah, let's take a look at D-Wack, right? I mean, the day traders love to trade this. D-Wack is such a fun stock. And this is a classic example of what's called a breakout. So everybody wants to know, like, how does stocks break resistance levels?  I thought it'd get out. My avicars told me to do that. Well, when you look at the NASDAQ BookViewer, the goal is you always wanna have a game plan. See, what's nice about it, it teaches you the future. You're not focused on the past. And it's all about those orders. And right here on D-Wack, which is a fun stock, there was a 240,000-share seller at $23. So as a stock, you can see it's been having a really good trend. It got to that resistance, and it stopped right there. Now, this is where the breakout kicks in. That 240,000-share seller got done. And the way you know that is by looking at time in sales, and you could see it obviously just trickling away, which a lot of people neglect and don't realize the value of that time in sales. But you sort on BookViewer, and you could see those orders actually dwindling down. And what happens? Stock goes up right to 25. That's really quite the run there. And you can even see, okay, when is the time to sell? If we look at the BookViewer there, and you see that 25, 24 price is what you need to be paying attention to. Because as you work your way down, Jill, you'll notice there are sellers at every single price level. And because we got away from fractions, we're at decimals, they're at every single penny. But you could see it when you work your way down on that BookViewer, boom, right there, 252,000 shares. So the game plan as a trader is like, okay, my next resistance level would be $2. Now, $2 is a lot when you're talking a short period of time. It didn't take too long to get there, maybe like 30 minutes. And $2 could be a very significant profit. But if you didn't get out there, Jill, look what ended up happening. Stock came right back down to $20.  And that's not what you want to do. And that's why the BookViewer is such a value tool. Let's look up and talk about XPEV, I believe this is an ADR. I mean, you can use it to trade anything. Absolutely. And what's very, I want to say the best and last, because this stock was a great stock that we traded the other day on the pre-market. Now, a lot of people don't realize that you can trade in pre-market and you could use the BookViewer. It starts really early. A lot of traders can't, depending on what kind of broker you had, they won't allow you to trade until the market opens up. But if you have a direct access broker, you can. But here on this stock right here, this was an awesome trade. So right around, what is this? Around $9.55, the stock had a big pop from $15.75 to $17.50. Now, it was classic about this stock that there was a 200,000 share buyer sitting at 1720. And he was sitting there for about maybe 20 minutes, right before the open. Now, I don't know, maybe he got an order, maybe some trader got an order and had to buy it there. But listen, if you have the BookViewer, you can see that because there was no other orders out there that substantial at that price. So it ended up happening. Once the market opened up, Jill, boom, the stock just took off and went past $18, shot all the way up to a price of $21 in a matter of 30 minutes. And it all was predicated on that big buyer that ran it up. And by you seeing those orders, and if you had access it to see what's happening pre-market, you could have had that game plan to get in. But once again, focusing on where to take that profit. You know, Faster, you would mention that you can trade pre-market, you can trade after-market. And for newer traders out there, is there a benefit to only trade from 930 to more or is it okay to trade pre- and post-market? Well, the advantage is that as without being a market maker and nice to trade, having access to that data before the street gets in or the general public to get in, remember they put orders in and they can't get their orders execute until the market opens up. But as a trader, we're out there like, let's say you had a guarantee order. I'm gonna go out there, I'm saying, oh, I got Jill wants to buy it. Well, I got to guarantee buy, I'll go out there and buy it. And everybody could see that who has access to that account. And then what happens when these orders start filling in, they have to get executed and that's how they get driven up the way they do. But when you have the NASDAQ book viewer, which basically controls maybe about, I don't know, maybe about 50% of the volume of the total market, that's a substantial amount of volume to kind of give you a sense of direction where the stocks can go other than, you know, everyone, general public should trade a couple of hundred shares. All right, I also appreciate the insight as always. Thanks for joining us on Trade Talks. And thanks for joining me. I'm Jill Malandrino, global market reporter at NASDAQ.


But how about the Bengals who represented in the Super Bowl just a year ago, finding their group went in like six out of the last six or seven on the last seven, dominating, but did playoff picture starting to get a little clearer? Darius, what are your thoughts on where we are right now, where we will be, and where we thought we would be right now in your eyes? I feel like we still don't have a flu, honestly. Honestly. I feel like anything can happen right now, especially on the NFC side, you mentioned it, because even when you look at the top teams, it's a bunch of guys who don't have a long track record of playoff success. The Eagles have constructed a team, they look awesome. Obviously Hertz is young, this will be a second time in the playoffs. But the Vikings, who knows, Cowboys, they always let us down. Niners, you got Purdy, so how will that go? So there's a lot of questions on that side, and the NFC obviously, you got the two top dogs with Kansas City and Buffalo. And then obviously, my preseason prediction of Chargers-Eagles, I'm still there with it. I like the Chargers, man. I think they're a completely different offense, so Mike Williams is healthy.


