OK, guys, I hope you can hear me and see me well. Let's just wait a couple of more seconds or a minute or two just so we are sure everything is working the way it's supposed to. And we can start this live session. Really confidant to it because, again, I'll be answering your questions. All right, man, you can hear me. Good. We did a test run before. I saw some people joining in because we had some issues with the camera. But now everything should work fine. Let's just wait for a minute or two, and then we're going to start if that's fine with you. Or let's do it like this. Let's wait for 30 seconds, and then we can start. That's the plan. From Libya, you're watching and following. That's awesome. That's kind of a far away from here. I hope you're doing well. I live quite close to Ukraine. I don't know if you're aware of that. It's just like 500 kilometers away. So it's quite scary, actually, to be totally honest with you, guys. Cool. So let's do this, guys. Thanks for joining. I'm really happy to see some people here. So again, I'm going to be answering your questions. I tried to support you. So yeah, whatever question you may have, just let me know. And I'll do my best to support you.  And I'm trying or helping people, trying to help people improve their brain performance, to achieve big brain performance, to boost focus, memory, and so on, to improve sleep as well, to reduce stress, anxiety. So I've been doing this for quite some time. And that's why I'm here today, to help you and support you on your way. So yeah, thank you, man. Thank you. I appreciate it. Yeah, whatever question you may have, let me know. And I'll do my best to answer it. As you may know, I've been researching brain performance for quite some time. But I mainly focus into nitropics in the past couple of years. However, now we are expanding this again because we know nitropics and brain supplements can help you a little bit. And they're very important, crucial actually. But they're not the only thing you can do or you need to do to achieve big brain performance. Without doing other things, like optimizing your lifestyle, yeah, it's really hard to get there to achieve this big brain performance. That's how it is. That's the reality. But yeah, let me know. If you have any questions, just comment in the comment section. Yeah, I'll try to come up with some good answers. Firstly, what supplements and entropics that play on different areas that I have to stuck with it? Firstly, what supplements and entropics? Zerfet, can you repeat this question? I'm not sure if I understand what you mean by it. Have I tried coffee pills? I did, actually. I tried coffee pills, so pills with caffeine. Sometimes there's LTE in it inside as well. What do I think? Well, if you need a quick energy boost, they're really good because that's what coffee does or caffeine pills do. When they combine it with LTE, it's actually even better because then you get this energy boost without the adverse side effects of caffeine. And that's even better. So if you do well with caffeine, sure, feel free to take them. And if you kind of suffer from side effects, if you sleep really badly, if you take caffeine pills or if you're very anxious like I am, for example, then it's better to avoid them. But feel free to experiment with them. Do take them in the morning, though, because caffeine's half-life is very long. As you probably know, this means that if you take them at around 9, 10 in the morning, that's fine. If you take them later, then the caffeine that is in the pills can, of course, negatively impact your sleep. And you don't want that to happen. So yeah, many, many brands actually contain caffeine besides many are in tropics. And some people love that others don't. Personally, I'm a fast caffeine metabolizer, which means I get this boost of caffeine. So this boost of energy that comes very quickly and I feel super high and excited and happy and so on. And then like two hours later, I crash very badly usually. So that's why I'm another big fan of caffeine. Yeah, what are the top supplements to buy and take daily and how to choose them? Well, when it comes to top supplements, it depends on your goals. What do you want to achieve? You should answer this question first, like what is your goal? And you can let me know and I'll then give some suggestions. But in general, you need to be complex, especially you need to take B12 vitamin. You need some D3 and K2. If you don't spend a lot of time outside, those are the basics. And then you need some omega-3. Omega-3 are crucial for optimal cellular function and brain performance. You need those. And then of course, it's good to take an additional nitropic supplement as well. So yeah, tell me about your goals. Like they want to boost your focus, memory, attention, energy and so on. And then I'll try to support you. What would be the best ADHD stack? I'm trying to stay away from the usually prescribed ADHD mess like Ritalin and Adderall. So