People used to think that the most dangerous game to hunt was Man. We have recently realized, though, that's not true. The most dangerous game to hunt is for a Netflix original movie that's good. Let's talk about Red Notice. The trailer promises three big-name stars are going to get together and compete for some artifacts or something. I don't know. I just remember Dwayne Johnson, Ryan Reynolds, and Gal Gadot were all over the promotional materials for this. They were the focus of this movie. And oftentimes, I find, especially with Netflix, they're kind of misleading when it comes to their talent. They'll put them on the posters, they'll put them on these teaser images, but they'll only be in the movie for like five minutes. Thankfully, not the case here. All three actors play a prominent role in this, and they work very well with each other. The big standout for me was the always stunning, always sexy beyond all compare Ryan Reynolds. Really playing up the jokes, really having a good time. No, seriously, Gal Gadot's really good in this. Although, to criticize, I'm still not sure she's a great actress. I think she's great in very specific instances, but sometimes when she's reading off her lines of dialogue here, it feels like she's reading off her lines of dialogue here. Maybe the accent's throwing me, or maybe it's helping her cause. I don't know. It's just once in a while not working for me. She's always working though, visually speaking.  Since Dwayne Johnson is in approximately 365 movies a year, he's done this formula many, many times. And at this point, I'm convinced he has a stipulation in his contract that he has to film in a jungle setting at some point. Since he's been in so many movies, I've seen his shtick before, where he doesn't work well with others. He has a partner that he can't stand. What movie was it recently that this just happened? Oh yeah, Hobbs and Shaw. This is basically Hobbs and Shaw, but it's more self-aware. Red Notice is a really tricky movie to talk about because the praises I want to give it can also be spoilers. So I'm gonna be very vague, but I will just say this. As I watched this movie, I kept just checking off all the boxes of cliches I've seen in action movies over the years.  That's like my bread and butter. So I've seen all of this shtick before. The good news for Red Notice is the writer and director know this. They too are familiar with the formula. And as the movie progresses, the formula gets kind of broken down. They kind of flip things on their head. That's as vague as I can get. So you too may have started watching this movie like I did and thought to yourself, why am I doing this? This is so lame. I've seen all of these tropes a million times. You might start rolling your eyes. You might get heavy sighs if you're by yourself watching like I was. Just sad, alone, miserable. And then you'll hit a point in the movie and you'll go, oh, okay. That's kind of cool. That's kind of fun what they did. At least that's what happened for me.  In which case, I recommend finishing it. It's not an overly long film. It's got plenty of action. Some of it's really bad. Some of it has some incredibly embarrassing CG work going on. The green screen stuff. Oh my God, what is happening in this film? There are points early on where I thought, wow, this is a really slick, highly produced picture. And then as we get to the middle part, specifically, there's a sequence that takes place in a bullfighting ring. Wow. We just had an afternoon, I guess, with the guys on set. We had a green screen and that was about it. Really bad. And then not much later, there's a car chase scene in a tunnel or a cave of sorts. Very Indiana Jones-ish. Complete with the horrid green screen work going on. At least Indiana Jones has an excuse. It's pulpy. It's supposed to be a little tongue-in-cheek. And also, you know, it came out like 30 years ago at this point. This movie doesn't have that excuse. I think they just ran out of budget. It looks bad. The basic plot is very simple. There's three MacGuffins in the form of eggs. Cleopatra eggs that have gone missing. They're worth a lot of money. Actually, one of them's not missing. It's at a prominent museum. Unfortunately for the museum, Ryan Reynolds is the best in the business when it comes to stealing these things. Second best, only to Wonder Woman. She comes and goes with little in her way. She's very good at what she does, arguably too good. I mean, this is one of those movies where people just, oh, they're here and they're in this country now and they're there and they all find each other instantly. I want to criticize that stuff, but then the movie acknowledges it. So it's weird. You're watching a film where the majority of the picture is, I've seen this. I know what's gonna happen. That's not possible. How are they finding each other? But then later it's revealed. And so you have to kind of reflect back on the movie. Go, oh, that was kind of clever. But in the moments leading up to that, you're not having a great time because it's a been there, done that scenario, but much worse. As an overall adventure movie, it's fine. All the characters have good chemistry. Plenty of stuff happening to keep you invested. As for a Netflix movie, this is like an A plus because we've been really scraping the bottom of the barrel lately. This was a refreshing change of pace. Still not great, but better than what we've had. I personally had a better time with Hobbs and Shaw. I thought the spectacle there was better. I thought the action was great. The chemistry between the two bulky action stars was on point. So I would recommend that over Red Notice if you haven't seen it and you're looking for something dumb and action-filled.  It's not the best way to spend an afternoon either, but here we are. All right, those are my thoughts on Red Notice. Let me know what you think in the comments. Did you love this film? Did you absolutely hate it? Did you give up on it before the final act like I almost did? Or did you push through and say, oh, that was kind of clever. I didn't see that coming. We'll talk about it a bit in the comments. Make sure to subscribe to the channel if you haven't. Like the video if you had a little bit of fun here. Maybe you had a lot of fun. I don't know. I don't know you. And hopefully I'll see you next time. Take care. If you're new here, I suggest sticking around. There's tons of movie-related content on this channel, lots of reviews, rants. Here's one or two right there. Probably just one. And then a picture of me that you can subscribe to. So check those out. All you have to do is push or click on that video. It'll start right up. That's the power of technology today. And the face, that's for that. Well, again, you can subscribe here. That's paramount to the success of this channel. It's getting people on board. Okay, I think that's, I think I said enough. I'll leave you to it.


My name is Esmeralda Bonn and I'm from the University of Nottingham. My research is about the communication and behavioural standards of public office holders in the United Kingdom. My name is Aisha Slamat. I'm from Bournemouth University. I'm trying to develop a machine learning analytical model specifically for SMEs in the private coach hire industry. Hi, I'm Oliver Exton. I'm a second year PhD student at the University of Cambridge. My current research is looking at how firms have changed their exporting decisions in response to the Brexit referendum. Being the UK DF Data Impact Fellow has greatly increased my exposure within the faculty. It's also increased the exposure of my work. It's given me the opportunity to travel elsewhere for training which will help me in my career developments. It's helpful to know that there are other peers out there who will be there to support me in my career. We had seven early career researchers here with us today who we funded to support them in looking different ways that they can impact with their research. It's been a really great opportunity today to find out about the different sorts of research that they're doing, to learn about some of the challenges that they're having with also the opportunities. And it's great that people have been able to share the sort of practice and stories and that we've been able to give them some input into thinking about some different ideas about what impact could be and also how they can develop their voice as an early career researcher. I'm Antonia Igemoniahelam. I'm based at a Beresuit University. I work in the project that's called Global Rural and the primary focus of the research is to examine the way globalization impacts on rural communities around the world. Being an early career researcher for me allows me to be able to make mistakes and learn from them and not feel bad about it. It's great that space for me to kind of be comfortable in my own zone. My name is Matteo Sandi and I work at the Centre for Economic Performance at the London School of Economics. My research is about school discipline and youth crime. I feel the obvious pressure of having to, you know, demonstrate my skills, probably at the end also one reason to believe my research more because, you know, it needs to be reliable and interesting. I started the fellowship at quite an early point in my PhD but I found it particularly useful as I started to think about how I can use money towards generating impact. We've been very fortunate to have the previous cohort been able to attend today and they've been very, very positive in their feedback. I see a lot of potential for collaboration and collaboration is what we really need. It's much better than competition and it's rather what we should all do.