Hi, my name is David Rosales. I'm the pastor of Calvary Chapel of the Chino Valley, California. It's been said, the more things change, the more they remain the same. This reminds me of something Solomon wrote in Ecclesiastes chapter 1 verse 9. He said, that which has been is what will be. That which is done is what will be done. And there's nothing new under the sun. The more things change, the more they remain the same. We're living a very difficult time. The faith of many is being severely tested. We need to remember that the times we are enduring are not unique to us. Over the centuries, there have been many times of difficulty that the churches face. From the very beginning, the enemy has done many things to undermine the work of God in the church. He's instigated persecution, infiltrated the church through bad doctrine, has provoked people to live carnally, has attacked through intellectual arguments, undermined marriages, caused a vision, corrupted the use of spiritual gifts. Though all of this has occurred, the church remained and has endured because Jesus clearly said the gates of hell shall not prevail against it. The church in the United States is clearly under attack. This attack has come from outside in a variety of ways, including government intrusion and a virus. It's also come under attack from the inside. I haven't seen the church this divided in all the years that I've been a Christian. Much has to do with the advent of social media. There was a time when you had a disagreement with something that had been said or a personal problem with someone. You would actually take the time to speak to them. This is not happening as much today. Some still follow scriptural guidelines and speak personally to the person they have a problem with, but most simply post their disagreements on social media for all to see and for any who might desire to jump in to the conversation to add their own opinions. This kind of thing is so common that it's now considered not only normal but proper. There seems to be no shortage of experts on any given subject who seem to think that the world needs to read their unfiltered thoughts. The ones who concern me the most are the ones who are so free to teach people concerning Scripture and how to serve the Lord properly. In the New Testament, to safeguard the church, a system of letters of recommendation came into existence. You see it in various books of the New Testament, including 1st and 2nd Corinthians, and it's alluded to in 3rd John. This system was intended to protect the sheep from being tainted by false or immature teachers. And to be honest, you ought to exercise the same concern that the early church had concerning the spread of misinformation or bad doctrine. It's almost daily that I read a post that may have good intentions, but it's simply bad or undeveloped in its theology. When pointed out, the result is not sober consideration of whether what is being said is right or humble repentance when the thoughts are demonstrated to be wrong. The common response is an argumentative defensiveness and accusations. I know that my remarks are going to be met with the response that I'm speaking about, but may I ask you to deeply consider what you're saying before you furiously type out your undeveloped opinions? We need to join together to oppose the efforts of the devil to bring discord and division amongst us. Let's decide to love one another, and if you have a difference of opinion then take the difference to the person you differ with. Refrain from writing your thoughts out and posting them online for all to see. Let's remember that united we can stand, but divided we will certainly fall. Take time to consider what I'm saying. This is David Rosales, pastor of Calvary Chapel of the Chino Valley, California.


Welcome to Effective Online Communications. Let's take a look at these words. Effective and Communication. Put them together and you have Effective Communication. Now we see that Effective Communication means the exchange of a message by speech that makes an expected effect. In this module you'll also learn more English. While you learn how to speak and write well with friends and classmates. So here are the main things that this module will teach you. What a message is all about your story. It's you knowing about you and putting it down on paper and speaking it so that people will want to hear. Painting a picture in people's minds so that they see what you see and hear what you hear. Knowing about the objective of a message and why it's important and also technology. We're going to be using some tools that work well and help you deliver a good message. But Effective Communication is also about methods and tools that work. It's about tools like PowerPoint, Skype and gestures and spoken language that serve to share your story and help others as well. You're also going to learn what makes people listen and want to know you. You're going to learn how to give your message well online. You just have to see, learn and practice. Some simple rules and strategies. So welcome to Effective Online Communication.