if you have ADHD, then there are a couple of things you can take. I will actually just open one file here because I have the exact protocol written here for people just like you that want to stay off Adderall and Ritalin and that's actually pretty good and really happy you want to do that. Just give me a second and I'll just give you the exact protocol that I use as a professional, a brain professional specifically designed for ADHD. Just give me a second protocols, ADHD. Here you go. So for ADHD, so here's the thing. If you're trying to stay off Adderall and Ritalin, then of course fish oil, so obviously Omega-free oil, take one to two grams per day, and DHA but try to find one that is high in EPA fatty acids. Then you need some either green tea extract, green tea extract is really good or L-tionine, that's also really good. And then it's good to take a dopamine precursor. I suggest based on the protocol, I suggest L-tyrosine or you can also take phenylalanine but L-tyrosine is usually the better version of the tube. And then a rhodiola-rosa is really good. That's what I recommend, rhodiola-rosa and maybe you can take a copper-moniaris as well. So those are kind of the basics. So those specific nitropics I just tell you are used in the protocol from my coach for example and myself and my team as well and we prescribe this, well we can't prescribe, we suggest this to people who have ADHD. So this is gonna do the trick for sure. Cool, my goal is to increase all my areas. So you take omega zinc, multivitamin D magnesium. Yeah, that's good. You should continue taking those, those are really good. Now I would then add a high quality nitropic stack. Now if you can afford it, I would go with quality of mind. Give me a second. I would take this, this is for me the best stack on the market. You can take mind-approved as well, that's a great one. I would choose one of those two. Those are the best stacks on the market in my opinion. They are a bit different, one more expensive than the other one, but yeah, you should just take one of those two. Cool, thank you, that's exactly why the coffee, coffee pills with alethenin, yeah, that's what you should do. Usually you have 100 milligrams of caffeine, with 200 milligrams of alethenin. That is the usual dosage and that works really well. Hi, man, I have a lot of problems with willpower and focus. They're starting to finish doing tests. They recommand in tropics to improve that. Of course they do. So focus, willpower, motivation, yeah, you need to boost your dopamine, increase your dopamine. You need some kind of a kick. In this case, if you're looking for a stack that you can buy right away, again, I would go with quality of mind. This is gonna do the trick. If this is something you don't wanna buy for whatever reason or if it's too expensive for you, then I would take Panix Ginseng. Panix Ginseng is really good. Caffeine with alethenin combined. If you don't do well with caffeine, avoid caffeine, just take alethenin. Ginseng, lance main mushroom, then take choline. So you can take citric choline or alpha-GPC both are really, really good. And maybe some rhodiola as well. This is gonna do the trick right away. And if you still want a stronger boost then, again, supplement like this, I'm not trying to promote them and there are no affiliate links anywhere. I'm just saying this is a product that I would take and I do take quite often. Hey, Mateo, it's good to see you here. Awesome, I honestly appreciate it, man. Now, do you guys have a pre-made stack or would I be buying this separate? No, right now we don't sell any Nutropics yet. We're working on something, then as soon as I start selling something like my products and people will probably think I'm kind of biased towards my products even though I would develop the best supplements, in my opinion. But for you, DJM films, just, you know what we can do? Just send me an email to greck at yourinception.com. I'll just write it down. Just send me an email. Just tell me that we discussed the ADHD stack and I'll give you some good pre-made stacks for you. Two or three, maybe I'll check which are the best. I have the list of all of those which you can buy and you can choose one of those that you find the best. So yeah, that's what we can do right away. Why do I choose specific ingredient? I don't like complexity because I would never know which one did the best for me and price it like you said. Yeah, look, with individual compounds. So for peak brain performance, here's what you need. You need EPA and DHA. So you need Omega-free. You take this already. So, you know, continue taking this. Then you need a colon compound. Coling, for colon, you can take city colon or alpha GPC, 200 to 300 milligrams of one of those, right? And that's what you need for sure. And now whether you're gonna feel the effects or not, that's questionable. You know, some people feel them, others don't, but that's not the point with contropics.  