All right, so the recording is on. Let's just pray together and then we will get started in our course, BC 308 on Revelation and Daniel. And let's just pray and ask God that as we look into His Word, we will receive understanding and also revelation and our lives will be strengthened. Could somebody lead us in prayer, please? As we begin? Father God, we come before you, join once again. We want to just say thank you, Father God, for everything. Thank you, Father God, for your Word, Father God. Thank you, Father Subject. Thank you, Father God. Give your wisdom and audits and peace and comfort, Father God. Give your revelation, Father God that we can understand the subject, Father God. Thank you. Amen to your hand every time, Father, thank you. Almighty Jesus, name be pray, amen. Amen, amen. All right, thank you. Good morning, everyone. We are going through our study in the book of Revelation. And just quickly review a few things. We are still in the early chapters of Revelation. Just to quickly review our approach to the book of, our approach in studying the book of Revelation is that one of the things we've said is that really the Lord Jesus gave the soul revelation to John, the apostle. You know, in three parts, he said, write the things you have seen, the things that are and the things that are yet to come. So we have broken or say, we have divided the book of Revelation into those three timelines. Things that you have seen are essentially what John saw right then, that is Revelation chapter one, things that are, which has to do with the seven churches that existed at that time when John was there, which is Revelations chapter two and three. And then things which are too come, that means things that are going to happen in the future, which is Revelation chapter four on till the end. So we divide or we break up the book of Revelation to these three simple things, just as the Lord told John. We are right now going through chapters two and three, just looking at those seven churches. What was happening with those seven churches and what lessons we can learn from those seven churches. So we are just going through that out quickly. Summarize some things and then we just, we are in chapter three, so we will finish that and then move on into chapter four. So, you know, as the Lord Jesus is revealing this to John and you know, John sees that the Lord holds the seven stars in his right hand, the seven stars, each of the star represents the leader of each of the seven churches, the human leader of the seven churches. So he holds the seven stars in his right hand and the Lord is also the one who walks in the midst of the seven lampstands. So each of the seven churches is represented by a lampstand and the Lord is walking in the midst. That means he is, you know, overseeing, looking at all those seven churches. Now what we mentioned last week is, this is only representative of what the Lord is doing. That means the reality is there were hundreds of churches at that time, all across, you know, the Middle East and across the Mediterranean, literally hundreds of churches, small, small, small towns, villages, cities. So there were many, many churches. But in the vision, the message that is coming across to us is the Lord is holding the leaders of the churches in his hand, both as a protection and also as a sign of accountability. That means these leaders are accountable to the Lord, they're answerable to the Lord. And secondly, the Lord is one who walks in the midst of the churches, meaning he is overseeing, he is looking into every local church, how it's working, how, what is happening, what is going on, he's seeing. You know, so it's, even though here we are seeing only, you know, seven stars, seven churches, seven lampstands, the reality is this applies to all of the churches of the Lord, right? So that's something we mentioned. And therefore it applies to us as well. You know, we are here 2,000 years later, but the message the Lord gives to each of these churches is a message for us today. That means we must also check our own lives as leaders and also our own churches as leaders because the Lord is looking into every local church, examining it, seeing how it's doing, et cetera, right? So just to cover the churches we have gone through our revelation to be the first church that the Lord speaks to is the church in Ephesians.  They're doing the ministry, they're doing the work and they're very diligent, they are very, you know, they are very discerning and so on. But yet the one thing he says is, look, you have departed from your first love. And so you need to repent and you need to do the first works. So first love, first works are very important and they have to be maintained first in our lives. First love for the Lord, first works towards the Lord that has always to be maintained. And if we depart from that, the Lord Jesus is telling the church in Ephesians, you have actually fallen. That means you have gone down. You know, you're not where you're supposed to be. Why? Simply because they have gone away from their first love. They have stopped doing the first works. So we learn from that, you know, how important it is to keep our love for the Lord Jesus Christ. Love the Lord our God, all our heart, all our mind, all our soul, all our strength and to do the first works, which is to seek him first, to pray, to read the Bible, to, you know, just focus on him in worship. Those are the first works, meaning that the works that express my first love, those are first works. So I must do that first, keep that always in our lives and then, okay, go about all the other things. It's a good things. So the challenge for you and me is, personally as leaders, we must maintain first love and first works and also make sure that congregation, the church as a whole, maintains first love and first works. That means keep the focus on the Lord Jesus Christ and don't let the ministry, the works, you know, they were doing a lot of work and they were, the church in Ephesians, very strong, et cetera. But you shouldn't let that become more important than first love, first works. The church in Smyrna, the Lord recognizes that, you know, they're doing their works, they're doing the ministry, or they're going through hardship. They may not be very well off. So he says, I know your poverty.  And spiritually, they're really good. So the church in Smyrna is a good church. It's one of the two churches that he doesn't rebuke. He doesn't tell them they have to repent of anything. He only wants them that they're going to face hardship for 10 days, but he's going to let them be tested. So the reason why he doesn't stop the hardship is it's part of their testing.  They're going to come out stronger. So he, you know, he's letting them go through those hardships.  The church in Purgamos and the church in Taitira, which we saw last week, both these churches had kind of a similar problem. The problem was they were taller. I mean, they were good churches. Again, they had, you know, they were doing their works. They were walking in love. So like he tells the church in Taitira, and I was 19, a revelation too.  So, you know, he's really commending this church. They're doing very well. But the common problem both these churches was they were tolerating wrong doctrine. They were tolerating it. So in the church in Purgamos, and they were tolerating the wrong doctrine of Balaam and there are doctrine of Nicoletians. Basically this doctrine was leading people to do things that are displacing to the Lord. So how do we test doctrine? What is the fruit of that doctrine? You know, how is it affecting people's lives? Is it drawing them closer to the Lord? Is it helping them become more like Jesus? Or is it causing them to go into things that are wrong, sinful, displeasing to God? So in the case of the church in Purgamos, the doctrine of Balaam, the doctrine of Nicoletians was just leading people away. In the case of the church in Taitira, they were tolerating a woman who claimed to be a prophetess. But what she was doing was actually leading people, her teaching, what she was teaching, was actually leading people into sin, doing things that are displacing to God. So once again, the Lord is not happy about that. So the learning for us here is, we have to watch over the doctrine, what we allow to be preached and taught and brought to the people in our care, right? We have to make sure that what we preach, what is taught, what is brought to the people should strengthen them and help them become more like Christ and not, give them a license to go do things that are wrong, things that are displacing to God. That's a test, we have to be watchful. And so in both these churches, he says, you need to repent, you need to get it back, get back, take these things out.   So it's a learning for us. Then we went into chapter three, the church in Sardis. We kind of did that very, very quickly. The church in Sardis had a different kind of a problem, but it is something we also must be very careful about. The problem with the church in Sardis was, they had a reputation, a name that they were alive, but the Lord said to them, you are dead. So we cannot go by reputation. We cannot go by what people say about us. As leaders especially, we have to go to the Lord and say, Lord, what are you saying about the church that I am posturing, I'm leading? Where are we? How do you see us in the sense, is everything okay? Are the things we need to correct? Because if you only go by reputation, which is what people are saying about us, like the church in Sardis, they may say, wow, what a wonderful church, what, this church is alive, this church is full of life, and they may say nice things, but we can't go by that. So that was a problem with this church in Sardis. People were saying, you're alive, but the Lord was saying, you are dead. And then he points out certain things. He says, your works are not perfect before me, before God, that means they were doing things, but what they were doing was not right in God's eyes. I mean, it wasn't really what God wanted them to do. And then he also points out, he says, you know, this is in verse three, remember, remember how you have received and heard, hold fast. So it seems like they had left the things that they were taught in the very beginning, and they moved away from it. So he says, remember how you received and heard, you know, what you were taught in the very beginning, remember those things and hold fast to it. So it teaches us how important it is for us as a church, that we need to hold on to the foundational, fundamental truths, and not depart from it. Hold on, remember how you have received and heard, and hold fast. Another thing he points out to the church in Sardis is, that most of the people that have defiled their garments, that's a phrase that simply means, they are now living compromised lives. They're living in, you know, worldly things. So they've defiled their garments, they become worldly. Only there