It's a pleasure to speak here. I've learned a lot already earlier today and yesterday. My presentation will be entirely on medically focused applications of what you could call synthetic biology, but I've tried to relate a little bit to what we heard yesterday as well. So in fact I've heard multiple times that we have 20 amino acids. I mean that is certainly at protein level not entirely true. We have a closer in the neighborhood to 200 different amino acids in proteins because of a process that is called post-translational modification. So 20 amino acids are encoded in the genome but then afterwards after the protein is synthesized or during its folding a lot of modifications are added to those proteins and my lab is focusing on the synthetic biology if you wish of those post-translational modifications. And so I just give you a chart here of the different types of post-translational modifications. I just got this from SwissProt yesterday on experimentally validated post-translational modified sites. You can see there is a whole zoo of modifications that can be added covalently to proteins. And I will talk about this third most abundant one there which is called enlinked glycosylation which is the addition of sugars to the asparagine side chains in eukaryotic proteins in particular. I will spend a little bit of time also on o-linked glycosylation which is the addition of sugars to serine and threonine residues again mostly in eukaryotic cells. So eukaryotic cells do perform these modifications by nature and it's our take on this that we might as well use these natural modifications and re-engineer them to introduce particular functionality into therapeutic proteins. So glycans are not just tiny modifications on a protein. Just to exemplify that this is the crystal structure of influenza virus hemagglutinin. And whatever you see on there which is yellow is enlinked carbohydrate. And as you can see this covers large areas of the protein surface. So this is the dominant antigen in the influenza vaccines that we use today. And every area that's covered by yellow here is basically not available for recognition by antibodies because of steric hindrance. And this is yet a very conserved picture because in crystallography very often the outer branches of carbohydrates are not captured because of large mobility of these structures. So what you see here is effectively the part of the carbohydrate that is rigid enough to yield definable electron density in the crystallography maps. So we're playing with these carbohydrate structures in effect about 55 to 60 percent of all therapeutics we have on the market today. Biopharmaceutical therapeutics are glycoproteins and that includes all monoclonal antibodies or molecules derived from monoclonal antibodies. So it's a very important post translation modification for the biopharmaceutical industry. So we've been talking yesterday about standardization and glycobiology is a fairly recent field in its current generation. And so the standards for nomenclature of carbohydrates have just been agreed upon about less than 10 years ago now. And there's just a few weeks ago there was an update published on this. So just like you have the four letters for the basis in DNA and you have the 20 letters for the amino acids in proteins, we now have symbology for carbohydrates which is universally accepted. And because carbohydrates are branched structures, we cannot suffice with a linear denomination of consisting out of letters. We need to capture stereochemistry in this symbology. And so this can be found it's defined by the consortium for functional glycomics in here. So effectively what this is useful for if you would have to capture the entire chemical detail of a carbohydrate of moderate complexity like this one, I think we could all agree that it's not very convenient to write that out every single time that you would have to talk about such structures. And so the symbology now effectively allows us to reduce that complexity to something more amenable also to automation and to database introduction. So you'll see a lot of this in my talk. Effectively every symbol is a monosaccharide. Squares have glucose topology. So this would be enacetylglucosamine. Sorry, green dots or balls, circles would be mannose. And mannose differs from glucose because of the actual OH hydroxyl functionality on the C2 position. And then this would be galactose which differs from glucose because of an actual hydroxyl functionality on the C4 position of the ring. And so these are the main topologies in the carbohydrates that I would talk about today. So as in other fields we do have models about the biosynthesis of these carbohydrates and models don't always have to be quantitative. In fact we have no idea at all about the quantitative nature of the glycosylation pathway. So this is an entirely qualitative model meaning that we know which enzymes are in which sequence after one another. This is a very simplified depiction of the eukaryotic n-glycosylation pathway. So I'll just walk you through it a little bit. N-glycans, so those that finally end up on the aspergine side chains of proteins, are synthesized as a tetradecaccharide precursor, so 14 monosaccharides. There is an entire sequence which I have not depicted here of enzymes that build up this lipid linked, dolicol linked precursor oligosaccharide. And then as a protein that is in eukaryotic cells designed for the plasma membrane or other intracellular membranes or for secretion outside of the cell, when such proteins all contain single peptides that's recognized by single recognition particle in the cytoplasm until the ribosome SRP particle docks to the translocon, the sex 61 translocon in the endoplasmic reticulum membrane, then the protein translation resumes and the proteins kind of pumped inside the lumen of the endoplasmic reticulum. And at that stage, so prior to folding these n-glycan precursors are added to the protein. And then this entire process in the endoplasmic reticulum of eukaryotic cells is universally conserved in the whole eukaryotic world. And the reason for that most likely is that these glycans play a very important role in catalyzing the protein folding in the endoplasmic reticulum. So, some of these carbohydrates, and I will not go in detail today, some of these carbohydrates recruit proteins like calnexin and calreticulin to the folding protein. And with calnexin and calreticulin you get protein disulfide isomerases, which assists in resolving disulfide bonds that have formed inappropriately in the protein. And so after that has happened, so in other words, you can't touch this very much. If you remove glycans by simply mutating the sequence to which they tend to be attached, many, many proteins will simply not fall anymore in eukaryotic cells. This is also one of the main reasons why so many proteins do not fold in E. coli, because E. coli does not have this