We have got a fun one for tonight in MLB DFS primarily because of the pitchers for tonight there are a lot of guys can go towards and you're not really having to force it as we did last night where you've got guys in several different spots you can feel good about both for cash games and for tournaments and on top of that there are enough stacks to get by it's not like a super plentiful offensive slate but a good enough one where you can potentially get a bit different get a read on the room decide where you want to go where other people may not go I think there are some good potential contrarian options available for today so let's dive on and get you set for this Tuesday night main slate welcome on into the solo shot that's right here on the fan dual podcast network and numberfire.com my name is Jim Sonnis I'm a senior writer and analyst for numberfire.com here to break down this 12 game main slate with lock set for 705 p.m. eastern for tonight a couple weather notes for today one of which is wind in Baltimore for the Orioles and the Cubs winds are out to center or out to left I should say at 11 miles per hour bump up batters they are keeping mind the the increased portion left but the wind should help not negate it but at least offset it a tiny bit finally there's a chance of rain in Pittsburgh for the pirates and the Tigers they should be good to go but check back on that one later there's a pitcher I do like there so we can talk about that in things to watch but just know there is some potential rain in Pittsburgh for the pirates and the Tigers we will get you set for this Wednesday Tuesday main slate in just one second but first a quick reminder to make sure you're subscribed to the numberfire daily fantasy podcast coming up later on today Branding a doula back from his vacation breakdown the PGA DFS slave for the RBC Canadian open but it's coming up on the fan of YouTube page at 10 a.m. and up on the numberfire daily fantasy podcast after that PGA MLB every weekday USC NASCAR all in the same place hit subscribe if you like what you hear leave us a rating and review as well the NBA finals are here and you can play a free daily fantasy challenge with a $10,000 prize pool it is a body armor inside edge challenge and it is easy to play just set your five player lineup while staying under the salary cap the MVP position will get double the points the star position gets 1.5 x times the points and the proposition gets 1.2 times the points you have two utility players if your team scores enough fantasy points you will be eligible to win part of the $10,000 prize pool the body armor inside edge contest is for game three of the series which is coming up on Wednesday so make sure to enter before then you can sign up for the free contest at fandual.com to enjoy this free DFS contest for your chance to win $10,000 pitching preview for this Tuesday Main Sleigh Alec Manoa is the highest salary pitcher on Fandall checking in at $11,000. Just in Verlander's 10,9 followed by Kyle Wright at $10,500 $500. Derek Scoobl is 10-4 with Carlos Rodan at $9,900. James and Tio 95. You Darvish 91 with Michael Kopeck, Jeffrey Springs, Cole Irvin, Jose Quintana, Jason Alexander, Taiwan Walker, and Brad Keller as the others at $8,000 or higher. I really love Kyle Wright for tonight. There are a lot of options for today, and you can go a lot of different ways. For me personally, Wright will be the guy I have as my SP1. It gives the best combination of floor and ceiling with ceiling being the more important factors here. I'm going to rank Wright first, and a lot of that is due to the matchup. He's facing the A's who have been what we thought they'd be. They have a 79 WRC plus against Wrightys, with a 111 ISO and a 23% strikeout rate. They are a plus matchup for Wrightys in all three of those categories. And Wright is a really good arm in a vacuum for DFS. He's made 10 starts this year. He has solidified the gains as the sample has expanded. The gains from last year to this year, 28% strikeout rate, 3.43 skill interactive ERA, really good bat at ball data. The one issue he has had is walks because the walk rate for Wright is 10%. The A's just don't draw a lot of walks. I'm not sure how much it matters. He has a 7% walk rate, or they have a 7% walk rate, which means it's kind of the perfect matchup for Wright, specifically given the low walk rate there. We have not seen Wright in many plus matchups recently, but he did have 11 strikeouts against the Marlins at home back in April. He followed that with eight against the Cubs. We've seen Wright get eight plus strikeouts in four of his 10 starts. I think he could get there against Knight, which is why the ceiling to me is still very good, despite the fact I think he's kind of a traditional cash game type play. He is a good ceiling too. I like his safety, even if he doesn't have the ceiling there. Wright is my favorite guy of the night. A lot of good competition, but to me, Wright deserves to be the number one arm for tonight. I'm going to put Carlos Rodan seconds, which might feel odd because things have been super weird for Rodan recently. I still think he has enough to get the job done though. He's facing the Rockies here, who is a team he absolutely mowed down back on May 9th with 12 strikeouts and six innings, a sick outing for Rodan, but he hasn't done much since then. He's had no more than six strikeouts in four games. He's had less than four twice and also had an eight earned run outing in there. He does have some injuries in this past, which might make you think that something is off with Rodan. I always look at pitch usage and velocity to get a glimpse at that. The Vivo did dip for a second for Rodan, but it was back up his last time out. These sliding usage changed a bit as well.  That's pretty in line with his full season number. We saw him get a 12.2% swinging strike rate in that most recent game. It is possible that Rodan had a blip in there at some point, but it does seem like he got over that in his most recent start. He threw 98 pitches there too.  I do think there is extra uncertainty here, which is why I think Wright is safer. Both guys have similar upsides. In a vacuum, I prefer Kyle Wright, but Rodan could easily bounce back in a big way here and I want to be in on him in case that does happen. To me, even on a loaded slate, I've got Wright won Rodan number two, and I think that I feel pretty good about in that ranking despite the fact we have seen Rodan slip a little bit more recently than he did earlier on this year. We'll also talk about Terry Scoobal and the things to watch section. I really don't want to be in the bargain bin tonight given how many stud pitchers there are, so I'd rather live there for the most part. If you do want to save some salary though, my favorite guy is Ranger Suarez, it's $73. It's been a rough year for him. He has a 4.690 IRA. His peripherals aren't a ton better, but we've seen things trending better for Suarez recently. At least the peripherals, not only were the results, but he's been using more forcing fastballs across his past five starts, and that is definitely a bigger strikeout pitch for him than his sinker. He has a 24% whiff rate against the foreseamer according to baseball. Savant versus the sinker at 12%. The strikeout rate is up to 25% in that time, and he still has good batting ball data. He is walking too many guys, but part of that could be the teams he has faced. In those five starts, we've seen Suarez face the Dodgers twice, the Braves, and the Giants. That's a tough string of teams to face. He did still manage to get five plus strikeouts and four of them. He's facing the Brewers on the road here. They have just a 64 WRC plus on their active roster against lefties, with a 24% strikeout rate, minimal flyballs.  That's not enough for me to use him personally because I want to live in those high upside guys in the 9,000 and 10,000 range, but it's totally understandable if you want to go here. It just doesn't fit my personal style. I think Suarez is the best value play probably not enough to be in my player pool for tonight. We'll talk about one more picture in Thanks to Watch. First of all, let's go through the stacks for today. I think the top one is the Astros, which is weird because Chris Flexen is facing them. He's had a lot of success against the Astros this year. He's had three starts, and he's allowed just five runs across 18 innings. That's pretty good. That's a positive for sure. He knows how to handle this team, or at least he has so far. But Flexen has really struggled against other teams. His overall ERA, including those good Astros games, is 4.55. The peripherals say he's closer to the rough starts than the good ones. I'm going to stack the Astros against him for tonight and see if they can write the ship in their fourth meeting against Flexen this year. The peripherals in general say we can stack against Flexen, and the results due to outside of those Astros starts. He has a 4.71 skill interactive ERA with a 17% strikeout rate, a 43% hard hit rate, and a 44% fly ball rate. All of those numbers are right in our wheelhouse from a stacking perspective. The Astros are still good. They have a 117 WRC plus against Wright. He's with a 179 ISO. It just makes it more confusing that they haven't done well against him. They did have some success in the first game. They scored three runs there. It could just be that he's been on his game the past two starts. That's what I'm banking on. I think they are due for some progression here, especially now that we've seen Flexen, or they've seen Flexen three times, two of which have come in the past five starts. A lot of familiarity there for sure. I do think that we can go at the Astros again despite the fact they've struggled against him thus far. Part of the reason that he's been good against them could be that Flexen struggles more the