are, there are only a few of them who have not defiled their garments. That's in Revelation three, verse four. So that's another problem we find. So maybe it's, you know, it's because of these things that their works are not right in the eyes of God. And ultimately, they have a reputation, they're alive, but they are dead. So it's a big learning for us. We have to be very careful as leaders and as a church. So God, please help us to be right in your eyes. Please help us to live wholeheartedly before you. Please help us to do the things you want us to do, and not worry about reputation. Okay, it's nice if people, you know, say nice things about us, that's up to them, but we can't live by it. We have to go before God, and say, what do you say about us? Okay, so we stopped there, and we are going to pick up Revelation three, verse seven. We're going to look at the church in Philadelphia. Okay, so let's please start reading from Revelation chapter three. Sorry, that was a long review. I just reviewed everything we did last week, but I did quite a lot of time. Anyway, Revelation chapter three, we're going to read from verse seven to 13, and could somebody read that passage for us? This is the church in Philadelphia. Anyone can read? Siddharth, you want to read that for us, please? Chapter three also. Yes, Revelation three, seven to 13. For the end of the church in Philadelphia, because the work of faith is holy and true, we hold the key of faith. What we open, no one can shut. What we shut, no one can open. I know you did. See, I have placed before you an open door, but no one can shut. I know that you have little strength, yet you have kept your words in a marginalized manner. I will make those who are, who are of the synagogue faith, to claim to be Jews because they are not, but are liars. I will make them come and fall down of your faith and of knowledge that I have loved you. Since you have yet no amounts to endure patiently, I will also keep you from the power of fire that's going to come on the whole world to the best inhabitants of the whole. I'm coming soon, hold on to what you have, so that no one will close their eyes. I want you to tell us, I will not have to look in the center of my heart. Never again will they lose. I will write on them the name of my God and the name of the city of my God. The new Jerusalem is coming back out of heaven from my God. And I will also write on them my new name. We are going to ask you a present word for the state church of Jesus Christ. Okay, thank you. All right, so the Lord is speaking to this church in Philadelphia. Now, this church in Philadelphia is, the other church to which the Lord does not rebuke. He doesn't tell them, you know, repent of anything. So church in Smyrna and the church in Philadelphia. These are the two churches. There is no call to repentance, there's no rebuke. But let's look at the church and let's see what the Lord is speaking to them. He's saying, he introduces himself again. This is another thing to see across the seven churches. How the Lord introduces himself to each one of the seven churches and what he promises as a reward for the overcomer because these are still, you know, that's who he is today. And so it's very much something we should look into. So he introduces himself as the one who has the key of David, that means he has the authority of the kingdom. The authority of the kingdom, right? David represents King David. He has a key of David, authority of that kingdom. He's just showing to them that he is ruler. He is the one with authority of the line of David. And he says, look, this is verse seven. He says, when I open, no one can shut it. When I shut, no one can open. In other words, this is absolute authority. So he's assuring the church, look, I am the Lord who has absolute authority. I am the one who has the key of David. The king, the ruler of the kingdom, absolute authority. No one can undo what I do. If I open, it remains open, if I shut, it remains shut. And from that introduction, he tells them, I have set before you an open door, and no one can shut it. Now, in what exact context this applies, we don't know, right? Maybe is it an open door for ministry? Is it an open door in their city to keep growing and reaching people? What exactly this open door has to do with that? Of course, the leader of the church and the church would understand, because it is, of course, relevant to their, do what's going on in and amongst them. But for us today, we have to remember that the Lord deals with us in a similar way. That the Lord, our Lord, is the one with absolute authority.  He says, I've set before you an open door, no one can shut.    Right? So that's the assurance he's giving this church. That's a beautiful assurance, I was praying, Lord, let that happen for us as well. Just like what he did for this church, do it for us, because God is no respecter of persons. And of course, our context is different. We are in a different city and doing a different kind of ministry here, but he's the same Lord. And he's the same Lord over each of our churches. And then he says, you know, if you look at the good side of the church in Philadelphia, and I will just highlight that, here is a church that has kept his word. You don't find that mentioned in any of the other churches. He says, you have kept my word. So this is in verse eight. So it seems to us that this church in Philadelphia was a church that was committed and was living by and was walking by the word of God. You have kept my word. So through all kinds of things, they've kept his word, they've not denied his name. And he also says in verse 10, once again, you have kept my command to persevere. That means you haven't quit. You've kept my command to persevere. They've kept going. So these are two things the Lord, you know, highlights about the church. You've kept my word and you've not denied my name. You have kept my command to persevere. Verse 10. So this church seemed to be a church that really stayed with the word of God, refused to deny Christ in any way and they persevered, they persevered. They didn't quit. They didn't give up. They kept going. And it is so amazing what he promises them. First, he had promised them. I said, before you're an open door, nobody can shut it. Second, he said, I will make the synagogue of Satan. I will make those people come and worship at your feet so that they will know I love you. The synagogue of Satan represents these Jewish people who were opposing them. They were actually, you know, these people who, or at least they claimed to be Jews and but they were opposing the church and they were actually, Jesus refers to them as a synagogue of Satan. People who are the devil is in their midst. They're an assembly or a gathering of Satan's people. What a promise he gives them.  I was praying, Lord, do this for us. You know, those who try to oppose the church, who oppose the work of God, let them come and bow before the Lord, who is in our midst and let them acknowledge the Lord loves us, just like what he told this church.  And this could refer to the great tribulation coming. It could refer to that. Some may want to take it in an immediate context which means something that happened right then and there and there at the end time, which the only issue is, he says it's something that will dwell that comes upon the people now on the whole world and those who dwell on the whole earth. So that's the problem. If you apply verse 10 in an immediate context, that means if you put it back in the first century, well, what kind of a tribulation is perfect to the whole world? We don't have any. So that is why Revelation 3.10, most likely, and I'm just saying most likely because I'm not saying very definitely, but just we're trying to understand it. Most likely points to that great tribulation, the trial that will come upon the whole world, upon all the people who dwell on the earth. He says, I'll keep you from it. Meaning I'll keep you from that great tribulation. And he also promises them other things.  This is what meaning, I'm gonna ask, what does it mean for a new name? It's a very, it's like, God is getting very personal.  That's a Revelation 3.12. It's a name nobody else knows, but he says, I will give you that special name. You know, it's like this between the Lord and you, right? So the Lord and the believer, who's all coming, I'll give you a special name. Just show that I love you and you're gonna be part of that new Jerusalem which will come from heaven. So that's all future, it's all in the future. This is the blessing that he has promised believers. Okay, so the key takeaway from the church here in Philadelphia is they kept his word. They didn't deny his name and they kept his command to persevere. And that was it, in response to that, he has promised them wonderful things, something that we can ask for our own congregations and our own churches. God, you said before us an open door, no one can shut. You make those who are opposers come and bow at our feet and let them acknowledge that you love us Lord. And God, you keep us, preserve us. Of course, you keep us and preserve us from the out of trial, okay? So the last church, this is Revelation 3.14 to 22. This is the last one. The church at Laodicea, can somebody read that? Revelation chapter 3, 14 to 22, please. And to the angel of the church of the Laodicea, write, this thing says, the amen, the faithful and true witness, the beginning of the creation of God. I know your words that you are neither cold nor hot. I could wish you so hot.  Because you say, I am rich and become wealthy and have merest nothing. And do not know that you are wretched, miserable, poor, blind and naked. I counsel you to buy from me gold, refine in the fire that you may be rich and white carmines that you may be clothed, that the same of your nakedness may be revealed. And anoint your eyes with eye slip that you may see as many as I love, Arabic and Cheston. Therefore, be jealous and repent. Behold, I stand at the door and knock. If anyone hears my voice and opens the door, I will come into him and dine with him, and he with me. To him who overcomes, I will grant to sit with me on my throne as I also overcome and sat down with my father on his throne. He who has an ear, let him hear what the Spirit says to the circus. Thank you. Thank you. So, to this church in Leodicea, it is a very, very strong rebuke, very strong rebuke. But there is a lot that we can learn and take for ourselves. Now, it is interesting how Jesus introduces himself to this church. He says, look, this is verse 14. He is the Amen, meaning the fulfillment of all the promises of God. He is the Amen. He is the faithful and true witness. He is the one who came into the earth, faithful and true witness, referring to his earthly life, the one who lived on this earth and bore witness to the truth. Faithfully, he never compromised. He was faithful and true witness. He bore witness to the other truth, the word he was called to preach. Now, this last part of verse 14 can cause a lot of trouble. It says, he Jesus says about himself, he is the beginning of the creation of God. So it can cause a lot of trouble because people can use this and some cults, evidently. I mean, like, for example, the Jehovah's Witness and others who don't believe in the deity of Christ in the sense of him being equal with God but who talk about Jesus as a created being would use this latter part of this verse because Jesus says he is the beginning of the creation of God.  