machinery at all. So that's why the biotech industry needs eukaryotic expression systems on top of E. coli for manufacturing purposes. This is the very single most important reason why that is the case. Then after this folding catalyzing functions have occurred, the protein is then processed to this mannus-8 click-nag-2 structure, and that's recognized for export from the endoplasmic reticulum to the Golgi apparatus. And in the Golgi apparatus, depending on which eukaryotic cell you're looking at, there will be an entire battery of glycosyl transferases, which I've just simplified here by this swarm of arrows, that will process that mannus-8 structure into rather complex end-glycan structures that are then secreted from the cell. And this is just depicting what a pathway in a very simplified way would look like in a mammalian cell, say a liver cell that produces the majority of protein in our serum. If you'd be looking at a yeast cell, this battery of glycosyl transferases would be entirely different. So the protein is secreted, modified with an entirely different set of glycans, and that causes a whole set of problems in the biopharmaceutical industry. And this is the reason why still today mammalian cells are the main expression hosts on the eukaryotic site in that industry. So this is the knowledge that has been gathered by many, many colleagues and giants on whose shoulders we stand for then engineering this pathway. Even in eukaryotic cells, in mammalian cells, such pathway will not convert with 100% efficiency every intermediate to every product. And so to build this, we need about 16 enzymatic steps starting from this mannus-8 structure. And all of that has to happen in the about 10 to 15 minutes that a protein has between the time point when it enters the Golgi and the time point when it leaves the Golgi for secretion from the cell. So in 15 minutes, you need 16 enzymes to work on that substrate in a sequential way to convert all of that to the final product. I think you can easily understand that this is not going to happen with 100% efficiency at every step. And the consequence is that then you get the synthesis of a large array of these glycan structures, some of which would be similar to the final product, but most of which would be biosynthetic intermediates along that path. And this makes it very difficult to assign structure-function relations to glycoproteins, to glycoforms. And so a goal that we've set for ourselves more than 10 years ago is to design a set of eukaryotic expression systems, expression hosts, that would produce particular glycoforms, single structures with a level of homogeneity that will be sufficient to be able to purify it from that mixture. And so that's what I will be talking about today. So as every synthetic biologist, one needs analytics, high throughput and highly reliable analytics to quantify what we are doing. And so what I did back in the days when I was still a graduate student is to use DNA sequencers, capillary DNA sequencers as the analytical platform to very quickly profile carbohydrate mixtures using capillary electrophoresis. And so that gave us the throughput that has been used to sequence the human genome to now be able to do clonal analysis of all these different clones that we generate along this engineering path. So we label, we basically take off the sugars from the protein, if that would be the blue line, it would be the protein. We take it off with an enzyme, then we label these sugars with a fluorophore called APTS, which imparts negative charge and fluorescence to the carbohydrates. Then there's a bit of cleanup steps, and then we go on the sequencer. And so the anode, the positive pole will be here, the negative one will be there, and we just separate them out using electrophoresis. And just like you would do for DNA or for proteins, we include size standards, which are depicted there. That's just a hydrolyzate of a carbohydrate polymer called starch. And so in this way we can very quickly at high throughput analyze these carbohydrates. Okay, sorry. So the platform that we now have built over the last 10 years allows us to make every single of the structures that you see here with a homogeneity of higher than 85%. And that has taken us many PhD thesis and many postdocs work to arrive at this particular stage. What it allows us to do, amongst other things, is to target glycoproteins. So this would be the protein that's the sugar, of course not in proportion here. But what it allows us to do is to target biofarmaceuticals to particular tissues, because different tissues in the human body express a different complement of sugar binding proteins called lectins. So by injecting proteins modified with particular carbohydrates, we can target therapeutics to particular organs. And so I will talk about one particular application that we've been working on, which was published a few years ago, which is the modification of carbohydrates with Manosix phosphate residues, which allows obviously to target Manosix phosphate receptors. And this is important in treating a category of human disease, which is called lysosomal storage diseases. And so I'll come back to that as we go. Our most recent addition to this platform is this. And that's I think what looks most like synthetic biology. We were at a certain point not happy anymore with what nature had to offer. And we basically reduced complexity of this pathway to its bare minimum, effectively allowing to produce just these very tiny dye and trisaccharides. And I'll also explain to you why that is important, at least in our mind, for therapeutic applications. Okay, so the first stage of this work involved the building, the construction, the transfer of the human angloic oscillation Golgi pathway, module, if you wish, to the yeast Golgi apparatus. And the reason why we wanted to do that is that yeast is tremendously more scalable in manufacturing than mammalian cell cultivation is. It's also much cheaper to do that. And so this is the scale at which yeast fermentation occurs in the industry. This is a factory for human serum albumin, which is built in the north of Japan, where human serum albumin is produced in literally metric ton quantities for use as a serum expander blood expander during surgery, where people just if you don't have enough blood as a surgeon, you can take just take the blood you have and expand it by diluting it with a solution of serum albumin. And so the doses that you have to inject there are in the hundreds of grams. And so you can make that in yeast and you can make it in yeast only at the for the time being. And so being able to construct things like monoclonal antibodies in yeast would change the economics of antibody production quite dramatically. But you need to get the glycosylation right. And so if this would be, in fact it is, the crystal structure of the FC part of human IgG. So for those of you who know for the