righties. A lot of the righties in the Astros have been in a slump. But I think they're coming around. Alex Bregman has a 47% hard hit rate the past two weeks. Hasn't translated to a lot of extra base hits, but it could soon. Jose Altube went yard last night, stole a base a couple of nights ago. I think that that could signal that he's starting to be more aggressive, maybe getting back in the good, bad at ball areas. I do still love the lefties. I will never talk myself out of them, but I do think that those two righties, Altube and Bregman, are showing signals of upside again. So I'll probably prioritize them more than I typically do. I'm typically your Don kind of guy and still will be, but I'll bump up Bregman and Altube more than I typically do against a rightie. As far as our second stack, the Angels have not yet confirmed who their starter will be. I assume it's Reed Detmerz. It is his terminal rotation. He was fine last time out. So I'd assume it's him. Just check back on that to be sure though. And Detmerz has had some good individual outings recently. Obviously had the no hitter. That's a pretty good one. He held the Yankees, scoreless or four to third innings last week. He's facing the Red Sox tonight. And I think we should stack them here mostly due to the underlying numbers in that no hitter. Detmerz started throwing a changeup more often. And obviously it worked. He had no hitter. That's pretty sweet. But in that one, he had just two strikeouts. He led up a lot of fly balls, but none of them fell in. We've seen that carry into other starts as well. Across the past four, starting with that no hitter, Detmerz has a 5.17 skill interactive ERA with a 15% strikeout rates, a 49% fly ball rate and a 40% heart hit rate. That's got him in trouble twice. Couple rough outings against the Rangers. And he led up a pair of home runs in both games. One of which was at home, which is where he is at for tonight. Plus right before this stretch, the Red Sox got to him with three earned in four innings. So it's never a sure thing to stack against Detmerz. He can have really good starts. But I think there is enough here where we should stack the Sox due to the number of cracks at long balls they will get here via the balls in play via the five balls, et cetera, et cetera. I think there is definitely a good case we made for making them a high priority stack again, assuming Detmerz is in fact the starter for today. It's hard to figure out what to do with Rafael Devers against the lefty. We see a lot of high profile lefties hit lefties. Well, Matt Olson, Freddie Freeman, guys like that, Corey Seeger and Devers though didn't do that last year. He had a 128 ISO. This year, the ISO is 228, but it's inches 57 plate appearances. He has a 28% strikeout rate, 20% fly ball rates. The fly ball rates was better last year, but still just 30%. So I've inclined to ding him quite a bit against the lefty. Obviously, that changes if everyone avoids Devers because it's a lefty on lefty. But I do think that ranking out guys within the stack, JD Martinez obviously above him, Trevor Story right now, Enrique Hernandez due to the salary, probably Zander Bogarts too above Devers. You could talk me either way on that one, but I do think he just lowers the prioritization. So I'm okay using lefty on lefty if the guy I think has overlooked, if I think that he has better numbers than you may think against the lefty. Devers is one guy where I do actually ding him a decent amount when there's a lefty on the opposing mount. Now I think that Kyle Bradish is a similar pitcher to Reed Devers, just in an odd way where both these guys can have really good starts. For Bradish, the good starts are due to his strikeouts, which makes him risky to stack against, but he keeps getting bad results. So I do want to stack the Cubs against him tonight. They are the number three stack here behind the Astros and the Red Sox. Bradish does have juice. He has a 24% strikeout rate in seven starts, not too many walks, which leads to a 3.67 skill interactive ERA, which is pretty good.  When people make contact, they are making contact. They are whacking that thing. Bradish has let out four plus earned runs in four out of seven starts that includes three of the past four he has made. And that's despite getting strikeouts in there, but he's just getting punished whenever a team puts the ball in play. The Cubs are not a juggernaut offensively, but they do have a 102 WRC plus on their active roster against righties, which can at least keep him honest, I would say. I'm going to keep Bradish viewing him similar to Josiah Gray, Hunter Green. Green was awesome last night, but we can have bad starts. So the upsides of stacking against them make the risks worth it. Gray, Green, Bradish all can have good starts, but when they have bad starts, you want to be in there for DFS. So I think the Cubs good internees for tonight due to the upside that they provide. It's been super impressive to watch the Cubs fill in for injured guys in their lineup. Christopher Morel has some power against righties, but a lot of speed. Ian Hap playing good ball, Patrick Wisdom hitting for power against righties, though he does strike out more than you like, but there's definitely enough here to feel good about the Cubs, even with some key really good batters being down. So the Cubs have lost some pieces. Obviously, you prefer those guys, like Zuki to be healthy, but I think even with the offense in its current state, still a good one to go to for stacking in the right spots. And I do think they have the right spot for today. Things to watch. My favorite stud we did not discuss yet is Terak Scubble. I'm going to put him above Alec Minoa and Justin Verlander. I just like the matchup more for Scubble. He's facing the Pirates. We have a 23% strikeout right against lefties, which puts Scubble at 6.9 projected strikeouts for today. That is actually the best number on the entire site. It's above Rodan. It is above right, but they're both at home in good matchups. And that's why I wound up going them over Scubble despite the projections liking him more for today. I still bet about those guys, but I would put Scubble above Verlander, above Minoa and my number three pitcher for tonight and the guy I will use for sure might not get to Minoa or Verlander just because I don't really want to use for. They're definitely worthy of using. I just don't really typically want to use for pitchers in most scenarios, especially when it's like good guys, like I feel pretty good about having a good floor and ceiling. Don't know who the twins will start today. Bailey Ober is on the IL, which means it could be Colsans. He started a double header last week on Tuesday. So would be his time up in the rotation. He's had rough results in both AAA and the majors. The peripherals are better. I would bet it winds up being Sands. And if it does, I would say fire away on the Yankees for sure. Definitely an okay stack. I'd probably put them below the Red Sox and the Astros, but they'd probably be a hair above the Cubs for me if we assume Colsans at the starter. Problem is that Yankees often still decently banged up despite getting stand back. So, you know, I think they're fine, but not a top stacking. We assume that it is Sands below the Astros and the Red Sox for me. I think a good contrarian stacked tonight could be the Dodgers. They're facing Michael Kopeck, who has a 2.20 ERA, but his underlying numbers don't really blow you away. He led a five runs with two home runs, the Blue Jays last week. So I think there's some cracks in there and the Dodgers are a team good enough to expose them. It's just for tournaments because it's not safe enough to use in cash gains, but I do like the Dodgers for sure. So you kind of got to work to be different on a 12 game slate. I think the Dodgers could be a good way to do so.  Let's finish up here. The Dinger calls on Dinger Tuesday. The boring one will be JD Martinez facing off, I assume, with Depmers. Again, a lot of fly balls, a lot of hard contact. Martinez, good against everyone, but especially against the lefty. So JD Martinez, the boring home run call for tonight. The fun one, I will go to Chas McCormick if he plays. McCormick about a 180 ISO against Reidy so far this year. I mentioned that Flexon has struggled with same handed batters. So I'll go McCormick if he plays, not a lock to play though. So I'll toss in Frank Schwindel for the Cubs against Braddish if McCormick does not go. Again, Schwindel better recently, better bad at ball data against Reidy. He's been trending back up ever since he got demoted. So I would say he'd be the alternative if McCormick does not play, but I like McCormick more straight up if I get him at my disposal. So JD Martinez and Chas McCormick with Frank Schwindel being the alternative for today. That's all we got here for this Tuesday main slate of MLB DFS. Like I said, it's a fun slate. A lot of ways you can play things. You got five legitimate, really good options at the top. I'd be okay with any of them. I gave my three that I like the most, but I think that if you have a strong conviction towards Verlander, strong conviction towards Minoa, totally justifiable despite good competition for today. If you've got any questions for me, I am on Twitter at Jim Sonnis, J-I-M-S-A-N-N-E-S. You can also follow the FanDuel podcast network at FanDuel Podcast. We also again do have a PGA podcast coming up later on today on the Number Fire Daily Fantasy podcast feed and on the FanDuel YouTube page. So go there, hit subscribe if you like what you hear. Give us a rating and review as well. Big thank you to everyone for tuning in for today. Good luck to you with your MLB DFS. Long as for Tuesday, we'll talk to you once again tomorrow to break down yet another slate of MLB DFS. This has been the solo shot right here on the FanDuel podcast network.