So he's not God, but he's the first one whom God created. He says he created being. But that is not an interpretation that is consistent with the rest of the scripture. So remember, one of the principles of hermeneutics that he learned is every passage in scripture has to be interpreted in the light of the rest of scripture. You can't just say things in isolation. The other passage in scripture referred to Jesus Christ as God, who was eternal, who was there from everlasting to everlasting. And he was, you know, he's the Word. The Word was with God. He was in the beginning with God and so on. So don't let this phrase or the sentence, the beginning of the creation of God, put you off in the sense through you off, man. It cannot be interpreted as he's the first one whom God created. But it has to be understood as he is the starting point of all of creation, that means all creation took place through him, by him and for him. This is where it started, or this is the one through whom it started has to be understood like that. It means in him, through him and by him, all creation began. And this is very consistent with what other scriptures say. One passage that I would point to is Colossians 7 to 1, and verse 16. It says 16 and 17, for by him all things were created. By him all things were created. All things were created through him and for him. So I'm talking about Colossians 1 and verse 16 and 17. So that's the way we understand this. So don't let somebody take this, this line, the sentence and say, oh, look, he was created. Now you have to understand that that's everything, all of creation began in him. And that's consistent with Colossians 1, 16 and 17. It's consistent with John chapter 1, verses 1, 2 and 3. All things were created by him and for him. And several other scriptures, including Hebrews 1, 3. Again, it says he, everything began in Christ. So Jesus introduced himself. Look, everything starts with me. The beginning of the creation of God, all of creation started there with Jesus. And it came out of him. And so he speaks to the church and there it is here. And he says in verse 15, see, I know your works. That means you aren't doing ministry as a church. I know your works, but it's lukewarm. It's lukewarm, meaning it's half-hearted. You're doing something, but your heart's not in it. It's passionate work. It's not wholehearted work. You're lukewarm. And I'd rather that you be on hot or cold. But you're doing this. You're doing works, but it's half-hearted. Heart's not in it. Now, why is that? Why is it that they're not through on fire? Because he says, and so verse 16 he says, because you're lukewarm, I'll get you out. Meaning I will just, I'll spear you out. I mean, I don't like that. It's utter disgust to God. So that's very important that when we serve the Lord, do the works he's called us to do.  Don't do it half-heartedly. Don't do it with lukewarm passion. Do it with the red-hot, fiery passion, meaning 100%. Heart is in it. Because he just doesn't like what's lukewarm. He will just reject it, spear you out of the mouth. But why is that? Why would they like that? Well, one reason, one thing that we can see here is, and Jesus points that out in verse 17, he says, you say, I am rich. I'm wealthy. I don't need anything. That means this church in Laodicea had come to a place where they said, look, you know, everything is fine for us. You know, we've got everything. And it could be, you know, and if you read some commentaries, they might point this out. But it could be that because here they were in a city where everything was going good. And they were in a city where, you know, the city was doing well. So the church was doing well financially. Church, you know, everything was going fine. No problems. And so even the church had come to this place of complacency, self-sufficiency. You know, we have everything. Everything is fine. So we don't need anything. And so they became, you know, okay, we just do our work a little bit. I've had it. Just keep going. They're not on fire. No passion. And they are in this mode where they're saying, I'm rich. I'm wealthy. I don't need anything. Now that maybe their experience in the natural put them like this in the spiritual spiritual also that became complacent. I'm not saying that just because things are going good in the natural that will make us become complacent. It doesn't have to, you know, we can always keep ourselves stirred up in the spirit with everything going well in the natural too. I mean, and that's great if we can maintain that. But spiritually, this started to come into a place where they said, we don't need anything. But the Lord is telling them, see, actually, spiritually, you are wretched, miserable, poor, blind, naked. Spiritually that is your condition. Your real condition. Poor, wretched, miserable, blind, naked. Spiritually that shape. But you're thinking, oh, I've got everything. Everything is fine. Spiritually you're in bad shape. So what is the solution? Of course, he tells them you have to repent. Repent. See, I love you. That's why I'm rebuking you. That's verse 19. I love you. So I'm rebuking you. I'm correcting you. You need to repent. You need to be zealous or become zealous.  So what was their problem? They were lukewarm. No zeal, no fire. So what is it? You be zealous. You become zealous. Stir up the fire. And he tells them something very interesting verse 18.  Buy from me. What does it mean? Buy from me. How do you pay God money to buy from him? What it means is you pay the price. So he's not asking for money, meaning offering or cash. That's not asking. You pay the price. Spiritually you pay the price. For what? He says you buy from me gold. You buy from me white garments. And you buy from me the anointing that will give you real revelation that you can put on your eyes. You can see. So you buy from me gold. Gold represents what is truly divine gold. So when you see God, when you see his throne, you see gold. Gold represents what is truly divine, what comes truly from God. Gold. You buy from me gold. You buy from me white garments. I mean, this is true righteousness. And you buy from me the anointing to put on your eyes. You buy from me the true anointing. Buy doesn't mean we're giving money. But means he says you pay the price spiritually. You pay the price to receive what comes truly from God as opposed to, you know, just these earthly things. You've got you're doing very well financially, et cetera. No. That is okay. That takes care of the natural things. But spiritually, you need to buy from God what truly comes from God. Second, you pay the price for true righteousness. And you pay the price for true anointing. You pay the price spiritually. So become zealous. Turn yourself up. And you pay the price for what really comes from God for righteousness. It comes through righteousness that you can walk into righteousness. And you receive anointing that brings revelation. You pay the price for it. So don't be in this complacent place where you say everything is fine. I am rich. I am wealthy. I don't need anything. Don't be like that because actually spiritually, you're blind. You're naked. You know, spiritually nothing is there. So become zealous. Stir yourself up and pay the price for this. And he says, he's rebuking them. He says, look, I'm telling you this in love because I love you. I'm telling you this. And then verse 20, Revelation 320 is a verse that, you know, you all know, but it's also very interesting verse because he's actually speaking it to believers. We often use that verse when we are doing an altar call or salvation to the unsaid. We say, you know, the Lord is standing at your door and knocking. And if you let them in, he will come inside. Okay. But actually, Revelation 320, he's speaking to believers, to the believers here in the church in Laodicea. So what's it telling us? It's telling us that because of this complacency, these believers have actually locked Jesus out of their lives. In other words, they're happy, they're self-sufficient. So they are like, I really don't need Jesus now. He's outside. And he says, I'm knocking. To whom? He's talking to the believers. And if anyone broke open the door, I'll come in and I will have this fellowship with him. So Jesus is missing that fellowship. These believers have become complacent. They say everything is fine. So that means they're not having fellowship with Jesus in their personal lives. So he's outside. So this is just a picture. Revelation 320 is a picture of their current relationship. It's like he's outside. No fellowship with Jesus anymore. They become complacent. Everything is easy. So he said, look, I long for fellowship. I want to come and I want to fellowship. So I'm knocking. If you let me in, I'm going to come. I'll fellowship with you. So, Revelation 320, if you look at it from its context, he's actually speaking to the believer. And it shows how deeply the Lord wants fellowship. Why else would he be standing and knocking? Stand at the door and knock? Because he wants it. He would desire to fellowship. So if you open, if you let me in, I'll come and have a meal.  Meaning this is a picture of that fellowship of communion with the people. What a beautiful picture. But that's the danger of becoming complacent, lukewarm, no zeal just going on. But he says, you need to become zealous. You need to stir yourself up and you need to pay the price and open the door. That's fellowship. That's how we come out of complacency. And then he promises, you know, where's 21? To the overcomer, you're going to sit with me on my throne. You're going to rule with me. So it's not like everything is over. You do this and you're going to sit and reign with me. This is real authority that you're going to have. This is verse 21. So even though chapters 2 and 3 were written, you know, 2000 years ago almost and was written to churches in those days, there's a lot we can learn. There's a lot we can take from these seven churches. And we can constantly pray for ourselves as leaders and for our churches. And so God keep us from making, you know, getting into those problems that they got themselves into. Keep us from that.  Okay. So it's a time for our break. So we're going to go for a 10 minute break. And then when we come back, we get into chapter 4 and 5, which is we are now moving into things to come, things that are up ahead. Right. So we'll move into that. So let's take a break now and we will be back in 10 minutes. Thank you.