mathematicians, antibodies are the molecules in our serum that help us combat infectious disease amongst other things. And they have a typically Y shape. Maybe there's some chalk here. So human IgG has this kind of shape. Sorry for that. A little bit like this. So there's a linker region here and what you see there is the bottom part of this. That's called the FC region. And this would be the FAP region of which you have two. These binds for example things on the surface of pathogens. This is where then the so-called effector functions are localized. This binds to other proteins on immune cells and that then triggers reactions like killing activity for the pathogen that sits here. Okay. So what you see here is the bottom part and you can see the blue squares there. Those are sugars and that's conserved in all antibodies that we have in our body. There's a universally conserved carbohydrate there and an n-link glycan of which the structures are normally like this. And what's been found about 10 years ago that if you remove this little triangle there, just one monosaccharide, which is called fucose, the antibodies become approximately 100-fold more potent in their triggering of killing activity for tumor cells. So this is called antibody-dependent cellular cytotoxicity. So if you remove that, you get much better anti-tumor antibodies. And so this is now being commercialized by companies like Roche. So their newest generation of anti-tumor antibodies are manufactured in mammalian cell lines that do not make such fucose anymore. Now yeast doesn't make it in the first place. So you don't have to engineer it out, which makes this somewhat attractive. Also 60% of all biopharmaceuticals today are antibodies contained in this part of the antibody molecule. So what we've been working on is to, you could call it B-cell eyes, the yeast. So you try and make a yeast which looks like a B-cell in terms of glycosylation at least. And so this is what yeast normally does. It takes man-8, and then it converts this man-8 into these hugely complicated, so-called hypermanus-related structures. Whereas this is what you need on the antibody. So at first I couldn't be more different, but it all comes from the same precursor structure. So by knocking out the first steps of this pathway and then, like we do here, and then building in the human pathway in a very optimized way, you could hope of arriving at least at this structure. Mind that this would also require you to have all the sugar nucleotides to build these monosaccharides on top of one another, which is also, certainly when we started, it was not obvious at all. Certainly for galactose, it was not at all clear that yeast made UDP galactose. In fact, it turned out it does, and we still don't know what it makes it for in nature here. So to do this, to build this pathway into yeast, we need to get all of these glycosyl transphases in the appropriate location in the Golgi apparatus, at appropriate expression levels and activities for this pathway to work with high efficiency. And so we do that by targeting the catalytic domains of these glycosyl transphases to the Golgi, by fusing them to transmembrane signals from yeast glycosyl transphases. So enzymes that, by nature, go to the Golgi apparatus of yeast. And exactly how to make that fusion, that's part of the part of the optimization work that needs to be done when you do this engineering. So this is the current results that we are getting from this after about 10 years of work, I must say. So this is what the yeast glycosylation pathway would look like, by nature, very complicated, and not all the structures you want. And then by gradually building in one glycosyl transphase after the other, we build up a glycosylation profile that looks like this, which is in fact better than what you would get from Choké one cells, which are the mammalian cells that are used today in biomanufacturing. So Bram Locans is working on this, finishing his PhD as we speak. So we introduced also a new trick, which unfortunately I can't tell much about yet, to remove all the yeast endogenous glycans, because if you build that pathway, there will be remaining yeast endogenous glycans remaining, about 10-15% of the total glycan pool. And that's really complicated for the pharmaceutical industry to get rid of. So you have to engineer it out, and we will be publishing very soon how we have done that now, effectively allowing us to make pretty homogeneous glycosylation profiles. Okay, we were talking yesterday about modularity and orthogonality, and so I just thought this is not published yet. But when you engineer these pathways, very often what we observe is that the new intermediates that we synthesize along that path, in this case, this Manos V structure, this has never been seen in evolution by the yeast glycosyl transphases, by the yeast Choké glycosyl transphases. So in other words, there also has not been selective pressure for the yeast glycosyl transphases, not to recognize these human intermediates. And sometimes they do, as you can see here. This is a glycosylation site, one site on a human protein called interleukin-22. And what we expected in this train was to get Manos V here, as we do on almost all other proteins that we've tested. With all of a sudden, there was like 30% or more of this new carbohydrate, which we'd never seen before. And so we did all the structural analysis, which quite a lot of work to do, including an NMR analysis of this, to find out that it was this Manos V structure, but now with a tetrasaccharide on top of it involving three, sorry, four different glycosyl transphases to build this. So all of a sudden now you make an intermediate from the human pathway, which yeast has never seen before, and the yeast-choké glycosyl transphases start building on top of that. They're just illustrating the kind of complexity you can get and the kind of non-orthogonality of these modules, these glycosylation modules. Fortunately, we could solve that problem now, because if you get this, this is likely to be very, very immunogenic. It's an entirely new structure. And so you certainly wouldn't want to have that in a therapeutic preparation. Okay, so we can build the human pathway into yeast after a lot of work that works, allowing us to make antibodies in yeast at like gram per liter quantities in a very short space of time. I mean mammalian cells, you need about three to four weeks of fat batch cultivation to get up to the gram per liter levels that people talk about. In yeast this takes three days. Okay, so you have a space time yield, which is at least in order of magnitude better than with mammalian cells. Okay, and then the second target that I was referring to is to try and re-engineer the glycosylation pathway to deliver therapeutic glycoproteins to the lysosomes of patient cells. Now whether the lysosomes, lysosomes are vesicles inside mammalian cells of which the main function