Hi Keith, I guess right out of the gate, I noticed on Sunday, I guess I know Roger had been ruled out and then he was carted back on the field on the side to kind of stand on the sideline, which I don't know if I've ever seen that before. Is that because he wanted to kind of watch history be there with his teammates, you know, in the event the team won? Do you know anything about why he did that? Yeah, actually, I don't, you know, I'd be speculating if I said one way or another, but you know, I have a feeling he just, he's a great teammate and he wanted to be out there with those guys. No, and it wasn't just about, you know, Derrick and being a 2000-yard rusher, which was awesome that Derrick was able to do that, but also because it was the conference, you know, the divisional championship on the line. So, important to him to be there. And as far as far, I know everybody kind of shares in everyone's success, but I know Dennis talked about after the game, Ques talked about leading up to the game. How much pride your group take in blocking for a 2000-yard rusher? No, man. I think it's special. And, you know, it takes everybody. It's, you know, from, you know, the GM to the head coach to the, to the, to Arthur, calling the plays the way he calls the game. And then of course, you know, all 11 on the field.   They want him to succeed. And so I think that's a really good, you know, a really good achievement for everybody that was involved, which is the whole offense and really the whole team. Terry? Keith, how much, how rewarding is it not only to see Derrick get 2000 yards, but to also see it come with so many adjustments and so many changes having to be made on this offensive line due to all the injuries, having guys like Quisenberry and Brewer and, you know, on down the line step in and help out.  I don't remember, you know, five, six games, whatever it is, to get to win the division and get that, get that for Derrick and then for a guy like quiz who's come as far as he has is just amazing.  You get a bunch of guys that like being together and like coming to work and and share common interests and common goals and, you know, it makes all the hard work and sacrifice worth it. And so I think it's really cool for those guys to be able to do it the way they did it. Teresa. Keith, in addition, the guys on your offensive line, you've had you've lost Taylor, Ty, Roger, Ben, Dennis, you know, they've all been nursing through injuries and finding ways to keep being available on Sunday. And when somebody has gone down, somebody's been able to come in and step in. How and what is allowing these guys to be this value available and continuing to be resilient and keep contributing. Yeah, it's a, it's a great effort, our great question and it's really such a team effort from our training staff to the way great coach variable manages these guys and, and then of course it comes back to the players.  Of course, you know, it's a job and of course there's ups and ups and downs and everybody wants to get paid and make money and do all those things. But at the end of the day, it's fun coming to work every day because these guys care about each other and it's really important to them. So, you know, it's, it's, it's pretty remarkable for someone like Brewer who, you know, is technically undersized and, and, you know, comes from, you know, quote unquote a small program and college and all those things to see him go and play guard and function and, and, and not look out a place out of there is just a huge testament to him and, and he loves football and he's willing to do whatever it takes to play at this level and he's just really fun to be around because he just loves football.  But at the end of the day, these guys are really good professionals and they know and you know, like, coach variable preaches this all the time just like john does Robinson and it's like, you got to be ready when your numbers called and they've been able to do that and it's it's something I'm really proud of the way they've been able to work, work through the different situations step in and and help this team win it's been it's been pretty fun. John Glenn, Joe rector. He was talking earlier about, you know, Derek's progress as a runner in terms of, you know, reading things no one went to hit something sneak through something. Just wonder what you've seen from your perspective on that and just the entire operation there with the run game is it is there also something from the offensive line perspective that has gotten better or tighter this year to increase that production. Yeah, that's another really good question. I just think, you know, in terms of Derek, you know, it's it's a funny it's a slippery slope with running backs because you need to, you know, you try and coach them and cut Tony does a great job about getting the landmarks right and how to read the defense and all that and then there's this other element that you, you, you need to let them run with instincts. And so, I think the best runners in this league have a really good combination of both and as coaches if you if you want to read and over coach the, the perfect landmark and footwork and all that and you start taking away the instincts, and then if you let them run too much on instincts and not and and don't get them right from a discipline standpoint having the right tracks and stuff and go the wrong way the other way so I think to me what's been really fun watching Derek grow over the last three years is just is just that, you know, watching his, you know, his comfort in what we're trying to do and and and come together in a way in which, you know, kind of discipline things work together and and that's been really fun and you know what's interesting about Derek is more he carries the ball the better he gets and and so it's it's, you know, whether it be throughout the season, or whether it be throughout a game, and he just is, you know, on a mission man and you know, it's, it's, it's fun to watch him do that and I think from a line standpoint I think you know, just like anything else, coach rabble and we all here just as the best we can preach just find a way to get better week to week and that's focusing on the here and now instead of the big picture and and these guys have done a great job doing that and have shown steady improvement throughout the season. Emily.  Who have you seen kind of step up on the offensive line to to replace that. You know, you lose someone like Taylor and and Taylor does so many things for us not just as a player but he keeps the room light and he keeps, he keeps, you know, you know, a certain I guess, what's the best way to say it just keeps keeps every and kind of moving in a certain direction and moving forward and and you know, never lets it get too serious but never lets it get too far off track and kind of just keeps you steady that way and I think with him being gone I think what's what's been really kind of interesting on the watch is it's been a collective group effort and you guys mentioned Dennis area earlier and some stuff he said in an interview but he's as steady as a rock I mean he just like you just know what you're getting out of him every day and he does a great of using all his experience and stuff to help everybody around them and hold people accountable and you got guys like Ben Jones who same ways are really good pro who who has been doing this a long time and he tries to help everybody as often as possible same thing with Roger I mean I think what happened what happens is is you know when Taylor got hurt it just instead of coming more from one guy to become a group effort and I think they've all handled it really well and have kind of shared that experience and I think they're all most of them have have done this a long time and then they know when to be led and when to be leaders and I and they've done a nice job kind of letting me giving each other room to do just that. And just to follow what can you say about you know the tight end group to kind of coming in and helping out and it's not as simple as just plugging in you know a new offensive lineman when one goes down. What can you say about that group to helping you guys out. I can't say enough. I mean just all those guys between John who and and obviously and crew and Jeff and and FERC and I mean it that's what that's what we really we really try to preach here that it takes all 11 people on the field to run a football and the tight ends the they are just as important as offensive line and I really mean that and so you know the tight ends just just it's been really fun for me because now you know we've kind of been together for three years and sometimes that's a luxury in this business and so it's been fun watching you know people like John who and crew kind of get comfortable like like Derek has in the line has and the different things that we do and ask them to do and kind of grow together so they are absolutely instrumental I think Todd's done a great job with those guys and and again they're just you know they're a bunch of guys that love football and want to be good and it's fun. It's fun watching them, you know take those baby steps, baby steps and keep growing. Last one Joe. Yeah, he'd also want to ask the offensive line perspective on on playing with Ryan blocking him for Ryan. You know him as a presence in the pocket but also as an athletic guy who gets out isn't afraid to obviously go throw his body around a little bit what's what's that been like for from the offensive line perspective. Yeah, it is. We have so much respect for him and I think you know from an offensive line standpoint, you know we whenever you block for a guy that's as tough as Ryan is who will stand in the pocket take a hit and deliver a football. It just it makes you want to keep him upright and clean even more and I think what Ryan has proven over and over again is that regardless of the fact he plays quarterback he's as tough as anybody on the team and the linemen love that and so I think they've really gravitated towards him and you know his ability to obviously stand in the pocket deliver accurate football is his steadiness and leadership he's been a great leader every day to because he again shows up and just put so much time and work into this thing. And you know I think they've grown to really respect them and trust them and you know and his ability to make quick decisions and get the ball out and not hold on to the football and stuff like that like I you know I just think he's done a great job and and I would. It's safe to say that the old line room really appreciates that and what he brings to the table.


beispiel iverso yondई जी मेंँया ठाव मैंजिन न शोत मेंटर फाची आ��crany glio gression yet ceremony in evidencer is about 我iami Cute Discovery ilst subsequently today i am going to deliver a video session on performance of steam condenser two at the end of this video session You will be able to calculate the vacuum efficiency ॐ and condenser efficiency ॐ also you will be able to calculate quality of steam entering the Debatte the contents of this video session are the numericals on performance of steam condenser we are going to study two numericals related to performance of steam condenser the first problem is the following observations were recorded during trial on steam condenser barometric reading equals 765 mm of mercury condenser vacuum 710 mm of mercury mean condenser temperature given as 35 degree Celsius temperature of hot well 28 degree Celsius condensate collected equals 2000 kg per hour quantity of cooling water collected is 60000 kg per hour temperature of cooling water at inlet is 8 degree Celsius and temperature of cooling water at outlet is equal to 24 degree Celsius this is the given data and we are supposed to determine corrected vacuum to standard barometer vacuum efficiency of condenser under cooling of condenser condenser efficiency and quality of steam entering condenser now let us see how we can find out each term one by one the first step in the solution is first we need to calculate the absolute pressure in the condenser and already we have discussed in my previous sessions the absolute pressure in condenser is given by barometric pressure minus vacuum gauge reading now in the problem it is given that barometric pressure is 765 and vacuum gauge reading in the condenser is 710 so 765 minus 710 gives us 55 mm of mercury this is the vacuum in the condenser in mm of mercury which we can convert into bar which will be equivalent to 0.074 bar we know that 1 bar is equal to 750 mm of mercury therefore 55 mm of mercury can be converted into bar by dividing by 750 mm of mercury so we get absolute pressure in bar as 0.074 bar now let us see how to find out the corrected vacuum in the condenser corrected vacuum is given by an equation barometric pressure minus absolute pressure in the condenser now let us pause the video for movement and let us recall why do we find corrected vacuum as we know that the barometric pressure varies from location to location and condenser will be