Welcome everybody to the BioXL webinar and today we have a student webinar, in particular is the student that won the poster prize for the summer school edition 2023. And the three winners are Daniels Surswequa, Eleonora Serra and Ricardo Scarin. And I'm hosting the webinar, I'm Alessandra Villa from the Royal Institute of Technology and with me it's Otto Andersson from the Finnish IT Center for Science. So, as you can see I was speaking come from different parts of Europe. Daniel come from the Eideberg Institute for theoretical study Eleonora come from the ITT, Institute Italiano of the Technology at Genova, and also she's working in Seca Melossin. And Ricardo also works for a pharma company Atlas Molecular Pharma SL and also for a research center CAC Bionune Bilbao. So, the webinar is recorded just that you are informed. In this webinar, you can ask questions to the speaker so I will ask you to use at one at two at three to address specific Daniel, Eleonora or Ricardo maybe you have a question for everybody don't put anything. And you have to use the function of zoom that you find at the bottom of the zoom application depends on which operating system you have you might see this symbol, or you might have this symbol. You just click and then you type your question you can type the question whenever you want. At the end of the three presentation, I will unmute you if you have a microphone so you can directly ask your question, or I will read for you the question to the speaker. There's something about the speakers of today. So Daniel will speak about how a stretching force difference to stabilize the chemical bond on a protein backbone, while in honor will be speaking about bidirectional path based non equilibrium simulation and in free energy estimation. And finally, Ricardo will speak about development of pharmacology chapter on for the treatment of tyrannism. Oh, sorry, Tyrocymenia type one. So, now I will start to give the word to Daniel. So can you see my slides. Perfect, we can hear you and you can see all your presentation please go ahead. Okay, so today I'm going to talk about my PhD project. And in this project we try to describe the effect of an external force that a pool molecule, and then it could desistabilize the bones in the backbone of peptide. And this is important to study because a basically stretch molecules are everywhere in material science, basically all materials who load load any external force are submitted to stretching of the molecules. And in particular in biology, when we moved any muscles, then our tissues are stretched, and that basically means that a hour. The molecules inside are also stretched, and then understand what's happening inside of this stretch molecule could give us some clues of some to solve some healthy problems and so. Now this is basically the broad problem but the more specific problem that we have to what we want to address is, which are the most unstable bonds in a molecule that is stretched by an external force. And then to do that, we start assuming that the change of energies when we stretch the molecule, the total change of energy can be decomposed to the change of energy in each one of the degrees of freedoms. So, we have here the expression that we are using to do such a discomposition. And then the index I respond to the different degrees of freedom and when I say degrees of freedom I refer to distances and goals and that he does, then our model is predicting how the energy is distributed in these degrees of freedoms. And the idea is to do this, a sort of solve this problem using QM approximation so the level of accuracy of the of DFT. And but the Q problem, the key problem is to solve this integral here, where this fi correspond then to the force in the direction of the degree of freedom and the Q would be the different degrees of freedom the values corresponding to the degrees of freedom. And then we have this equation and for solving this question that we have two options. One of them is called Jedi that uses harmonic approximation. And this one basically we assume that around the minimum we can do an harmonic approximation and have some prediction of the distribution of energies. And the other solution that is a solution that we propose is to use this method that we call safe. And in this one basically we do smaller stretching, such that we have many intermediate stretch configurations. And for the final distribution of energies and we do a numerical approximation of the distribution of energies. So we are going to use this method, the sift method because if we compare then the distribution, the summary of the distribution of energies, the summary of this term that it will be equal to the total change of energy so basically, we can compare the result that we are taking from each one of these the composition method with the total change of energy from the DFT. So basically here, the method using sift is way closer to the reference value, and then we are going to use this method to do this description of the distribution of energies. So for example of the kind of result that we have, then I will going to show the example of three Alanine, this is small peptides with three Alanine's. And the normal procedure that we do is that we start from the optimized configuration, we optimize the structure using DFT. And we basically increase the distance of the stream atoms by a value of delta D, and then we constraint that distance, and we re optimize again using DFT. And then we repeat and repeat the process a number of times until we have different stretching, and then we can do the numerical integration that I showed in the previous slide. So the result that we have is something that I show here. So here I show the change of the energy or the energy store in each one of the degrees of freedom, respect to the degrees of freedom here are all the degrees of freedom. And then correspond to the degrees of freedom of distances. This one are this part here is the degrees of freedom of angles and this one of the heat rights. And as you can see, then the gradient of colors correspond to the stretching step. The gradient stretching step then the energy store is completely zero. That is what we expect if we don't stretch the molecule then there is not any distribution of energy but then when we start to increase the stretching of the molecule then we start to see the distribution of this external energy that is being introduced to the system. And here we can see something like the first or the most made for results. That is that the distances stored most of the energy. While data he draws a degrees of freedom doesn't store a, a much, much energy. And also we obtain we continue this stretching steps and step of stretching until we got a rupture. So a rupture means that the distance between two pair of atoms increases a lot and the rest of the molecule got relaxed. And then we obtained that the rupture happened always in the distance that it stores more energy. So basically what we are telling is that the degree of freedom obtained that stores most of the energy is the most probable to be to suffer a change in the distance is that it would mean that the distance is storing most of the energy is then a is going to suffer the rupture in some point. And we do this kind of analysis not only for a three Alanine three Alanine as I said before it's just an example of the kind of procedures that we do, but instead of that we can have a different combination of amino acids, in particular we are having three order basically three order amino acids and do remove the effect of the capping atoms, then we only consider the distribution of energies in the amino acid of the middle. So in this case, if we do different combination for example in this one, LKD, then we will have only the distribution or we are going to analyze only the distribution of energies in the K amino acid. And the reason again is to remove the effect of the external of the capping atoms with this database or this basically this set of different combinations of amino acids then we want to create a machine learning algorithm that predicts the distribution of energy for different stretching steps for different configurations. And then we can apply this method this analysis method for larger system because of course, in nature, we are never going to find or not like a very often a such a small amino acid but we such a small peptide but we usually find something like way larger systems, like proteins and so with the machine learning algorithm, we expect to have a prediction of the distribution of energies for larger system and the scales of a molecule proteins and so this the database that I mentioned before this being under construction yet, but still we have some preliminary results. So what I am showing here is that the energy store, specifically in the degree of freedom corresponding to the distance between the alpha carbon atom of the background and the nitrogen atom of the back bone again, and the alpha carbon and the C atom of the back bone. So, as we can see here, basically, the first result that we obtained from the comparison of one and the other is that the C alpha C bond, this is sort of way more energy that would mean then that this bond is more probable to be broken in comparison with this bond. And another result that is also a basically a one reason to or so to say a highlight of this result is that for different amino acids, each line is a different configuration, a different set of amino acids, different can store different ways, the energy in the same bond. That would mean that different configuration, if we have different amino acids, then we will have some specific amino acids that are more probable to be broken. And then what we have is that we implemented a method to obtain the distribution of energies in a stretch peptide. In particular, we can predict then with this distribution of energies, which bond will be broken so for example in this case we have for every stretching a different distribution of energy and the bonds that is storing most of the energy is the first to be broken or the most probable to be broken. With this information with this method, then we are creating a data set to train machine learning algorithm. And in the near future we expect to have some results with this engine to show to the world. And, yeah, but so far with this analysis with this data set we have, we can conclude that for different configuration of amino acids, different order of amino acids we have different distribution of energies. And finally, I want to say thanks a lot to the MVM group, led by Professor Pralki Greta, that is the group where I'm doing my PhD, and especially thanks to the bio Excel organizers of the summer school. So, yeah, that's it. Thank you. Thank you very much. And now we go on with Eleonora, please Eleonora. Daniel, could you stop sharing. Thank you. Yeah. Okay. Can you see the screen. Perfect Eleonora we can hear you and we can see everything please. So, good afternoon everyone. Today I will present my PhD project and that is done in collaboration between it and the second center. And it is titled the two directional pathways non equilibrium simulations for binding free energy estimation. So here I did the little introduction about the central topic of my research. So binding free energy is the central quantity in the drug discovery field, because it's able to quantify the strength of interaction between a legum and the receptor. The protein legum process, the complex