is to degrade biopolymers that the cell doesn't need anymore. Okay, this is in a very simplified way what lysosomes do, at least one of their functions. And so if for this degradation of biomacromolecules to the monomer building blocks, you need depolymerizing enzymes and there's about 40 of them in the human genome. So genes that code for such acid pH optimum degradative enzymes and just one disease is illustrated here of what happens when you don't have such degradative enzyme. So this is in the case of Pompey's disease. There you don't have the alpha glucosidase in the lysosomes and this leads to the accumulation of glycogen in the muscle cells of these patients and gradually this becomes toxic to the cells and the cells lose their functionality. So Genzyme, one of the main biotech companies, one of the oldest biotech companies in the world, contributed tremendously to the outlook for these patients by realizing that the targeting system for these lysosomal hydrolysis involved carbohydrates. Normally when they come from the goji to the lysosome there is a receptor that recognizes Manus 6-phosphate on the carbohydrates. Now this receptor that normally targets these enzymes to the lysosomes is also expressed at the cell surface of almost all of our cells. So if you inject a glycoprotein, such a missing enzyme from the lysosomes, if you inject that in the human bloodstream, if it's modified with Manus 6-phosphate these receptors will capture it and endocytose it and transport that protein into the lysosomes of patient cells. So these molecules are amongst the most expensive therapeutics we have today. So the alpha glycosidase for pompous disease cells at about 300,000 euro per year, life long, for these patients. And in fact because these molecules are made by mammalian cells today, their levels of Manus 6-phosphate are very very low. On this particular protein is less than 5% on the currently used formulation. So that means more than 80% is cleared by delivering the first few minutes after injection and this is not a very efficient therapeutic. Although at the high doses that is used today it has changed the life of patients but you know there's clearly room for improvement on the manufacturing side. So what we've done is to take the yeast glycosylation pathway and yeast by nature builds Manus 6-phosphate like structures into its cell wall. The only difference being that there is another Manus residue sitting in the way, substituting the phosphate there and that blocks these sugars from recognizing the human Manus 6-phosphate receptor. So what we've done is to build the to pump up the levels of Manusylphosphorylation in yeast cells by overexpressing the genes that are the rate limiting catalytic rate limiting steps of this pathway. And then the question became can we now convert these kind of sugars into the kind of sugars you need for lysosomal delivery. We have no enzymes to do that you could do that chemically without any problems whatsoever but there is a problem there is a protein here very labile protein that you have to keep intact during that conversion. So we figured we needed new enzymes to do that. And then so this is just I'll skip this so this became the question how are we going to do this right. So as always as biotechnologists I think our first reflex is to go out in nature and think about where in nature there might have been evolutionary pressure selective pressure for such enzymes to originate. And what we realized is that actually fungal cell walls are very very abundant carbon sources in the biosphere. So we thought there must be bacteria that have evolved to grow on fungal cell walls as their sole carbon source. And in fact just going through the biological literature you can easily find a number of bacteria like that. One is this particular species cellulose microbium cellulose. And so when you grow these bacteria on yeast cell walls as their only carbon source you get the induction of a whole array of carbohydrate hydrolytic enzymes including some enzymes as we found that can indeed catalyze these reactions that we were looking at. So this has been done by these two very very good scientists in the lab. There was a project that took quite some time as you can imagine. So this is just showing you that we indeed could find such enzymes. And so I'll just walk you through this. So we have here are the carbohydrates that are made by this yeast cell that we've engineered to express very high levels of mannose 6 phosphate mannose. And those two peaks here would be structures that contain that. So when we take one of these enzymes that we found from this bacterium which we have coined with this name here. When you treat that they all of a sudden in capillary electrophoresis move faster which means that either they are smaller or they have more charge. And then when we treat those with calve intestinal phosphatase an enzyme that removes end standing phosphates now they run slower again showing that indeed these phosphates are now terminal and you can remove them. And we found a second enzyme that then also can remove these mannoses there and you also need to get rid of those for these ligands to be recognized by the mannose 6 phosphate receptor. So this organism effectively makes two of these enzymes. We also crystallized one of them this one here to explain why it worked and it's all in the paper if you're interested in that. So with two E. coli produced enzymes you could basically convert the yeast produced mannose 6 phosphate containing carbohydrate into what we really needed for therapeutic delivery. And so this was the effect then if we exposed patient cells so we took cells from pompous disease patients fibroblasts in this case because that's the only cells you can easily get from patients. And then we exposed these cells to increase in concentrations of our recombinant enzymes and we looked at how much enzyme got into the cells. The current therapeutic drug had this behavior here and then our yeast made drugs we needed about 15 times less of that enzyme to reach similar levels of activity intracellularly. And this is just because we have about 15 times more mannose 6 phosphates on these on these drugs. This is also translated in a mouse model and unpublished yet in a non-human primate model in increased clearance of glycogen from the muscles of of these experimental animals and it's now moving into phase one slash two clinical trials because this isn't genetic disease you can actually do phase one and phase two at the same time because a precursor drug I mean the current drug is the protein part is exactly the same. So there's less issues with suspected toxicity of these kind of products. So this is being commercialized in this company called Oxyrane and the R&D division is in in Ghent. Okay how much more time do I have right now? Okay perfect. So everything I've said so far is basically taking existing pathways or more