They're gonna make sure, they're gonna provide building blocks for your neurons to operate properly. So that's what you need. And then, what's really important is to take, or I suggest taking one of the adaptogens to one of the adaptogenic herbs, like rhodiola, like panagrygenic or bacaupaminari for brain performance. If you take one of those, you should feel the effects for sure. And that would be my starting point. I would start with those. And then of course, yeah, of course, L-taining, L-taining is great. You can feel the effects of L-taining. If you don't believe me, just drink a cup of really strong green or black tea and you'll see what I'm talking about. So those are the individual compounds that I would take right away if I were you. DJM films, no worries. Just let me know. You asked me about supplements. There are things to do to boost your performance that support supplements. Some are even more important than taking supplements. I mean, you should take the supplements, but there are some other things we can do and I can send you some suggestions as well. I mean, I have lots of people that come up to me that have ADHD or some one of the types of ADD and we have fantastic results because we know how a brain performs and or how brains perform and what are the protocols to fix those things. So yeah, that's why I'm here to support you guys. Cool. By the way, for those of you who have ADD or ADHD, did you know there are like seven types of ADD available or that we know about seven different types of ADD? It's, you can Google it if you don't know which type of ADD you have. This is actually very important to know because yeah, the protocol for each of the type is quite different actually. And that's a funny thing. Not everyone is aware of that. And then people will take Adderall or Ritalin or some are stimulants, also Modafinil for the wrong type of ADD and they don't get the right results and yeah, that's a big problem. There was a study about alpha GPC and increased risk of heart attacks. Does city calling pose the same risks? Did you have a negative effects from it? Yeah, I'm aware of the study. So the problem with alpha GPC is and any other calling compound is if you take it too much, right? So where, which foods contain high amounts of calling? So X are number one.  So that's the problem. And not just that, you get lots of fat and then yeah, that's what's causing the problem. So calling to too high levels of calling can be problematic but not getting enough calling is also problematic. So yeah, you have to kind of balance it. That's why when I suggest taking calling either alpha GPC or city calling, I suggest supplementing it, not getting it for food and or well, not getting it too much for food. And secondly, I suggest supplementing a small dosage that that's very important. Now personally, I never had any negative effects from calling, but I have to tell you I eat mainly plant-based foods, not entirely but mainly. And I generally don't eat X for several reasons and therefore I don't get too much calling for eating food. So yeah, just so you're aware of that. But I don't know any people that had, sorry, that had issues with calling and a lot of people who take it. So if you take a low dosage, I wouldn't worry about it, honestly. I tried all those and really all that was amazing. Ginseng, we take quite a high amount of Ginseng but calling 5X, I should just take a couple of tips here should I stop. Yeah, man, don't, so first of all, yeah, I'm happy that Rodeola works well for you. It works well for many people. Actually, it's a really cool metropic, quality best, one of my top five metropics actually. Ginseng is really good as well. Ashwagandha is amazing for stress. But yeah, if you eat five plus X per day, I would not take calling because you get it too much already. I would decrease the amount of X right away if you can because yeah, you get too much calling already. Love LTNN, I take it. Yeah, yeah, that's good, man, LTNN is really good. Oh man, yeah, if you were diagnosed at age six, I don't know how old are you actually, but I assume you're older than age six now. So you probably, yeah, my assumption is they didn't tell you which type of ADHD you have. And this can be problematic. Maybe you don't have ADHD actually. You may have it, but maybe you don't have it. Did you ever take any stimulants? So did you try actually Adrenal or Ritalin? And if you're 22, so this was like, yeah, that was a long time ago, right? So 16 years ago. If you took Adrenal or Ritalin, did you see the benefits? So did it work for you? Like did you feel more focused and so on? Or did you suffer from any side effects? That's an important question. Okay, try Adrenal, okay. So here's the thing, if Adrenal worked for you, then you probably have ADHD, but not necessarily, they're still like inattentive ADD. The people with inattentive ADD, they're also prescribed Adrenal, for example. So yeah, I stopped caffeine now. I do that every year for like a month or so, but what can I take instead? I know about caffeine, yeah, I slept caffeine from a year ago, yeah, okay. So here's the thing, if you need caffeine, okay?  