between the legum and the protein is an equilibrium with the free form of the protein and the legum. And starting from this equilibrium, the gift sign in free energy can be computed. This quantity is related to the KD, so the dissociation constant of the complex that can be evaluated experimentally, but it can be also evaluated computationally. And among all the computational tools that can use all atoms molecular dynamics are the most suitable. In this way, the binding affinities and the free energy that can be evaluated from simulations can be used to optimize promising compounds along the drug discovery field. However, evaluating computationally binding free energy pose many challenges, starting from the complexity and the size of the systems that we have to simulate, but also because of the wide range of the time scales of the phenomena, starting from the femtosecond for the bond vibration and reaching also the seconds for the protein legum binding process. For this reason, we cannot use a standard molecular dynamics, but we have to use an ensemble sampling methods because they are able to accelerate their events and to expand the approachable time scale. An ensemble sampling methods can be divided into different families, method based on physical path and method based on physical path. The first family of methods relies on the fact that the free energy is a state function and they follow alchemical transformations. They are often used to estimate relative binding free energy. They are in some way simple to be implemented, but they don't offer any kinetic or binding information. On the other side, the method based on physical path required identification of a collective variables. They are used to define the potential of mean force and they reveal kinetic and mechanistic information. However, the definition of the collective variables can be very difficult. And among these two different methods, we decided to use method based on physical path. Then after the definition of the methods that we use to sample their event, we have to define the estimators and the free energy estimator binding free energy estimators can be divided into class equilibrium free energy estimators and non-equilibrium free energy estimators. In the first family, we can find the free energy perturbation approach or the Bernat acceptance method. While in the second family, we can find the Georgianski estimator and the Cooke's identity. And what we decided to use was to use this kind of estimator. So Georgianski equality is a simple equality and it's able to relate the work done during a transformation to the free energy of the transformation itself. So in our case, they work done during the unbinding transformation to the free energy. The Georgianski estimator is fairly simple to be implemented, but it can be biased, give an immediate number of observed work values and exponential average is sensible to our events. For this reason, we want to use a two-sided estimator as the Cooke's fluctuation theorem because it's known to be more accurate. And here I presented the Cooke's fluctuation theorem. And as we can see, it's simply a relation between the forward and backward distribution of the work values and the intersection point between the two distribution. In our case, the work values for the unbinding event give us the binding free energy. So after the definition of the sampling approach and the estimator that we want to use, we have to define a computational pipeline for computing and binding free energy. And what we decided to do was to take a pre-existing pipeline, published some years ago in our lab, and to improve and modify it. So in the first step of the pipeline, we use the non-sensamic methods to generate a preliminary path describing the relevant of interest. So what we decided to use was adiabatic bias molecular dynamics with an electrostatic-like collective variable to describe our unbinding and binding events. Then, among all these preliminary paths, we select one trajectory which bears the smallest simulation time and it is mechanistically sound. And we call it our initial guess path. The initial guess path was then used in conjunction with two algorithms, the principal path algorithm and the distant waypoint algorithm. The first one was able to clean the putative minimum free energy path and it was composed by consecutive conformations capturing the relevant. And then the second one was able to define a minimum free energy path with uniform spacing in terms of density between the consecutive frames. In this way, after this step, we obtain an optimized minimum free energy path describing our unbinding event. Then in the original pipeline, they decided to use world-temperate meta-dynamics in conjunction with the path collective variables. That is an in-rently serial algorithm. But what we decided to do was to use instead of world-temperate meta-dynamics, steered molecular dynamics to perform simulation following the path for the unbinding and binding simulation. Because we know that steered molecular dynamics is trivially parallel. Then after the use of steered molecular dynamics and performing multiple replicas, we computed the standard binding free energy as a sum of two terms. The binding free energy from simulation and the standard volume correction term, where the binding free energy from simulation was simply computed as a ratio of the partition function. Okay, so here I try to represent what does it mean to use path collective variables. Path collective variables are a specific type of collective variables published some years ago by Bernoulli and coworkers. And they're called S and Z, where S describes the progress of the system along the path, while Z describes the distance from the path itself. So here I try to represent better. So after the use of our algorithms, we define a minimum free energy path and describing our unbinding and binding events. And then by using the path collective variables, we were able to perform steered molecular dynamics for the unbinding and binding events. Okay, so which systems we decided to use? We decided to use three different systems, increasing the complexity and the size of the system. So a simple host gas system as a toy model for fine-tuning and validate our pipeline. Then trypsin-vendamidin as a benchmark system for free energy strategies. And finally, a BLT-Rosin-Chinas and Glevec complex, because it is an interesting therapeutic system. For CB8GA, we follow the stage of our pipeline. So we've tuned in the robotic bias molecular dynamics. Then we optimize the minimum free energy path. And then by using the path collective variables, we performed 50 replicas of binding and unbinding events at five different simulation times. So 10, 25, 50, 100 and 100 in a second. After the use of steered molecular dynamics, we reconstruct the work profile of our transformation. We applied our estimator and we reconstructed the potential of mean force. To reconstruct the potential of mean force, we applied the estimator for each value of the S variable. And in this way, we obtain a point of the free energy surface along our collective variable and so a point of the potential of new force. I have also to specify the fact that instead of using the simple Krux-propagation theorem, we realized that he has the same mathematical formalism of the Bennett-Septown ratio method. And so what we really implement was the Bennett-Septown ratio method replacing the work, the internal energy with the work values. Okay, so we applied our estimator, as I said, for reconstructing the potential of mean force. And then from the potential of mean force, we had to define a discriminating frame that was able to discriminate between the bound and the bound states. And we did that through a visual inspection and analyzing our potential of mean force. After the definition of the frame, we were able to integrate our potential of mean force. And finally, we compute the binding free energy as a simple ratio between the partition functions. Okay, so the first results that we have for CB8G8 are here reported. And what we can see is that considering an experimental binding free energy of minus 13.5 kilocal mole, we had a good agreement between our computed results and experimental data. And increasing the simulation time, the Krux-propagation theorem was able to converge towards the experimental value. And considering our second system, trypsim and zamidine, we follow the same stages of the pipeline. So we performed the 50 binding and unbinding replicas. And then considering the results, considering an experimental value of minus 6.2 kilocal mole, we could see that the free energy computed by the Georgianski estimator were an upper and lower limits of the experimental value. While the Krux-propagation theorem had a good agreement with experimental value, and we could see a good equivalency and a good converters feature for the Krux-propagation theorem. Moreover, what we tried to do was also to define the sensitivity of the binding free energy with respect to the discriminating frame. And we could see a low sensitivity of the binding free energy with respect to the discriminating frame that was used to define the bound and nonbound states. Finally, for Abial-Glivak system, that was the most complex one, we did this initial stages of our pipeline, while the 50 replicas of binding and unbinding events are still ongoing. So as a conclusion, we could say that for small and moderated sized protein-negan complexes, we found that the Bernat-September ratio estimator coupled with the possible active variables is able to converge. And our approach gives accurate binding free energy values in conjunction with mechanistic information from the past. So the integration of the past collective variables and two-directional steel molecular dynamics enables both much faster converger rates and trivial parallelization of simulations, significantly reducing the overall time required to obtain accurate binding free energy estimates. Finally, for bigger systems having pharmaceutical interests as our Abial-Glivak complex, we are now in the process of challenging our approach. So I want to thank my supervisor, my collaborators, and thank you for your attention. Thank you very much, Eleonora. And now we go further. If you could stop sharing, thank you. And now we have our last speaker. Ricardo, please. Okay. Can you hear me?  