or less existing pathways from other organisms and putting them into another one which in our case was yeast. And the second story involved the discovery of new enzymes that we needed which were not present in the human pathway but which we needed to convert the yeast pathway into the human pathway. So the next part and this is something that started about five six years ago in the lab is when talking to biopharmaceutical industry people very often they they'll tell you that they have to live with carbohydrates on their molecules because that's that happens to be how nature modifies these proteins and then you have to engineer them to such an extent that they don't harm your function but if you actually could get rid of carbohydrates altogether in eukaryotic cells for a large number of applications that's what they would prefer but they can because it introduces so much complexity to the manufacturing processes and it introduces heterogeneity in the pharmacodynamic behavior of these molecules but you can't get rid of them so far because you need them for protein folding so that there has been this catch-22 for a very long time and and so we figured perhaps it is useful if we could find a solution for that and so I'll skip that if you have questions on what applications would be useful we can come to that I'll show you a few. So this is what wild type cells mammalian cells do they'll make n-glycans of the very complicated type that they've already shown you they also make oglycans which can also introduce quite a bit of complexity there's about in the range of about 200 different structures known in humans here there's about 500 actually known on the oglyc hospitalation site so there's tremendous complexity that can be introduced so what we made is something we call glycol delete where we re-engineered this pathway short-cutted it basically so that we now construct n-glycans of this trisaccharide shape that is identical to the outer branches of what you normally get in mammalian cells and then we've taken it one step further and that's as yet unpublished where we then completely remove heterogeneity only keeping one glucic residue at the n-glycol oscillation sites and entirely destroying oglycol oscillation because oglycol oscillation is not important for folding with very few exceptions so you can't get rid of it actually and from mammalian cells and keep the cells alive this is rather drastic and it was not known at the moment when we started whether this would be compatible with cellular life so this is one of the I think a small example of how sometimes this kind of engineering can teach you more about what is really necessary for cells and for from unicellular life okay so I think the concept was simple you recognize this from the ER we get this manus-8 structure and then we wanted to kind of put a pair of scissors in the in the goji an enzyme that would take off the glycans entirely but for the last residue here and then we were hoping that the endogenous glycosyl transphases galactosyl transphase and cialyl transphase would take that as a substrate and build this trisaccharide which is identical in structure to what you normally get on the outer branches so this was done by Leander in the lab who is still with me right now so then we started playing and you need selective pressure like everything if you were doing engineering you need some kind of selective pressure to select for what you want and for sugars such tools are less elaborate than for proteins and nucleic acids but what we do have is cytotoxic lectins sugar binding proteins that have a toxin domain like rice and toxin there rice and toxin binds terminal galactos right and that if that binds it will kill you as you know rice and is very toxic now if you re-engineer such pathway and you make a mutant that does not make galactosides anymore then the cells become resistant right and in this way you can select for a glycosylation phenotype in your cells that you want and we wanted to have these mannose 5 because the scissors that are available and only recognize such structures they wouldn't recognize the wild type carbohydrates that mammalian cells produce so then we got the scissors from this beast which is hypochrye jack arena that's a fungus that's used for making cellulases nowadays in the biofuels industry and when we studied the secretome of this fungus we found that it actually secretes its glycoproteins modified with just a single glutinac residue indicating that it must secrete an endoglycosidase of the type that we were after and so we clone that gene and it has this gene structure if you wish open reading frame coding sequence structure so it has the intermal pre-prosequence for secretion and the catalytic core and it has a c-terminal prosequence a peptide that as long as this protein is in the endoplasmic reticulum falls back onto the catalytic core and with the crystal structure in the meantime falls back on the catalytic core and inhibits the enzyme basically protecting the glycosylation pathway of these eukaryotic cells from this degradative activity and this enzyme this propeptide only gets removed in the trans-goji apparatus then fully activating the enzyme at the goji which means after the stage where glycans are important in the cell for folding catalysis which is in the ER so it kind of activates in the trans-goji and then removes the carbohydrates when they're not needed anymore for folding catalysis so again we used the lectin trick to select for the cells we wanted in this case we used concanavalin A which binds terminal mannocytes and there again when we remove these terminal mannocytes with this n-dot as we call it this enzyme pair of scissors we get the gluconate we don't get any terminal mannose anymore and that makes the cells resistant against concanavalin A selection so we analyzed that using this particular protein which is an important pharmaceutical human granulocyte macrophage colonist stimulating factor GMCSF and so it has two and glycosylation sites which you can separate from one another using cleavage with trypsin protease so that allows you to do simple mass spectrometry on these glycopeptides and so on the parental cell line where we got the high mannose sugars you get these of course then when we looked at the same glycopeptide from glyco-delete cells all of that is gone literally everything there's nothing detectable anymore and we get new carbohydrate structures which are the intermediates towards what we want to build here and now moldy mass spectrometry is now very quantitative I'll spare you the details on how we quantitated this but we have about 70% of this about 20% of that about 10% of this just without any further engineering so we're now engineering this further to pump it towards this structure and so we have good ideas on how to do that right now so this reduces heterogeneity quite tremendously on glycoproteins so this is gmcsf