My brain doesn't work, I have brain fog, I have no energy, I need a kick. Then you're doing something wrong. Then something is wrong, okay? So either you don't sleep long and well enough, that's one option, or your diet is not optimal for brain performance, because once those two things are optimized, once your diet is optimized, and once you sleep well or long enough, you don't need caffeine anymore. You don't ever think of caffeine. You wake up like I do, I have lots of energy, I'm like ready to crush the day, and this is my small studio, so I get into my studio, I start helping people, I do some coachings and so on, and I'm shooting videos, and I don't need caffeine. And if I do drink coffee or take caffeine, then sometimes after one day, that's fine, but if I take it a couple of days in a row, I crash because I'm too anxious, because I get too much of energy and too much of everything. So if you need coffee, then you need to figure out why is that you need it?  It's actually a really good one. You see it quite quickly. If you wake up, you need caffeine. Ah, something is not working well. Hello to India, man. It's good to see you. Okay, so fifth grade, C-grade, it turned me into a zombie, yeah. So, man, I cannot tell you if you have ADHD or not, but if I were you, you know, I would just go see a psychiatrist right away and ask him or her to do an art test, because now they have better technology and they will tell you what type of ADD you have. And please do that actually before you start taking any stimulants or any supplements, because you're gonna save money and you're not gonna take something that may cause some damage, all right? Losing emotions with phenylpyracetam. Yeah, that's why I don't recommend it, man. I don't recommend resitams. Man, I'm one of those honest guys that say, you know, don't take resitams, not because there's something wrong with that, but because of lack of studies. There's a huge lack of studies on all types of resitams, not just phenylpyracetam. And there are usually some side effects from taking those compounds. Now, if more research comes in the future, I'll be super happy about it and I'll be the first one to read it and maybe even change my opinion, but as it currently stands, there are better tropics available in the market, safer tropics that don't cause any side effects and I would just stick to those. And that's an honest opinion. And plus, keep in mind that resitams are illegal in most developed countries. Well, they're legal in the U.S. still, but they're illegal in most of the countries and it is mainly because of the lack of research. For studying, how do they smudge drugs? What kind of smudge drugs do they want to take?  It's like in tropics or are you thinking about something else? Let me know. I got really bad headache without coffee. Okay, you got bad headache without coffee because once you're used to drinking coffee, it's really hard to live without coffee. Now, if you get really, really bad headache, here's what you need to do. You have to stop drinking it obviously as soon as possible because you're addicted to coffee and that's how you know. That's where your headache comes from and instead of coffee, drink green tea or drink gerba matiti. So I'll just write down gerba, wait, gerba matiti, which also has caffeine, but a much smaller amount. And then, first two, three days will be kind of a bet. I can tell you that and I'm sorry about it, but there's another way around it. But after a couple of days, what's gonna happen is you will start feeling better and after a week, you will be like a new person and then you won't be addicted to coffee anymore. You don't have brain fog or anything. I don't like I have to take coffee, but the issue is when I stop today, I stop in daytime and that's why I get angry. Yeah, I mean, again, if, so it's really good that you don't need coffee and you don't have brain fog, but yeah.  It has lots of antioxidants and we know that people who drink coffee actually live longer, but coffee also causes lots of side effects like we're talking about right now. And in this world where we always seek this additional and dopamine boost, we are always so anxious and so alert and so on. Coffee even makes it more anxious and more stressed and that is the problem. No one's talking about it. Why? I don't know. So yeah, if you don't need coffee, please have it. Ultra brain, yeah, ultra brain is good. This is an Indian brand. I know the founder and I know what they're doing in India and it's the best probably you can get in India and I recommend it for sure. And about Ashfa Ganda, Rodeola. What is the best tolerance break? Cause I don't feel anything anymore from those and it was fantastic. Yeah, with Rodeola and Ashfa Ganda, I just had a discussion about Ashfa