located at a particular point where barometric pressure will be different than standard barometric pressure standard barometric pressure is given by 760 mm of mercury hence we can find the corrected vacuum by using standard barometric pressure as 760 mm of mercury minus absolute pressure in the condenser 55 mm of mercury which we have decided in our previous step and this will be obtained as 705 mm of mercury now let us see how to find vacuum efficiency vacuum efficiency is given by an equation it is the ratio of actual vacuum to the ideal vacuum actual vacuum in the condenser is given by the relation barometric reading minus absolute pressure in the condenser barometric reading given in the problem is 765 mm of mercury and absolute pressure in the condenser we have obtained as 55 mm of mercury so difference of these two will give us the actual vacuum in the condenser equal to 710 mm of mercury now let us see how to find out the ideal vacuum ideal vacuum is nothing but the vacuum in the condenser when air is totally absent which is given by barometric reading minus saturation pressure of steam now barometric reading already we know that it is given as 765 mm of mercury whereas saturation pressure of steam is obtained from the steam table corresponding to mean condenser temperature of 35 degree Celsius so by using the temperature tables in the steam table we get corresponding to 35 degree Celsius absolute pressure of steam equal to 0.056 bar which we can convert into mm of mercury as we know that 750 mm of mercury equal to 1 bar so 0.056 bar will be equal to 42 mm of mercury therefore ideal vacuum is obtained by taking difference of barometric reading of 765 mm of mercury minus saturation pressure of steam corresponding to mean condenser temperature of 35 as 42 mm of mercury which is obtained as 723 mm of mercury therefore using an equation for vacuum efficiency which is ratio of actual vacuum on ideal vacuum we can put actual vacuum as 710 mm of mercury and ideal vacuum of 723 mm of mercury we get it as 98.20% now we are going to find out the under cooling of condenser means when the condenser steam is cooled below mean condenser temperature it is called as under cooling now under cooling of condenser heat is given by the formula mean condenser temperature minus hot well temperature that is the temperature of condenser heat in the hot well which will be equal to 35 minus 28 equivalent to 7 degree Celsius now let us see how to find out condenser efficiency condenser efficiency is given by an equation actual rise in temperature of cooling water divided by maximum possible rise in temperature of cooling water now actual rise in temperature of cooling water is given by TW2 minus TW1 which is nothing but the outlet cooling water temperature minus inlet cooling water temperature which is equivalent to 24 minus 8 ... only when the temperature of the water is given is as high as possible, the saturation temperature of the steam corresponding to the absolute pressure in the condenser which is obtained as that. We can see that a higher saturation temperature of the steam corresponding to the absolute pressure in the condenser, of 0.074 bar is equal to 39.95 °C. उसी आई� trust the condenser efficiency,jin उदविय Money , you mindls , 150 नुआ तो ं explicar the actual heard is in temperature of the cooling water, mayor । किचिन एक बाई किचिन खिलिग गभा movimiento ज़ीरhm । किचिन भी नान्गट equations , i find न�giggling which redef maker ==== उ को skipping heedcm वहार नछ time ज़ीर home विस धो � Veter gucken उऊठका서도 थ दोंं कून ऎन C now let us see how to find out the quality of steam entering the condenser for finding out quality of steam entering condenser we will be using the energy balance equation and heat lost by the steam equal to heat gained by the cooling water now heat lost by the steam is given by an equation mass of the steam multiplied by enthalpy of steam entering the condenser minus enthalpy of condenser leaving the condenser which will be equal to heat gained by cooling water will be equal to mass of cooling water into specific heat of cooling water into rise in temperature of cooling water now the enthalpy of steam entering steam is given by an equation h is equal to hf plus x into hfg since the steam entering the condenser is weight we have to use the equation for enthalpy of weight steam formula where hf and hfg are obtained from steam table corresponding to absolute pressure of steam in the condenser which is equal to 0.00704 bar therefore hf and hfg are taken from steam table corresponding to pressure table in the steam table with the reference to pressure of 0.0704 bar and hc is enthalpy of saturated liquid at hot well temperature of 28 degree Celsius which is obtained as 117.3 kJ per kg so using the values of hf plus x into hfg and hc as well as mass of steam entering condenser mass of cooling water supplied and specific heat of steam and rise in temperature of cooling water when we put these values we can obtain by solving this equation the value of x that is nothing but the dryness fraction of steam entering the condenser which is obtained as x is equal to 0.819 now let us see the second problem in which the problem statement is the vacuum efficiency of condenser is 95% and the temperature of condenser is 40 degree Celsius if the barometric reading is 75.4 cm of mercury find the vacuum gauge reading of the condenser so here we are given with the vacuum efficiency mean condenser temperature, barometric pressure and we are supposed to find out the vacuum gauge reading of the condenser that is vacuum in the condenser let us see how to find out the solution now already we have defined that vacuum efficiency is actual vacuum upon ideal vacuum actual vacuum we can denote it in terms of head of mm of mercury or cm of mercury as hv and ideal vacuum is nothing but barometric pressure barometric reading hv minus saturation pressure of the steam in terms of cm of mercury therefore we can write this as the saturation pressure of steam corresponding to mean condenser temperature of 40 degree Celsius is obtained from steam table corresponding to temperature table at 40 degree Celsius we get saturation pressure of 0.0738 bar and converting this into cm of mercury as 5.535 cm of mercury we can write now the value as vacuum efficiency is equal to given as 95% that is 0.95 equal to the actual vacuum that is vacuum gauge reading which we are interested in to find divided by barometric pressure given in the problem is 75.0 cm of mercury 75.4 cm of mercury minus this saturation pressure as 5.535 using these values and solving this we get the vacuum gauge reading in the condenser as 66.37 cm of mercury which we can convert as 0.88 bar these are the references thank you


As we started discussing about reverse phase protein arrays and Dr. Ramesh Umani a guest scientist provided you an overview of how to use this technology for various applications. So today Dr. Umani will continue his lecture and he will talk to us more about this novel technology reverse phase protein arrays. In today's class you will learn about how to choose the correct samples for the experiment, what are the methods for sample preparation, selection of antibodies, spotting methods and signal detection methods. So let us have Dr. Ramesh Umani to continue his lecture now. First is samples there is no limitation any sample can be analyzed by RPMS or any sample which can be analyzed by western blot can be analyzed in RPME. Only thing is you have to follow certain methods to prepare your clean sample to make it ready for RPME approach. It is a high throughput technology that performs protein assays on thousands of samples simultaneously including tissue lysates just microdesected tumor samples there at least you will have a chance to get some microgram level proteins, some couple of milligram level proteins and then laser capture microdeception LCM. There you end up with very very low quantity of sample you cannot perform n number of western blots to validate n number of targets and PFE fixed tissue lysates also you can do. So there are separate methods are available to make the protein extracted from formalin fixed tissues to use for RPMS. Sometimes those samples cannot be analyzed by western blotting approach you see smearing effect which can be avoided here. The cell lysate particularly our tumor cell lysate every one of you might be handling some of the cell lines in the lab so the lysates can be prepared and small number of isolated stem cells are the other rare cell types. So now stem cell biology is a very very attracting word but they will deal with always thousands of cells not even millions of cells. We do millions of cells in cancer research serum also can be spotted there plasma or any body fluids such as cerebrospinal fluid or urine can be directly printed on the lysate by following specific methods. So to print the samples how to prepare the samples is very simple cartoon here can take a lysates buffer put the tissue of the rest and homogenate and spin down the tube and take the supernatant and go for printing or can kept in the minus 80 go home and come back tomorrow. This is a way but it is not that as simple as just preparing there are multiple steps has to be taken care. So you have to plan experiment and take all the parameters into consideration before executing the experiment. So in that way this is also one of the critical point. So this critical point basically highlights what kind of buffer can be chose not all proteins in all samples can be solubilized by single buffer. This is a major limitation in array approach. So lysates buffer should be optimal to work for maximizing the RPPS signal. So it should not interfere with the visualization of signals using any labeling system you follow either secondary antibodies like IR labeling, HRP label or alkaline passphrase label or directly Psi3 or Psi5 label and it should be compatible with the detection methods like a calorimetric amino luminescence and fluorescence methods should not interfere with substrate on solids and absorption of lysates. See for example you might have performed washing steps. What if the buffer is not compatible and washed away along with the proteins spotted on the glass slide end of the day you will have a red face no signals. So definitely should not interfere with the substrate on the slides in this case nitrocellulose substrate and should not interfere with adsorption of the lysate. I mean that means attachment of the or immobilization of your protein sample. Extraction efficiency may vary with the different lysate reagent and is dependent on the tissue being examined. Let us say for example if you are taking a prostrate tissue which will have a prostrate some stones will be there then you need a stringent buffer. Let us say for example you are lysing the cell lines simple rippa buffer is sufficient or ampere buffer is sufficient. So lysate reagent should be supplemented with freshly added protease inhibitors. Of course if you wanted to understand the post translational modifications and you need to have the inhibitor cocktails like phosphatase inhibitors and also some of the protease inhibitors not to degrade the protein of interest. So with my experience at least I worked with these four different buffers definitely these four different buffers behaved entirely different with the different samples believe that any one buffer will help is not true. In my hands we had a different experience with different buffers and different tissues and different cell lines.  So this there is no hard and fast rule that so and so teacher explain this concentration it will work it will not work. So you have to optimize please keep this in mind. Then second point highlighted was the essential requirement is the good quality antibodies. So here you need to spend at least some couple of months to years to establish antibody panel in your lab. At least we could establish I mean with my postdoc supervisor we could establish 190 antibody panel in one year from 3000 antibodies purchased from the market. You can see that 2800 plus antibodies just went into dustbin of the German Cancer Center that time. So now those antibodies we are repeatedly purchasing and we are using it but this effort one year effort helped in the long run definitely how it is I will highlight you here. Our approach for antibody validation is like this starting point take that western blot through western blotting approach take 10 different cell lysates or from more cell lines run 10 different lines then do western blot. If the antibody gives a single band say yes go to next step and then you pull the lysates from 10 or more different cell lines run a single line that means in a complex mixes also antibody has a capacity to recognize a protein of interest without any non-specificity. If it gives a single band it will go to the additional validation steps by western blot using knockdown experiment and stimulation experiments and if it is and if it is yes then it will go to the validation by RPPA. In RPPA we will validate by serial dilution of the samples whether antibody is able to detect the serial dilution the 2 fold 10 fold dilution properly or 5 fold dilution properly. If it is yes then this correlated is if the RPPA data is correlated with expect results from protein of interest then we declare that it is useful for RPPA and also every antibody will get a 3 codes go ahead use with caution and not suitable.  