Is it better now? Yeah, perfect. And we see if you can go on full screen, we see your presentation. Yes. One second. I just, okay. I'll try to share again then. Yeah, sure. Yes. All right. Okay. Okay, I want to speak about the development of pharmacological chaperone for the treatment of a tyrosinemy type one. So to understand this project, we have to look at the basis that were created in the lab to understand the pathological phenotype of tyrosinemy type one and the characterization of the target and the fragment screening that was done. The conclusion was in rational design and evolution, but to understand it, let's look at the background. So, and what is tyrosinemy type one, if we look at the catabolism of tyrosin, we see that several enzymes catabolize the final leases of tyrosin to acetate and fumarate. And if this enzyme failed to express this function, we will have different type of accumulation of metabolites that can lead to tyrosinemy type two or tyrosinemy type three. And the worst one is tyrosinemy type one if the last enzyme failed his function. Because these lead to the accumulation of fumarine acetate acetate, and the accumulation of a byproduct that is such a new acetone, this molecule is highly toxic is in use epatococcinoma and the people that as these mutations can die in two years old. So, I think there is a drug that block the production of susanacea acetone by blocking the catabolize of the tyrosin at the very beginning. So, in our lab, my colleagues characterized the target that is FAH, fumarine acetate acetate in relays. This is a dimeric protein of 96 kilodaltons. In our lab, colleagues find that mutation does not impair the catalytic side or better, most of the mutation are destabilizing the dimeric form to the monomeric in increasing the aggregation does taking out from the equation, the protein in the cells. So, in fact, modern patients that is a glycine 337 to serine. With case per cast model on hex cell, we see that with this mutation in Western blood, we don't have any protein. This is valid also for mice, we have mice model in which we see that the mice after 20 days die in agreement with the phenological pathology in the humans. So, let's go in the more computational part we did the fragment screening. And we like make a list identify catalytic side and a dimer interface and they screen several fragments with docking. And the best hits where the screen, mainly with the contribution of john jim Martinez by methyl drossi, and this give an idea of where it is the binding side. Which are the best find. In fact, he could co crystallize one of these fragments, and that's where we start from the fragment evolution. So, my do for this fragment evolution we use a symbiosis between docking ITC STD NMR and crystal. So, we start by molecular modeling we look at the catalytic site, we see that the substrate natural substrate is a polyket of acid and the coordinate calcium. And our compound is quite different in nature is hydrophobic. We have a hydrophobic bind as a hydrophobic patch, neither for patch where it does five by stacking with the finish line. So we have a crystal structure with the part of the substrate and our fragment, and we characterize the binding by ITC and it actually confirmed that the binding is mainly in a entropic. We see at the delta G contribution, the red part is the entropy of binding is quite high. So we want to create more specific binding, and we do that we do so by copying nature. So we try to enrich the scaffold by adding carbonyls and moisture that can be that the protein develop a taste for during the years of work. So, what we do we select some compounds, and from the same database, we screen for compounds that have 20 around 20 atoms and repetition coefficient between two or three, and we select the bench of these compounds and dock them and look at the pose to understand the pose can have a rational meaning. And then we select the 10 compounds only so it's a low basic screening because we will look for interaction by striking region in the from the patch and distance in between nine to 13 Amsterdam. Like the one that is in between the substrate and our heat from it. And we select plausible pose, we buy these 10 compounds, and we screen by STD. Luckily, four out of 10 of this compound shows STD, but still, STD can be displayed at five millimolar KD. So we are not happy, we want to verify that this compound actually improved the binding. We don't met him to see which part of the compound goes inside the pocket. Why STD you can see that the carboxylic acid was the one going more inside the pocket. So, we have a confirmation by ITC that the enthalpy contribution is increasing the green, the green column. And also the binding is the pattern the KD is lower is 120 micro molar. And so we are happy about that. And we will keep the same rational design path. We reproduce the same exact selection of fragments and docking, and the input fragment from the docking the best pose will be used as an input fragment to search for other similar compounds that the Tani model coefficient 0.7 and at the end we select, because we want a low budget screening other seven compounds that maintain the pie stacking and display the carbon is in a in a way that can make sense in the binding post. So, one of these compound we five out of seven display STD and one of them display a lot of STD effect in an MR so we tested by IDC and we actually got to the non molar range. Well, micro molar non molar range. And the binding is a contribution is a more entalpic than before. And in fact, this compound. We have a recap of the process and we see how did we increase the binding so they are the affinity. We lower the KD and we increase the entropy of the binding. And moreover, we see that this was the original crystal structure. We can soak the compound in the crystal structure and we can see that it fit exactly how was the docking pose the pie pie stacking with the freedom in 141 and the coordination with the carboxylic acid. The crystal structure as a round square deviation from the docking of only 0.55. So we can say that the docking model predicted the crystal structure, and we are definitely really happy about this because as you see there is the electron density and the binding diagram. And the resolution of the crystal structure with 1.2 Armstrong, and we want now to assess if, if this model is working like the stabilization of the protein is real, because it can bind, but we don't know if it gives a sharper effect. So we use an experiment that is those experiment diffusion oriented and MR, and in which we look at the rotational coefficient of the molecule in solution. So we take the proton in between 0.85 BPM to one, and we check how fast the movement solution. So the bigger the rotational coefficient, the smaller is the molecule. So of course, the, the blue one is the mutant. And at 41 degree, we see that the mutant is is turning faster in solution. So it means that is in the monomeric state more the population is shift to the monomeric state, but the wild type is not true. The violet one is stabilized in a dimeric state, because it has lower rotational coefficient. So, when we add the compound to the mutant, the red bar, we see that the regulation is shifted through a dineric state. That means that dynamically, our compound stabilized the dimeric state. So we can prove the kinetic of this disabilization by doing aggregation assay by checking the percentage of four ratio using time. And we see that without our compound, the mutant is degrading in a certain rate, but when we add the compounding solution, the decay of the signal is quite slower. So we have 70% more of the signal after four hour in three replicas. And we confirm that as a shaperon effect, at least in vitro. We also did the collaboration with Gonzalo group. We did the screen of two million compounds in which three compounds were successfully characterized in the lower micro molar range, and two of them were co crystallize. The future perspective of this study is to find results in vitro and mice model that I already show and create a new chemical entity with the good pharmacological propriety. I would like to thank you for your attention and the riser of by excel, and also the group in which I am working and is a really big group of metabolism and precision medicine. Thank you. Thank you very much. Ricardo. Thank you, Daniel. And now we can open for question. So there are some questions. Please go on to ask questions in the Q&A. We started with a question that we have and I will unmute the Antonio so you can ask the question. I just let me time to pick up the participant list or maybe opto can help me. Yeah, so we can, I can allow you to speak Antonio if you can ask directly your question. Hello, do you hear me. Yes, if you can speak a little louder, it will be great. Please. Okay, do you hear me. Perfect. Okay. Now I was just wondering if a little I could explain again what was the past collective variables and how did she define it for in her system. That's the question. Thank you. Thank you. Okay, so hi Antonio. Past collective variables actually are implemented in plund. So what we did was to use our algorithm to define the path that is simply consecutive frames describing your events. So in our case, the unbinding and mining event, and then the past collective variables implemented implement allows that allowed you allows you to define the fact that you want to follow the specific path. So s is an equation that identified distance from, let's say, in simple word, the frame in the path and you're the real position of your system during the simulation. And so you define this distance and you put the wall, a potential that allow you to have to follow the real path. So it's like you are defining a tube and your system will follow the tube and the same for that. So as some is to follow the distance in this way while that the tool, the minimize the distance in the other direction. But they are simply equation implemented in plund and you define a force to keep the system follow your path. I don't know if it was clear. Okay, so plund pretty much and make the path for you right and you define the path and then with plunder you follow the path by using s and z. Okay, okay, perfect. Yeah, that's not so thank you for your talk. Thank you, Antonio. And now we go to blood. I will unmute you wait just give me a moment. Yeah, you should be able to speak, please. Hi. Thanks a lot for this very nice talks. I was wondering from the second talk about influence on the pms of several parameters for the SMD simulation so the pulling speed for instance, the number of SMD simulations. Also, if you have a path that is kind of has a large volume, for instance, let's see you have a large channel of this sample. Will that also influence your pms and how, how will they do fancy. Okay, so thanks for that. Regarding the number of simulations we started with a small number of simulations and replica for the mining and mining event. And then we want to reach a convergence point. So we perform good strapping and we see when we could have a good number of replicas that tell us to reach convergence. So yes, of course, the number of replicas influence your results. Also, because when you apply, for example, Georgiansky identity, you have an average ensemble average so you have an influence. You have the right number of replicas by performing strapping and by defining the number of replicas that could allow us to reach the convergence of the value. While the second point was the velocity. If I remember, and yes, the velocity is very important because if you go very fast, for example, during a still molecular dynamics, you could have a very high value of dissipated work. And this could be a real problem when you are using out of equilibrium estimators. So we started with 10 or second simulation, for example, but we had a very high value of dissipated work. And for this reason, we decrease the velocity so we increase the time of simulation. So these affect a lot the potential of reinforce and also the final free energy. And then I don't remember the last point, sorry. If you have a system where you have a large path with a large volume to sample, so it's not a direct route, but you have a lot of degrees of freedom along the path. And I was wondering if the methods are applicable for that situation. So in this moment we are using the two path collective variables and we define as I told before, a sort of tube. So the volume, it doesn't really affect in this way, but it affects in the way that when I, I don't know if I was clear, but when we want to compute in the