Ganda today on Reddit where I'm also pretty active. You can take Ashfa Ganda for three months and then stay three months off. So three months on, three months off and you can do the same thing with Rodeola. You can also do something else. You can take Ashfa Ganda every second day. This is what I do every second day and then extend this to about six months and then take a month or two off and then you repeat this process. It depends what you prefer. Awesome, and yeah, just retest it, yeah, really. That's really important. What's your opinion on using an NMDA antagonist to reduce stimulant tolerance? Don't know if some magnesium form like Alzheimer's would make a significant difference. Yeah, I generally don't recommend just taking stimulants at all. It's a good question, but I don't know. Yeah, you could take them and see how they work on you, but yeah, I'm not like a really, really big fan. Like magnesium alternate is great as well, but I don't think it's gonna make a significant difference at all. Ultrabrain is not a smart drug. Ultrabrain is an atropics type. So make this different because it's very important because then people will think like you're taking drugs and you're not taking drugs, you're taking just supplements. But yeah, those supplements will have a long-term effect and that's good, not just when you take them, but when you take them for a couple of weeks to actually experience those benefit. Love from India, love back to India. I mean, India has a lot about adaptogens. That's where I learned a lot from an Indian doctor, from a doctor of Arvindic medicine.  That's what's gonna happen, nothing else and that's the problem. What if I stop taking new tropics again? Is there a chance to get back, back our backstage? Yeah, why not? Of course you can. I recently bought CityColin and have started taking 250 millions. I take 500 DHA and 250 EPA. How long do you think we'll take to see some effects? I have fish and chicken and my dad also wedges. Well, good question. I think you should see effects after one month or so. That's a reasonable expectation and I would probably take two pills of EPA and DHA because 250 milligrams of EPA is kind of on the low end. So maybe I would increase this to two capsules and CityColin is an optimal dose you take. So yeah, let's talk again in about a month. Just send me an email and tell me how you feel. Lance, we need a break too. Not that we know of, not that we know of but since it is an atropic, I generally recommend cycling on atropics, including lancement, not omega-free. You need to take omega-free daily, but lancement, you can also cycle it. I usually do five or four to five weeks on one week off. What kind of effects should the effects, should the cycle it? Yeah, so you should cycle CityColin five days on, two days off. Well, CityColin increases, so it basically provides colon which is the precursor of a CityColin, which is an important neurotransmitter for learning and memory and attention. So you should feel more focused and I don't know if you're gonna feel that but you should have a better memory as well, but your brain in general should work better. Altars are under the tongue. Actually, you know what? I never tried it. Sublingual. Will this make a difference? It shouldn't, I think. Not in terms of the effects maybe, because altars are not like a stimulant, so it's not gonna give you this boost of whatever, so yeah, it doesn't really make any difference, I think. But yeah, if you have it in such a way, I mean, you can take it like this. It's not a big problem. You follow me since three days and watch my videos. Awesome, I really appreciate it. What's the best supplement in India for effective in studies? Ultrabrain, for Indians, ultrabrain. What are taught on green juices like athletic greens? I have some at home, athletic greens. I have another one that just got from InnoSubs and another one from Adam, but I don't know what's the name of the product. Yeah, what should I say? I don't like the taste. So that's the first thing. I don't like how they taste, but yeah, they can support your health. For sure they can do that. I mean, they contain lots of antioxidants and lots of veggies and lots of fruits. So yeah, why not? If you can afford them, drink them a couple of times per week, they can be beneficial. Not needed, but can be beneficial. Can I take Lansman mushroom every day? Yeah, you can take it. Generally, as I said, I suggest cycling it, but you can take it daily. Sorry to confuse you. I like to ask if I stop the tropic, my brain will back to normal stage. Yeah, if you stop taking tropics after a while, of course your brain will need those nutrients for peak performance and then if you don't deliver those nutrients for peak performance, eventually, of course, you're gonna go back. That's how it is. How much Lansman mushroom should you take? 500 milligrams to one gram per day is good. Can I take GABA and DMAE? You can take GABA, DMAE, I don't recommend. This is an active compound in central phenoxin. It is similar, but I suggest avoiding it due to the lack of research, to be honest. Helmet, what's the best entropic for focus and memory? Sam, you mean like an individual compound or you mean as a metropic stack because if it's an entropic stack, quality of mind or mind lab pro, I have them here because someone asked me about those before.  