You can see that this is a like a pain we used to run these kind of western blots several hundreds to thousands for validation purpose you can see that some of the bands are very nicely giving single bands some of them are like a non-specific bands and here you can see that this is completely crap you cannot use them and mostly with my experience I am not as again brand ambassador for any antibody companies here at least I had a good experience with cell signaling very bad experience with Santa Cruz. So, I do not get offended please I just it was my experience I am sharing with a very lighter note with the student community here. So, you can see that if you have a single band here directly it is yes then we will proceed to this pipeline here. So, once we reach this step we need to follow additional steps this kind of antibodies we will grab them first step itself we do not spend any time and we will directly buy the another antibody. So, next round of validation is that again using western blot we take a suitable cell line and stimulate them with some kind of factors where your protein of interest gets modulated you know it prior information already and you look for this known information in the western blot results. In this experiment what we have done is we have taken HEPG to cell line and we stimulated with EGF and insulin these two factors will stimulate the phosphorylation of AKT and phosphorylation of ARC protein and those phosphorylations have been observed or not these are also mutually exclusive you can see that EGF is stimulating here phosphorylation insulin is not doing. So, this kind of approach we follow this is one example I am showing you. So, with this approach it is passed then it will go into the next line. Then we will not leave that story there itself again we take those antibodies and this pass for specific antibodies choose the different cell line whether they are behaving in the same way or not. So, here you can see that with the insulin pass for drug to pass for relation is going up with EGF only EGFR is going up.  So, once we have this we will not leave the story there again we have now SIRNA available for all the proteins expressed in the orpheum we buy the SIRNA down regulate them transfer to the cells make the lysates random on a western blot whether this antibody is really detecting the down regulation or not. So, in this case we have given to the cells SIRNA against EGFR and our antibody is detecting the down regulation of EGFR. So, it has passed through the this step here knock down or perturbation experiments. So, after this then we validate them by reverse phase protein or we simply take very pool of lice small pool of lices print on the glass slides and use this antibody and probe it see that whether it is really detecting the signals or not. So, when you plot the results like concentration versus signal intensity. So, as increasing the concentration. So, the signal is going up blank signal is at the base level the signal intensity is fine here and also to see that the detection levels are really in the range of detectable or not. So, target signal versus blank signal if the target signal is very near to blank signal there is no point we do not get a linearity there. So, then we have to drop the antibody at this step after reaching here if we see no here then we have to go here again. So, this is a problem after this process if we for process these two filters we have a like mono specific antibodies in this example I am showing Paso P38. This antibody is go for RPPA approach and antibody is approved for reverse phase protein or a approach you also need to do one more step you need to see the correlation between western lot and RPPA results. Because many of the why I am emphasizing this was that one of the RPPA conference many people did not believe that RPPA is better than the western lot. So, that was the reason. So, this paper was published in clinical cancer research where the RPPA data is very well correlated with the western lot data. So, the same sample set the RPPA data is generated compared with the western lots generated from the same sample set. So, then it is clearly understood that the community accepted that the RPPA data will correlate with the western lot data. So, that it is useful to validate large number of proteins in a smaller quantity of samples. So, here we have spotted the I mean this group Gordon Mills from MD Anderson they spotted serially diluted tumor lysates and it western lots and normalize the data this outcome here. So, now we have the antibodies suitable for reverse phase protein array approach. So, at this point to educate you in a different way not all antibodies agnest one target will behave the same way. Let us say for example, if you search cell signaling website for a EGFR or PASFO EGFR antibody target will have a 10 different antibodies with the different catalog numbers. That means, they are they are from different clones or different poly different monoclonal antibodies and not all the antibodies behave in the same way how it is now you look at this picture. So, in one lysate we used for downregulation of EGFR from the same lysate we did a western lot in a serial dilation manner and used two different antibodies to detect the signals here. Two different antibodies nicely showed the down concentration dependent signals for EGFR and also western lot results shows that 80 percent downregulation happened for EGFR after adding SARN. This data is completely wrong how it is when you did RPPA with the same samples can see that this is the anti EGFR one antibody detecting the lysate where EGFR is downregulated and here are all control samples. In this case this is antibody 1, antibody 2. When you look at the correlation this first antibody detects 48 percent downregulation second antibody says 83 percent downregulation. Now again question mark. So, when we deal with this antibody related approach or antibody based approach we should be very very cautious in choosing the antibody. Otherwise end of the experiment we not able to conclude or not able to infer any meaningful data. So, this is what again all antibodies cannot detect the single protein in the same way like for example, in this slide you can see the stat 3 antibody different stat 3 antibodies detecting the stat 3 in the same sample in different manner. You can see LOD this limit of detection some numbers are written here definitely I think I will circulate the slides also to you if you cannot note the numbers you can go through later on. So, this is green is a polyclonal red is a monoclonal blue is a blank values you can see that. So, different signals are going in a different scale along the Y axis. So, you also need to consider if the protein is very very less abundant in your samples you should use more efficient antibody. If it is highly abundant you may not worry much. If it is really low abundant protein particularly phospho proteins very few copies will get phosphorylated than that total protein right. So, in that way we should be very cautious in choosing the antibody. So, all in all I would like to say that if one stop database is available for antibodies which are approved for RPPA approach will be a ideal situation. So, the community or the society is working towards that they have formed a consortium with the different labs and all the labs will share the data and they will they wanted to fit it in the computer. So, this data is accessible to open anybody then they can use the antibody information from which company which catalog number and so on then they can use for RPPA approach directly. So, this is now in the process, but another approach the protein at last company is included in their catalog of validation steps is by RPPA also. So, if you look at the data sheet of any antibody if you purchase then there will be different form of applications like compatible for western blotting, immunoestochemistry, immunofluorescence and all. So, this company included another validation point that it can be used for RPPA or not. So, if the single peak is there that means it is useful for RPPA approach. They also have the catalog indicating that whether it is go, no go or use with caution for RPPA approach this protein at last. So, next is a preparation of protein microarray. Here actually it is important to show you how to print and everything since I do not have instrument to print and show you all. So, definitely it is similar steps exactly same steps it follow as Sanjeeva explained in the morning the glass slide with a nitrocellulose membrane in white color and the sample is printed by a small pin from the machine. So, there are different methods are available to print. So, protein microarrays are typically prepared by immobilizing the proteins onto a microscope slide using a standard contact spotter or non contact spotters. So, there are different methods are available one is a robotic method which is a contact microarray method. Contact microarray method means needle will go and touch the membrane and disperse the liquid in that. So, that sample will not disperse in a big size of the spot it will accommodate sorry small area on the membrane will accommodate the larger quantity of sample. These are the remaining three methods are the non contact methods ink jet method piezoelectric spotting and photoliography. So, maybe many people know about the ink jet method it will spray the sample near to the glass slide. So, there is a chance of big wider spots than the concise spots. So, that you can accommodate less number of proteins on one slide compared to the contact now contact microarray methods. And also you can use different wide array of substrates, but in RPPA approach we need a matrix coated glass slides. So, this matrix can be anything glass directly or gold or nitrocellulose or hydrogel. So, every substrate or every surface has its own advantages and disadvantages. So, in this case so glass is moderate reactivity towards a substrate and gold is low nitrocellulose is moderate and hydrogel is moderate. So, compared to this nitrocellulose is better because it gives low background and low fluorescence while detection methods are applied. So, that is the reason most of the people are using nitrocellulose coated slides whom I know at least may be others are using hydrogel methods as well. So, surface absorptions very low with the glass gold is very low and these nitrocellulose and hydrogel are very high that is why I preferred to use nitrocellulose coated slides.  You might have done thin layer chromatography in your masters right. So, exactly this thin layer is formed by silica gel in the thin layer chromatography. In this case nitrocellulose coated coating will be there that is it that is the difference. Once we have a glass slide. So, then you can decide the array. One slide can accommodate 2000 samples. Will you wait until 2000 samples will be accumulated from different studies? Not necessary right. So, then you can design the array into the miniaturized fashion. It is quite easy and possible. These chambers are custom made in our lab now are available in the market. So, one pad means it is like this. So, you can use one antibody for the entire slide for 2000 samples. If you have only 1000 samples you can divide them into two groups. So, 1000 here and 1000 here you can use two antibodies on each slide. And if you have very less number of samples you can also divide the chamber into 16 different chambers glass slide into 16 different chambers. So, that you can put 16 antibodies in one glass slide with maybe couple of hundreds of samples. So, in that way you can miniaturize your assay. So, in this case I will show you the examples. Here are the two pads. Here are the three pads. Here are the four pads. You can see this is the same sample set with the two different antibodies. Same samples with three different antibodies. Same samples with four different antibodies. That is it. It is just like a kind of miniaturizing your array. You can custom made array. So, once you have the array printed your protein of depending on the protein of interest you wanted to analyze and you can add antibodies and then probe and further next steps to be processed. So, after primary assume that in the RPPA also now you printed you added the primary after blocking and washing and everything. Here also nitrocellulose membrane empty spaces need to be blocked by using the blocking buffer. We use Rockland buffer which is a low fluorescence and after blocking your primary antibody hybridized. Then the second after washing to remove the excess antibody excess primary antibody bound to the membrane