end the standard binding free energy we have also to make a volume correction. So we define one correction term, and to do so we have to define the sample volume. Maybe I have a slide that could be, could help what I'm saying. Because, yes, here I don't know if you can see it, but when we want to do the correction, the volume correction, we, sorry, sorry, could you put it in full screen so we see. Yeah, of course. Thank you. Yes, so when you want to perform standard binding free energy, as I told at the beginning, we have to define a standard volume correction term. And here the volume is very affecting because we want to define the sample volume for the binding and unbinding. And so we use the software that is able to define this volume and then we put the, we did the ratio. So in this way, the volume could affect because we want to make the ratio of the unbound volume and the zero, that is a standard volume, but we did that for computing standard binding free energy. So I don't know if he's answering your question or if you want another point to maybe I lost your point. I don't know. In this way volume affected the our results for sure. So if you have the possible reorientations of the, of the weekends, for instance, within that volume. And that's sorry, could you speak a little loud because we couldn't hear you. Thank you. So I was wondering more about the case where where your legal would reorient so you think of a legal that it's more like a tubular shape and goes out but then during this process and kind of reorient right because it's possible. But would that be kept, would that be possible to simulate or you will just track the league and be in a certain orientation out. Our, during our state molecular dynamics actually the league and follow the path, thanks to past collective variables. So if there is not a real orientation along our path, if we put high force to follow the path. That during the steering molecular dynamics we could have a bigger orientation of the league and because the system is forced to follow the initial path the minimum energy path. Thanks a lot. Thank you. Thank you. And so while people maybe think about other question we have 10 minutes still, I have a question for Daniel. I'm wondering if you have looked on the fact so you say that you use triplet three residue. There is to calculate the value for the middle one. Did you check if you get different value and depending on which are the neighboring in your case. Yeah, indeed. That was one of the test. I wish I had my screen again. So, yeah, so for example, in this case, we not only tested the system for three learning but for larger system, not only Alanis but as following the previous example then I show here also larger Alanis. So this is a fight Alanis. And this basically we want to obtain something like the effect of the kept atoms, the cabin atoms. And if it's an infinite Alanis then we expect the same distribution for the old Alanis. And as we can see here, the three of the middle, they have the same distribution, but this one, for example, that is closest to the cabin atom then it has a different distribution. And basically this result concludes at the same for this one. So with this result, we can conclude that these cabin atoms is only affecting the first one because the rest is basically invariant to the effect of this part here. But this is the limit, something like how far. Okay, but so that you mean if you have an alanine with the between two treat the funnel you have an alanine two between two feet feet and in. Did you expect to have the same to find the same value for the alanine. Because because basically we obtain a we made the same kind of calculation for other system not only. Yeah, we obtain basically the same that the middle is unaffected by this part or whatever we change in here, something like the largest, the largest change could be with the first neighbor, not put it on that. Okay, okay, okay. Thank you very much. And I have also a question for Ricardo. So at the end of your presentation you speak you say that you want to do as I understand that you aim to have a two million compound screening. We already did that. Yeah, yeah, I was wondering how much time and do you need for such a screening. I'm from Gonzalo. We have a cluster that has several GPU. I don't remember exactly. Exactly the capability but it's quite large. The storage I think is one petabyte of storage and the GPU I think 100 and 150 and 12 Titan. Yes. So in this setting. It was quite fast one week. And it was flexible docking so semi flexible. Okay, so it was playing semi flexible docking you took one week for for two million compounds that are compounds like the one that you show so they are still drugs. Yeah, yeah, it was like, it was actually divided in, it was divided in a different patch. So you went from 10 to 15 from 15 to 20 atoms from 20 atoms to 25 25 to 30. And then back it was run and several times on the base of this crystal structure that I obtained with my best heat. Yes, but at the end, my screen was smaller. My screen was really smaller. I think in total what this process of iterative processing which I look at every post one by one, because I stress the scoring faction is no, it's not giving you really prediction of the binding or is is a weight in more the barred surface area than real meaningful connection in the pocket. So I look one by one and it's, I think it's been 300 molecules and in total from two million. No, no, no, this before the two me before that. Okay. Okay, I thought that's from the two million you will end up going to think some 300. Yeah. The two million. In order to, to search for the best hits, we of course like some filter based on the previous finding. Okay. So, but in between 15 and 25 atoms, the molecule that I have to crystallize was in the best hundred molecules. So this does like look always to the post because it's the best. Yeah, yeah, did you also plan to improve your scoring function. I did. I did try a different algorithm and different scoring function. So, gold out of being a MOE plants. I saw some point I saw like which one was reproducing the extra traction better. And I use that there's more in this case was. Okay, but you think it's not an absolute choice. It depends on the system that I am always. I, I think the correct approach is because at the end I use it as a tool and not. Yeah, no, you didn't develop me. I understood. But the correct approach would be to do a benchmark of different. If you have a crystal structure, you do a bunch of benchmark and you find which algorithm and scoring function predicts better. Your pause your real pause and then with this algorithm use it to find new molecules. Okay, yeah, yeah. Thank you very much. So I don't see any other question if someone has a question or put a raise and in the meantime, I will just share my screen and announce the following webinar. If I still have my presentation or just give me a moment with that pickup. So the next webinar that the next by itself webinar will be the 14th of November at always three o'clock but this time, not summer time and winter time. And it will be, we will have a Giovanni Bussi from CISA speaking about thermostat and barostat. Okay, so if there are no other further question. I will, I guess I close this session. And I, I thank you everybody, both the three speaker and all the attendees and see you in the next occasion. Bye.


Guess where we are. You're not gonna believe this. This is a famous American store. Target! This is like an upside-down nice stuff. People love Target. People spend a lot of money in Target. A little bit nicer than the Walmart. And they have everything. For food, to clothes, home wear, everything. Let's check it out. We've got baby stuff over here. We have home stuff over there. We have all your tech and TVs, cell phones, computers, all down here. Fraps! So, when I say that New Zealand doesn't have the Frappuccinos, they do in some places, but they have like one choice. But I guess when I'm thinking about it, I'm thinking about, you know, you get all the different flavors. We have the vanilla, the caramel, the mocha, and then you got all these. You got those just the shot espresso's. Look at this. Look at these choices. White chocolate mocha. Sparkling blackberry. I've had these last summer. So good. So good. Let's check out the cereal aisle. Look at all this. Forever. Both sides. Let's check out some crazy ones. You've got Willy Wonka's Fruit Loops. We've got Reese's Puffs. Have you guys seen the Cinnamon Post Crunch Cinnamon Rolls? Or are the Tricks Minis? Look at, we have Kit Kat cereal. Kit Kat cereal. Cookie Crisp. There it is. Sugar galore, everyone. Sugar galore. Okay, so we're in Target, and you can get full-fledged alcohol. We can get like Jack and Cove, like right here. We've got all these wine coolers and deliciousness right in Target. And look at those. Come here. Come, come, come, come. Look at these bottles of vodka. Look at this. Look at all this. Look at the wine. Look at this. Okay, look at what I got at Target. So these are called chocolate bombs. So you just put the bomb in your coffee cup and you pour the hot water and it makes the delicious, beautiful hot chocolate. We should try one. Should we try these at home? These are American marshmallows. So these are what we use for s'mores, and they're very different than the New Zealand marshmallow. Like, personally, I like the New Zealand ones better, but this is what they do, and you can get them, like, in the mini. So this is how we make rice crispy bars, which are way better in the US than New Zealand, because for some reason you just put, like, butter or something in it. Okay, look, you can even get lucky charms. Marshmallows blown up. Here is everything that you need for s'mores. We've got the marshmallows and then the graham crackers, and you can get off-brand, regular brand. And look at the different choices of first you get. Regular, we've got popping candy, and cookies and cream. Regular, way to go. Let's talk about Mexican food, but even at Target, you can get everything you need for Mexican foods. We've got the soft tacos, the hard tacos, we've got even the whole wheat tacos, we got beans, we got guacamole, salsa. Look at all those salsas, beans, everything. It's great. Oh, natter, butters. Dottie buddies. Okay, so you're always asking me what are cremers in America? This is it. Look at all these flavors. This is what you put in the coffee to make it actually taste edible. You've got cinnamon roll, peppermint mocha, pallium sweet cream, hazelnut, french vanilla, and then these are the more like all natural ones. Then we've got the duck and donuts brand. We've got, I mean, it's crazy what we have here. This is something that we don't have in New Zealand. These are cinnamon rolls literally in this can. So you take this can, you go, it's really scary to do the pop, it's actually I'm scared of it. You go pop, and you place them on a pan, and it comes with a little thing of icing in the top, and you bake them for like 10 minutes for the icing, and you have hot fresh cinnamon rolls in the morning. I mean, it's like baking for dummies. It's like cheating. Okay, here is real biscuits, guys. This is what I'm saying. Look at the layers of the biscuits. These are cookies. Salute to service. Pillsbury. Let's go see how big the produce is. What is this? Literally we have sweet cinnamon caramel apples. If it doesn't taste like that, I'm going to be very unhappy. What color you could ever want right here.