If they will grow in terms of what, popularity? Yes, definitely, the market is expanding. GABA every day, GABA is a, yeah, you can take GABA. I mean, you need to, if you take GABA, the problem with GABA is it doesn't cross the blood-brain barrier, right? So based on the research, the use of GABA in supplement is questionable. Maybe you can combine it with some other compounds like peppering or something else. But yeah, if you take too much GABA, the brain will reject it. That's the problem. So I mean, it's really hard to get some really good with the effects from GABA. That's the problem. Low libido advancement. I do business and I have time to study in the mind. So please just mind taking tropics. So if an entropy contains caffeine, you need to take it at least 10 to 12 hours before you go to bed. If it doesn't contain caffeine, then you can take it like six, eight hours before sleep. It kind of depends on how they work for you or on you, what kind of effects they provide. So yeah, at least four, at least six to eight hours before sleep if you don't take any stimulants. How do you cycle citicoline? Slide this on to this off. World domination tropic. I haven't tried that, but I'm in contact with the company and hopefully they will send me one box soon and I'm gonna try to make a review. So expect my review soon. I mean, can I take them together and what can I take for Lucy dreaming? Yeah, you can take GABA and DMAE together but I don't recommend doing that. I don't recommend taking DMAE. And what can I take for Lucy dreaming? I don't know, honestly. I have to check it out. Sam, look, if you're a student, while I would suggest taking mind leprosy, I think there's a better stack out there for you. For studying and it's called, it's called quality of focus or another option is quality of mind essential. Both are really good. Both are from the same brand in your Hacker Collective. Why do I recommend those more? Because they provide more instant boost of brain power whereas mind approach is more designed for long-term brain performance. If you can afford quality of mind, go with this one. You can take a caffeine free version or caffeine. If this is slightly too expensive, which usually is for students, then you can take quality of mind essentials. It's really good quality of focus. It's also pretty good. My review of quality of focus is coming next week. If you wanna wait and check it out, otherwise you have the reviews available on my channel for other supplements. Best sleep substance, many are good. Magnesium, Ashfagandha to calm down. I would take 5-HTP or L-Triptophan, that's good. Then you can take melatonin, a synthetic one, even though I'm not such a big fan of synthetic compounds, or you can take Montmorency tart cherry. So it's a tart cherry extract. And this one is great for sleep because it needs a natural source of melatonin. Those then some passion, you can take a passion flower or hops extract. Those are a couple of really good sleep metropics. You can check performance lap sleep. This is one of my favorite sleep metropic stacks or qualia night, that's another one. Those are really advanced stacks. D-Repose is a tropic and Tarine is a tropic. Tarine and amino acid, yeah, not really. I mean, yeah, it has a glutaminergic activity. It may reduce depression and anxiety, but that's about it, you know? So if you're anxious, yes, you can take it, but otherwise don't expect too much. It is in qualia mind though. In India, mind are pro not available, I already ultra brain. Yeah, then take ultra brain. I don't know the recommend daily intake of ultra brain, I'm not sure. I think it's like two capsules or something like that. So that's what that would take. Just follow the recommendations. And then, you know, with ultra brain, let me just check it, ultra brain. Give me a second, I'll check it right away for you because I want you to know what to do. You can take, yeah, you can take ultra brain, like try it like eight hours before sleep and see how it works. If you can fall asleep after eight hours, that's fine. If you can't, then take it earlier. And I'm happy to, you know, that I can talk with you as well. So thank you, appreciate it. So for students who believe qualia mind pro or qualia focus, no, so it's qualia focus or qualia mind essentials or qualia mind. Qualia mind is the most expensive, the best one qualia mind essentials is slightly cheaper and kind of in the middle and qualia focus is the most affordable one, but also, I mean, still good enough, more than good enough for studying. I would take one of those, to be