then you will add the secondary antibody. While choosing the secondary antibody you should be also very cautious. So, this choosing the antibody for detection is very critical and the outcome of the RPPA is based on the sensitivity and specificity of the primary antibodies. So, you should worry about the sensitivity and specificity, but also the signal detection method since the samples are delivered as a very very tiny droplets. And no protein separation is possible in RPPA. At least in western blot. So, your proteins are separated into small different bands. It is like a partial purification way, but here we are not following any separation methods right. So, the protein of interest may be expressed at a very low level and their visualization requires signal amplification. So, if the abundancy of the protein is very less, if your primary antibody is also very weak then still you have a chance to correct it in the secondary antibody detection method. As I said. So, how we can do that we will see. Thiramide signal amplification method has been widely applied to increase the sensitivity of the reverse phase protein arrays and calorimetric and communal limitations detection methods have very very high limitations in terms of linearity for quantification of signals. See for example, to get into your mind that what is the linearity means, if the if you use the fluorescence method the detection limits between 10 to 50 microgram. If you use calorimetric or chemiluminescence method if the signal gets saturated after 25 microgram you cannot have a proper quantification between 10 to 50 microgram range. But infrared detection system will have a very nice linearity between lower to high level of high level abundancy of the proteins. The fluorescence based signal amplification methods have been improved introduced to avoid issues with the signal detection. I will explain you one method. So, there are different methods are available. So, are applicable to development of the protein microarrays. It is again non labeling and labeling. So, I do not want to get into explain more about the labeling and non labeling methods and differences between these two. You can simply go through here and you can already see that labeling methods have labeling methods have little less usefulness in RPP approach than the non labeling approach. So, nowadays some people are using surface plasmon resonance as well. So, how it is a signal amplification method? You can see that simple spot is there and at primary antibody and labeled secondary antibody is added then you got a signal output. So, if you do not see any signal in this approach still you have a way to get it out. This is called AMSA method which was developed by one of the PhD student in my postdoc supervisor's lab, Heiko Mansberger. AMSA means antibody mediated signal amplification and you have a spot here. You can clearly see that you can have a spot here. This is a protein of interest and first antibody is added. Once the primary antibody is detected let us say for example, assume that it is a rabbit. The secondary antibody will be added anti rabbit raised in a goat. Then again we will select the third antibody which is raised against the goat in rabbit. This is again. So, now imagine that one label molecule became two label molecules. One molecule of infrared flow is attached with the one round of secondary to secondary antibody will have two fluorophores there. Then again if you repeat this for 10 times 10 up to three or four antibodies five antibodies one protein target it attached to five different signaling molecules. Then obviously, your signal will be increased by five times. So, this reiteration step will increase how it is. For example, in this example, in this cartoon you can see that the first direct detection you do not see any spot here. In the amplified detection first round of amplification we can start we can look at some of the spots in the right corner. After two rounds to three rounds of amplification then we can see almost all the spots in all the blocks here. So, that means, we are increasing the binding of labeled molecule to the protein of interest by repeating the signal amplification methods. So, only thing is cleverly we have to choose the what kind of antibodies to be used in the cycle primary, secondary and tertiary, tertiary to quaternary. So, this is a kind of cycle we have to repeat it. So, this is a reference if you wanted to go through how it is done here and you can clearly see that this red line is shifted from x axis towards to top. So, this shift of signal indicates that sensitivity has been increased, the signal detection has been increased. So, that we can visualize or we can detect the low abundant proteins in a large pool of proteins in the lysate present. This is a way and also linearity you can clearly see that. For example, here you can see this is a length. If you use infrared detection system, you will have this much distance to detect from lower concentration to higher concentration. If you use some of the other methods, you will end up only in the between the middle of the graph here up to maybe half of the concentration of the protein can be detectable. I am trying to just get you into the imagination, not able to show you the how signals can be amplified and all. So, this is a way. Once we have the array, once we have the signals here, definitely we need a soft ways to analyze. Nowadays, RPPA pipe which is a kind of open source software has been available and some of the companies have developed soft special soft ways to analyze the RPPA data generated, but it will have always commercial soft ways will have a lot of limitations. You cannot play with the data according to your biological question to be asked. So, keeping that in mind. So, that time myself also involved, Heiko Mansberger with Ulrika Korflab developed a RPPA analyzer which is a open source platform deposited into the website then you can use. So, there are different steps available here. After image analysis, the data can be collected in the form of GPR files and you will also give the file sample description, what kind of samples you printed there and slight description, what kind of antibodies probed like a 10 or 20 different antibodies against a set of samples and then you process it, the process data will be here and then automatically software look for quality controls means whether blank signal is really blank signal, the signal is really signal, what is the difference between blank signal to the real signal and also it will calculate the signal to noise ratio and if necessary to drop some of the arrays it will suggest what to drop, what to include then we can proceed further. Then obviously, once we have the quality control check, we always try to print a pool of sample in a serial dilution method. So, then the serial dilution will tell that whether antibody is detected the serially diluted samples, the linearity is 0 and the r-value is at least above 0.9, mean 1 is always ideal which will never achieved. So, at least 9 is the cutoff. Once we have this, then we will go ahead with the analysis further like a box plots, time dependent and all. So, when you have the box plots like here, you will see that whether the protein is upregulated or downregulated and also you can understand the signaling dynamics. Let us say for example, your samples are collected for a period of time, 0 minute to 10 minutes time. So, from 0 minute to 10 minutes time how the protein expression is going up or going down, simply you can see that the first few minutes signal is going up then started going down. So, signal dynamics we can measure and also we can fit this information into mathematical model to make a kind of network or to make a platform or to devise a pathway, regulation over the period of time, how it is getting stimulated, activated or inactivated and so on. Then obviously, you can design or you can draw a heat map, you can see that what are the different cluster of proteins are altering among the proteins I tested whether there have any relation or no relation. So, I hope today you have learnt about the importance of each and every step involved in RPPA workflow. You have probably now got a good glimpse and understanding of how different buffers behave differently with different sample types, their optimization is of utmost importance. Dr. Omani also emphasized on how it is important to establish good quality antibodies. And I must say this is one of the major issues in the entire proteomics community or the life science community to have the good standard antibody availability. And that is where the human protein atlas project along with several commercial companies have really provided the very high quality reagents which could be used for many implications including the one which we are talking today about reverse phase protein arrays. Today you must have also learnt about different detection methods and how each method has its own advantages and disadvantages. Dr. Omani further discussed about AMSA in detail. In the next lecture we will continue this interaction and talk about how to analyze reverse phase protein arrays microarray based data set. Thank you very much.


episode of In The Studio at Davis Media Act. My name is Lynn Weaver. Today I am Destiny Garcia. Thank you so much for coming in Destiny. Thank you for having me. You are a PhD candidate in the department in the US, UC Davis department of mechanical and aerospace engineering. Yes. That in itself is very impressive. Thank you. But what is striking is that you're also a former foster child. That's true. And that your background must have, in your background, you must have faced many challenges. So could you tell me, Destiny, how does your background as a foster child impact your life today as an adult and a wonderful scientist? Well, since I was in foster care at a really young age, I felt like I was like a sponge.   So I feel like being in foster care at a young age and just seeing everything around me, a lot of my values come from when I was in foster care or at that early age. I feel like I'm more empathetic. I can empathize with many people across different ranges and boards. I know how to think outside of the box related to engineering, but also just in life. I know that I had a different background, but I made it to the same spot as other people who are here in the same space as me today. So it's really helped shape me like how I think, how I motivated and just how I encounter and talk to other people around me. As a human being, yes.  Yes. Were you close to your parents, to your foster parents? Yes. So I was in foster care twice. When I was around three years old, around that age, I went into the foster care system for the first time with my other siblings as well. I'm one of seven siblings and at the time, only six siblings were here. And I went into the foster care system and I lived with my older sister and my older brother, my biological brother and sister. And then there was also another foster child there in the house that I was at. And then we got reunited with my parents and then they kind of messed up again. So we got back into the system. And then that time, my little brother was born and they wanted me to go with him to a foster home. And I didn't want to go with him because I didn't know him. He was just born, he was just a baby. And then I ended up going with my younger sister and my younger brother in a house that had other foster children as well. Where was this? In Fairfield, California. Are you originally from California? I'm originally from California. I was born in Woodland, California, so not too far from here. And I lived most of my life in Fairfield, California. And did you speak English at birth with your parents? Yes, I spoke English. And then just with like my family members, like my grandma, my aunts and uncles, Spanish. Spanish, yes. Would you say you were well treated as a foster child? Yes. I was very lucky and very blessed that I got put into the two different foster homes that I was in. They were great foster parents. I'm still in contact with them today.  