you see the mass here from about 14 and a half kilodalton to about 20 kilodalton all of that as heterogeneity introduced by the carbohydrates so it took me half a day to actually get any spectrum from that because there's so many different forms sure so many different forms here that it all tends to go into the baseline now in glyco-delete you have remaining heterogeneity but most of that is because of the O-glycans which are also present on the protein this sequence here is O-glycosylated there so Francis who's in the audience in the back took this one step further well he first looked at an application of this again here this is the antibodies which have this conserved glycan so if we produce a human monoclonal antibody in these cells we were looking at would that be helpful in any way for pharmaceutical applications what we found is this if you inject these antibodies in mice so this is in mice that must admit we haven't done it yet in larger animals but at least in mice when you inject these proteins from the wild app cells or from the glyco-delete cells what you'll see is that immediately after injection normally you lose about and that's true for all monoclonal antibodies you lose half to two-thirds of your antibody in the first half an hour due to so-called bio-distribution but if you actually look in the literature about what the mechanism might be for that there's absolutely nothing known there's no rigid scientific experimentation on that and so you lose it but we don't know how at the moment and so exactly at that stage we lose our glyco-delete antibody much less than the wild app-produced antibody resulting in about double circulating levels of the antibody with the same dose and now this is huge extrapolation okay if you'd be able to do this in patients what it would allow you to do if this were your therapeutic threshold you know your trough level that you need to have for getting therapeutic efficacy it would allow you to reduce dosing with a factor of two and like for antibodies with anti-TN where you treat against rheumatoid arthritis against TNF alpha these patients have to go to the hospital every four to six weeks for injections now if you could double that to every eight to twelve weeks this means quite a bit for for patients and we're certainly not at the end of what we can achieve there so i think we could get this even much further reduced and as we go forward in human medicine monoclonal antibody therapy will be much more driven by patient comfort rather than efficacy and so this might turn monoclonal antibody treatments into something that you can inject maybe three or four times a year all right so i'm almost done so this is the one step further that francis took it so francis also got rid of oglycosylation entirely in these cells and that's yet to be published but i can show you the result of that what we did is basically tackle sugar nucleotide metabolism for this so this is a western blood which is a method to analyze protein heterogeneity molecular weight heterogeneity and you can see here from wild lab cells this gmcsf is hugely spread out as i already showed you in the mass spectrum as well in glycol delete it's quite a bit reduced but still has remaining heterogeneity due to the oglycosylation and some level of the n-glycans which are not entirely the trisaccharide structure and now in glycol delete cells it's effectively becoming one band and the remaining heterogen glycol double deletes and the remaining heterogeneity we have is due to site occupancy of the n-glycan sites so n-glycosylation sites can be can or cannot be occupied with an n-glycan so if you take off the n-glycans entirely what you'll find is not modified and modified with a single glutinac residue so i think for the first time now we can actually make glycoproteins in mammalian cells with the same level of homogeneity as we could make non-glycoproteins in E. coli for the longest time and what we still have remaining is a single glutinac residue and so that sugar residue which sits on the n-glycans is actually chemo orthogonal to the protein backbone so we can do chemistry on that carbohydrate on that monosaccharide residue to couple whatever you wanted to do it there so that's hence my question of last of yesterday you know what could you do with more than two or three i mean this is another one that we can add to it and it's made by nature in case you're wondering this doesn't do much to the physiology of the cell glycol deletes adapts completely it's just behaves like wild type cells glycol double delete suffers a bit more but as you know eukaryotic cells and living cells go you can evolve them to adapt to that new glycosylation phenotype and we are still doing that so we're you know passaging it through normal cultivation conditions and it's improving still almost back to normal levels of cell growth okay so i had one more story but i'll skip that i guess i just will tell you that we have implemented glycol delete technology now also in plant seeds and this has been done together with and the picker who is in the department of mark from muntagu so mark as you may have known developed agrobacterium tumor fascians transformation of plants in the very early days of plant biotechnology and we were struck i think as all of you by the the dreadful situation of the Ebola epidemic which happened last year where we had antibodies that could prevent the disease but they couldn't be manufactured fast enough because they were manufactured in a transient expression system in tobacco and so to do that in tobacco you have to grow the tobacco plants which takes months right until you have the doses so what we figured is that if we could implement antibody production in legume seeds in this case soybeans if we could do that you could decouple the production stage from the purification stage of pharmaceutical manufacturing so these seeds as you all know i mean legume seeds you can store them at room temperature for years and they will still keep their germinating power which means that all the proteins in there are intact so we can actually store monoclonal antibodies at room temperature for many years in huge quantities using legume seeds and then having them available when such an emergency happens to purify the proteins and done the one problem was that plant glycans or allergenic to a large section of the human population if you inject them in the bloodstream of course we eat plants all the time and that's safe but that doesn't mean if you inject them in the bloodstream that it's safe and so we engineered out this allergenicity from the plants in a very simple pathway which is implementable in crops okay i'm done this is the funding there is a poster from our group that i want to point your attention to by morgan and then we have a similar meeting to this one in january so if engend is not far away from here if you want to join feel free all right thank you