So they're your family. They treated me so well, yes. They're my family, yes. So you were one of the lucky ones as a foster child. Are you still in contact with your biological parents now? Yes. After the second time of being in foster care, my parents fully buckled down and did everything that the court ordered them to do. And then we finally got reunited for the second time and that time being the last time I was in foster care. And so I'm mostly in contact with my biological mom since then. I got out of foster care when I was about five to six years old. So I was in there for a short period of time. And after that, I've been with my biological parents and siblings. And it's been an interesting family dynamic. When you take everyone, you split them apart and then you come back together. I would say I have a very interesting family dynamic. And when you say interesting family dynamic, is it you had to make a lot of adjustments? Oh, yes. Yes. Adjustments, the relationships that you have with your siblings or older siblings, your younger siblings. The first time around when I was in foster care, I was with my older siblings. So I was the baby, the youngest in that group. The second time around, I was the oldest with my two younger siblings. So I was the older sibling. So once you get put back together, once the puzzle is re-put together, I kind of felt like, am I an older sister or a little sister? Or where do I fit in now in this family puzzle? It's a fascinating story. But I'm so glad you were lucky and that you were reunited with your parents and your parents decided to become responsible and take care of their family. That's wonderful. Were there any challenging moments? In foster care? Oh, yes. For example. So for example, when you're a kid who is taken away from everything that you know, even if your parents are bad or they do bad things, for some reason, you still want your parents.  So being in foster care, at least the first time around, it was hard being a little kid, just wanting my parents. I just wanted to go with them. And at the time, I didn't know that maybe the things that they were doing were bad. I just wanted mom and dad. That's all you knew. Yes. Yes. And I can hear a little bit of emotion in your voice as you say that. So it's still marking you. Yeah. And just think of kids who are really young, separated from their families. They don't understand. Well, we have a very cruel example at the moment with the immigration debacle, that it's being caused by the administration and separating the children from the families, which is really unthinkable. Yeah. I can totally feel for them. Well, I understand. Yes. And I hope that they will soon be reunited. That's the main thing. And did you tell anyone, or maybe I should ask, what brought you to UC Davis? You must have been a really good student. I was. I was just one of those kids who was very tenacious. I wanted to just learn and be successful. In my head, success was like not being in jail, not being in drugs, not being in a gang. That was like success for me. So if all I had to do was do my homework and turn it in the next day, and I got good grade, I was going to be fine. It was bottom line for you. It was wonderful. Yeah. And so it just came a second nature to just do well in school, succeed. I did sports. I did extracurricular activities. I was always just staying out of trouble. I knew there was something more to life than just being in Fairfield, California. Oh, wonderful. Wonderful. You must have been very good in math. I was. And you still are. I still am. So what made you apply to UC Davis? So UC Davis was that one university that was on our way, you know, if we went to Sacramento, I always saw it on the freeway. Yes. And I don't remember which age I was, but I told myself, I'm going to go to that university. And I did. I applied right after high school. That's very, very interesting news. I mean, UC Davis should advertise more on the highway. Yeah. I just remember seeing the water tower from the freeway when you're passing UC Davis. It says UC Davis on it. And I just always pictured myself going to UC Davis. Did you tell when you arrived at UC Davis, did you tell anyone about your background? No. It was kind of one of those things. Why not? You just kind of like kept it quiet.  That's what I heard from other former foster children. Yes. Because there is that feeling that you were somewhat abandoned by your parents. Yes. And I also think that not a lot of people really understand what it means to go through foster care. So they think that like, oh, my parents are horrible. And yes, you know, they have all these judgments. They assume the worst right away. Yes. That is so illuminating, you know, what you're saying. And what advice would you give to a child in foster care at the moment? At the moment, your struggles of today will only make you more successful tomorrow. Like going through trials and tribulations just make you a stronger person. And it makes everything more valuable. Yes. Graduating high school, more valuable. Graduating college, more valuable. Going to graduate school is more valuable. And that is because it's a motivation that comes from within rather than being forced upon you by parents or your surroundings. It's something that either you have it or you don't have it. Yes. And I would say, if you're a form of foster youth, you have it. Yes. Or if you're in foster care right now, you have it. You have it. You have it. Yeah. And it's lovely to hear your story because I hope it will be an encouragement for other children or adults who have been in the same situation as you and who perhaps have doubts, self-doubts about themselves. Yeah. So that is, what are your plans for the future? When will you become a Dr. Garcia? Well, I'm hoping that this is my last year in grad school, so finishing up my last year. Hopefully next June, I'll be Dr. Garcia. My goals, like my ultimate goal is to become a professor in engineering, mechanical engineering. Just to be in that platform where I can represent a lot of different backgrounds and have a student look up to me and say, there's a brown woman and I can see myself in her. I would love to be a professor reaching out to other students. That sounds wonderful. Anything you'd like to add that you would like people to know, especially? Sometimes I just tell people I'm just an ordinary person.  And I just have a smile. If more people would smile more, I think the world would be a happier place. That's lovely. And also, one final question because I'm afraid our time is wrapping up. It's amazing how 15 minutes go by very fast. Have you heard of a lot of abuse in foster care? I've heard stories. My older siblings, their second time around, they didn't have as happy a story as their first time around. And to this day, I've heard stories where the foster parents abuse the resources that are given to them for the children. Yes. It's been a problem because I believe the law is they're given a stipend for the child, but sometimes the stipend goes... They keep it as an income. They try to use for themselves. Yes. But I think there's a lot more awareness now. And there are a lot of people working towards making the laws more just. Well, I'm afraid our time is up. Thank you so much. Thank you for coming in. And good luck to you. Thank you. May all your dreams come true. Thank you. And thank you all for watching from all of us here at Davis Media. See you next time.


Good afternoon. How are you doing good? How are you guys doing today? Very nice. Very well Good looks like we have about eight people watching and I know people have been tuning in afterwards So that's fun getting a lot of responses to the question So we're counting down to wikitree day y'all and we are at 21 days today Woo! It is fly by So if you haven't registered yet for wikitree day, I'm putting the link into the chat Go on out and register of course it with uh everything at wikitree. It's free It's gonna be a great uh set of speakers Sharing lots of information starting on thursday the second of november. So that's I can't wait Crazy crazy. Wow. Yeah, that's great All right, well Do we have a little Who's it? Who's it? Here we go. Here we go. Who could it be? Okay, and Kathy's with us. Yay, Kathy. Hi. Congratulations. Okay. If she's driving don't go off the road, Kathy You may you may swoop and holler, but yes, that's right So Kathy just email Aowyn to get to claim your prize She'll get that taken care of for you I love give it out prizes Nice if she says she won thanks to my niece whom she loves dearly. That's sweet Oh, is that is that because she was a nurse? A nurse for a doctor. Yeah Nice Very nice All right, and then let's share today's question Oh Oh Scandinavian roots we have 21 days to go That's the question Even if your answer is no it counts Right. Does it count if ancestry says you have 2 scandinavian? Sure And you can't find a single one in your family tree The DNA says so, but the paper trail doesn't Exactly All right, so Is there anything that we forgot to cover? I think that was I think that was everything you you're super efficient azure Well, now I've been doing it, uh, nine days. I think now so I've gotten I've got a little Script to follow so it goes pretty pretty good. Was there any questions about? Anybody had in the no chat? Everybody knows they can just go it out to any social media and respond there comment to this video So and that's not that's not in the comments below. Yeah the comment to the actual video. Yeah All right. Well, thank you Greg and Betsy for joining me today Always fun


It is an absolute pleasure to be here today, to be with all of you here in Boston and especially because Boston was really the launching pad for the American Revolution. And the American Revolution, as you all know, was not just a revolt of one group against another. It was a logical and intellectual revolt that succeeded in undermining the world's number one military and economic superpower. So I think this location has a lot of meaning for me. And it's very ironic that the event occurs right now at a time when politics is really threatening to undermine American society. It's breaking us apart. And I think if you really understand the Austrian understanding of politics, it all makes a lot of sense. And if you want to understand the Austrian meaning of politics, you just have to break it down. In Polly, what does Polly mean? It means many. It means numerous.  In tics, well, tics are blood-sucking parasites. And if you keep that definition, that is a scientific definition, if you keep that definition in mind, you'll know how to interpret things like the presidential race, the primaries, the debates, and ultimately the election. It's also a great pleasure to be here because I was kind of a last-minute addition to the program, not actually last-minute, but I insisted with Jeff Deist that I get onto this program because most of you probably don't know this. But in Auburn, it's been kind of warm and sunny and dry. We've got sort of wonderful summer-like conditions in Auburn. Not really any rain at all, and I insisted that I go to someplace where it's cold, rainy, and overcast to get a better feel for the great weather that we've been experiencing in Auburn, Alabama. And as a bonus, I've had an opportunity to use my first multi-gender bathroom here. I saw the sign in it. I didn't know what to do. Should I stay or should I go? So thank you. Is another skyscraper curse coming, is the talk I'm going to be giving here today. How many people in here have heard of the skyscraper curse? Wow, that's the first time that's ever happened.  But first, I want to lay some groundwork because the skyscraper curse is known to many individuals as a forecasting device. And that's not the most important reason for you understanding what a skyscraper curse is or the economics behind it because the skyscraper curse tells us a lot about what's going on, not just in a single building, but what's going on throughout the economy and what's causing those changes, which will ultimately lead to a skyscraper curse, but it's not just one building. It's something that's happening throughout the economy. It's happening in my hometown. It's happening here in Boston. It's happening in Asheville, North Carolina. It's happening in Toronto, Canada. Every place I've been, I've seen the same thing happening. So it's happening throughout our economies. And hopefully you'll be able to better recognize it as a result of my lecture. And it's also something that helps you understand the difference between the real economy and the perception of our economy. So a lot of people out there, including mainstream economists and the mainstream media, are telling us one thing about our economy. They're talking about the unemployment rate. They're talking about GDP. They're talking about economic growth. And then on the other hand, what we look and see in the economy and what other people are telling us is that things are really not that great, that things are really quite negative in many respects, that a lot of people are really, really upset. And that in turn helps us explain why does somebody like Donald Trump have such a message and have that message resonating with so many people who weren't even involved in the political process? And Bernie Sanders too, a socialist, something that Americans are like, no, no, no, no, no. But all of a sudden he has an appeal with millions of Americans, where he got just as many real delegates as Hillary Clinton, where millions of young people are saying, you know, I supported Bernie, I like Bernie because of his anti-establishment message, and I'm not going to turn over and go to Hillary Clinton. So that tells us that economically and politically that a lot of people are very upset and that that real economy that the other group is talking about really does not exist. So the skyscraper curse analysis is going to tell us a whole lot about why there is a difference between that mainstream message and that upstart Donald Trump Bernie Sanders message. Okay, the skyscraper curse. Currently the skyscraper curse is an historical association between the building of a world record-setting skyscraper and the coming of a world economic crisis. That's the association. Now that's not directly causation. In other words, the building of a skyscraper does not actually cause an economic crisis. Now you know, I hear you laughing, but three economists from Rutgers University spent an enormous amount of time collecting an enormous amount of data to refute the hypothesis that building a skyscraper causes an economic crisis. And not only did they come up with some empirical results, but those results were published in a widely respected academic economic journal. Well that really hurt me. But what was worse was that they sent their publication to the Economist magazine which is one of the flagship mainstream economics magazines, and they agreed. They said, yep, I guess skyscrapers don't really cause these economic crises based on this empirical information. But of course skyscrapers don't cause economic crises. The real causation which I've been writing about and talking about is that monetary policy in the form of artificially low interest rates is what actually causes things like an expansion in the economy. So it's stimulative at first to expand the economy. It also leads to people making investments in things like housing, retail, wholesale, technology. So those interest rates do have those effects and ultimately once they get the economy revved up enough, people have expectations of those profits and that growth continuing. And so they start to undertake projects like world record setting skyscrapers. So it's interest rates that cause an expansion of GDP. It's those artificially low interest rates that ultimately cause the building or lead to the building of world record setting skyscrapers. And ultimately that whole process is what brings about an economic crisis. So it's not construction leading to crisis. It's interest rates leading to both construction and economic crisis. And what hurts me even more is that those three economists from Rutgers University, that's what their results actually said. It's just that their interpretation, so their equations, their empirical, their statistics all validate the theory of the skyscraper curse, but their narrative, what they wrote in their article said the opposite. Okay, the history of the curse. Andrew Lawrence is a financial analyst in real estate, put together the skyscraper curse in 1999. That's when I found out about it, was very excited about it and been writing about it ever since. He showed that the panic of 1907 was surrounded by the building of the Singer building in New York and the Metropolitan Life building in New York, both of which were new record setting skyscrapers. The Metropolitan Life building is now named the Trump building. In 1929, the building of 40 Wall Street set a new record. The Chrysler building in the following year set another record. The, of course, the Great Depression started at that time and in 1931 the Empire State building opened up. So we have, in the case of the panic of 1907, two records. In the case of the Great Depression, we have three records. And then fast forward to the late 1960s and early 1970s. World Trade Tower One set a new record in 1970. World Trade Two set another record the following year. And the Sears building in Chicago set a record, I think, in 1972. So that preceded the stagflation of the 1970s where we had both, for the first time, we had both high unemployment and high inflation. Things were so bad that we had to go off the gold standard, impose wage and price controls on the American economy. Fast forward to the late 1990s where we had the Asian contagion in places like Singapore, Malaysia, Indonesia, which was a tech bubble. And then later, of course, we had a tech bubble here in the United States. And we also had two new record-setting skyscrapers in the form of the Petronas Towers and also Taipei 101. So again, multiple records associated with world economic crises. And then fast forward to 2007, we have the housing bubble in the United States, record-setting stock markets in the United States, ultra low unemployment in the U.S. And also we had the building of the Burj Khalifi Tower in Dubai in the United Arab Emirates setting a new record. It officially got to the height of a new record in July of 2007 at a time when everything was seemingly great. But by the time the building opened, of course, we were in a worldwide financial crisis. And there are others too, you can look at national records, you can look at continental records. As a matter of fact, many of the tall buildings in Boston were built during these same boom times so that if you look at a list of tall buildings in Boston, you'll see a very large number of them were open for business in 1970 and 1971. Right before the stagflation of the 1970s going off the gold standard and all of that. Right now, in Saudi Arabia, which is in a lot of economic trouble these days with low oil prices, but they are building out in the middle of the desert the Kingdom Tower. And it's being built by the Saudi royal family. And they have one of the largest construction firms in the world. And they are building this Kingdom Tower. It's going to be one kilometer tall. Now they wanted to build it one mile tall, but when the construction engineers got done with their survey, they told the king that we think that this thing would be so heavy that it would just plunge into the middle of the earth because the bedrock underneath it couldn't hold that much. So they brought their plans down from a mile to a kilometer, which is still going to be a record. And if you see pictures of this, all these buildings are very secretive. And the Saudis have an advantage in secrecy because instead of building this record in downtown Manhattan or downtown Boston, they're building it in Jeddah, which is a relatively small city, and they're building it north of Jeddah out in the middle of the desert. So the place that's least likely where you would want to build really tall is out in the middle of the desert. Where you really want to build tall is typically where land is very scarce. So the Kingdom Tower is coming. It clearly tells us the type of economy that we're in, that this is not real sustainable economic growth, but it's an economic expansion based on male investments. And that's M-A-L, it's not men investments, it's bad investments. So it's coming and we've got to be worried about that. We've got to be looking at our economy in a whole different way. Because again, the skyscraper curse fundamentals tell us that it's not just the one building out in the middle of the desert, but it's permeating the entire worldwide economy. So this is an indicator. It's an indicator of trouble. And I think it agrees with fundamental analysis on the ground, on the street, what's actually going on. It's a train wreck waiting to happen. And we shouldn't be the least bit surprised. Central Bank monetary policy has been unprecedented, radical even by mainstream standards, and it's really been reckless. Zero interest rate policies. Well, that might be conceivable for a very short period of time in a normal economy, but not for seven or eight years. That's never happened ever. And that didn't work, so they used quantitative easy. So typically they'll buy bonds from banks to reduce interest rates. They did that, they got them down to zero and it wasn't good enough. So then they just started buying more bonds. They started buying mortgage-backed securities. They inflated, for example, the balance sheet of the Fed from less than $1 trillion to more than $4 trillion in a very short period of time. And it's not just our Fed. It's the European Central Bank, it's the Bank of Japan, and it's virtually all other central banks keeping up with the Joneses, basically buying more securities. The Japanese are actually buying Japanese stocks, which is unprecedented and beyond the pale. I mean, central banks, the old lessons about buying short-term government securities, which were absolutely almost a gold standard in terms of being able to be sure that they're going to hold their value, now they're buying long-term government bonds and the Japanese are buying Japanese stocks. And they're a big holder of most of Japanese companies now. So that tells us there's also trouble ahead. So what we have is massive mail investments driving up GDP in our economy. So that tells us why the mainstream perspective and our perspective, how they match up. Our perspective is that the Fed is generating massive amounts of mail investments, and that explains why GDP is going up. And it's not just these mail investments driven by the Fed and other central banks, it's also government debt. Government has been spending beyond its means for years. I think we're borrowing over $600 billion to finance our deficits in the U.S. this year, and that's a record low since the financial crisis. We've doubled our national debt since the financial crisis. We've gone to less than $10 trillion in debt to almost $19 trillion in debt. So government has been spending about a trillion extra dollars each year. Well, when government spends a trillion dollars extra more than it takes in, that's going to add to that number of GDP. So we see national debt as a problem, is mortgaging our future, as literally throwing out the baby with the bathwater. Future generations, mainstream economics says, we're just raising GDP, we're just putting people to work. So again, the stories can be solved or resolved using those analysis. And there's nothing to suggest that government has used this opportunity to reform itself. It's made healthcare much worse with Obamacare, it's made banking and finance much worse with Dodd-Frank. Obviously the national debt is much worse, entitlements are much worse, war is much worse. We find ourselves in a quagmire in the Middle East, locking horns with Iran, with Turkey, with Russia, with Syria. So they haven't solved anything, they've only made things much worse. I'm working on a book on the skyscraper curse, and I just wanted to conclude with a paragraph that I tapped out of the manuscript because this problem is now baked in to the cake, unfortunately. There's really nothing Mr. Trump or Secretary Clinton can do about it. It's in all likelihood going to happen. I suppose if we made Lou Rockwell president and made Congressman Ron Paul, Speaker of the House, and we put Jeff Deist in charge of the Federal Reserve, we could probably make it turn out a little better than it's probably going to turn out. But we've already made those male investments, we've already weakened our economy, we've already increased the national debt by an enormous amount. And so this paragraph addresses my mainstream critics who say that everything is really doing okay. We've gotten ourselves out of the hole, we've prevented a great depression. So get off of our backs. So I did a little calculation, and I'll quote myself, however, as a best case scenario, let us make the heroic assumption that all the government spending was really valuable. In other words, all of that additional government spending that they borrowed the money for was really worth the money they spent it on. So there are no bridges to nowhere. There are no baby-killing bombs being made. It's all really good stuff that they're spending that money on. Let's make that assumption. And that all those investments made between 2008 under these low interest rate policies and the present will turn out perfectly well. In other words, all this construction is going to be used. It's going to be great. We're going to be great again. If we take the government's measure of the economy, that is gross domestic product, and adjust the government's measure for their measure of price increases, CPI, Consumer Price Index, or the GDP deflator, and then we adjust that figure by the increase in population. So we'll adjust the government's measure of the economy by inflation and by population. So it's their measure of how well we are doing on a per-person basis. We find that the U.S. economy grew at less than 4 percent over the entire period from 2008 to the last quarter in 2016. So that's 4 percent over that entire period. So 4 percent over eight years, maybe a half of a percent. And if we compare that to previous experience where GDP overall grew at 3 percent, or if we go back to the antebellum period where we had a free market economy and a free banking system, we didn't have any government regulation of banking or money or anything, and we adjust that economy for inflation and population, GDP growth per year per person was growing at over 3 percent per year. We get a more truer picture of what we are experiencing versus what we could be experiencing if we had an economy based on Austrian economics and free markets, and that is stark reality. Thank you very much.