honest. There are other supplements on the market as well, you know? But I trust those, you know, that's nearly every single stack on the market and they're really good for students. I have lots of students. I was a student myself back in the days when, you know, qualia mind was not available. And I wish I had it, it would help me a lot. Okay, give me a second. I mean, D is a ribose is a carbohydrate, right? It's a simple sugar. So I wouldn't take this as an entropic. I think it has some effects on the mitochondrial function, but I think this is very early research. So I don't know much about it, honestly, because it sounds really used in those stacks. And I think it's in a very early stage of research. Also, I started taking Ashwagandha for anyone asking the stress reduction is 100% also, but by KSM66, yeah, that's really good, Sam. KSM66 is the most potent and most well-known Ashwagandha extract. You can take this one, or you can take Sansaril. Both are equally effective, but they're really good. And I take them, I take KSM66 as well. For me, it's very important to improve the memory and cognitive function because I have clear some exams. Yeah, you said you are taking, you said you're taking ultra brain, didn't you? Yeah, so taking ultra brain. Yeah, ultra brain is a great stack. Follow the dosage, the recon deli intake, and just take it daily, like for a couple of, so you cycle it, so take it for five days on, then two days off, but take it for a couple of weeks or months, don't take it just for five days and then say, you know, this doesn't work. Take it for a bit longer period. What's the time? Okay, we have a couple of more minutes, so we have three more minutes till the time is gone, so let's see. You're still like Madafinal? Great question. I still take Madafinal, and then whether I like it or not, it's hard to answer. So look, Madafinal is a potent stimulant, right? And if you are smart enough, then of course you can take it because you will control yourself, right? You will know when to take it and how often to take it and how much to take it and also when not to take it. But generally I don't recommend taking it because it's such a powerful stimulant with so many side effects, you know? And people generally abuse Madafinal and other stimulants, that's the problem. And once you try Madafinal and then you take something like that, like this doesn't work. In reality, it does work, you know? So yeah, that's my opinion on Madafinal. If you're smart about it and if you can get the quality Madafinal, then you can take it now and then. But be aware of the side effects. Is it necessary to take multivitamin to enhance other intravic effects? Not really, no. It hasn't been proven. Brain stack plus ultra brain can affect, yeah, that's a good stack.  That what I know of is that it does make you addicted, even though it's not a physical addiction, but it's this, how should I call it, a mental addiction. So once you get used to it, it's really hard to live without it. There are some side effects as well, other side effects. So, but I'm not right now aware of if there are any related to brain performance in general. So no, I wouldn't worry about that, but yeah, I mean, they can cause severe headaches and stuff like this, which I'm sure you read about. While you have a prescription, you are good to go then and you have a quality Madafinal, but yeah, so be smart about it, all right? Please. So how much does quality focus or quality of mind improve memory? Is it noticeable? Yes, great question. I tried the dual and bag game. This is a special game designed to train your memory and I tested all those supplements. So I played the game for a while, got a score, then I took the supplements, I played the game again, I checked the score again and so on and I tried to see if there's an improvement and yes, I noticed an improvement every single time I took those texts. The improvement was from eight to 15%, something like that. I think in one case, even up to 20%, but you have to know that obviously by playing the game, you also train your brain, right? So you also get better at playing the game. So the difference was not just because of the supplement, but also because of me playing the game. But yes, quality of mind and many other quality stacks contain vocabulary and vocabulary has been proven to increase, to improve memory, shorter memory in healthy individuals so you can expect to have a better memory, for sure. Okay, I'll take three more questions today and then we do this again soon. I love quality of mind and quality of sleep. Yeah, it's good. It's quality of night, I think, not quality of sleep. I think I always, or is it? I think it's night. I always say quality of sleep and I have a good relation with the brand and they reach out to me like, hey, it's not sleep, it's night. I was like, yeah, sorry. Do you think taking all seven capsules is needed? Sorry, I was just thinking of a question. Seven capsules, yeah, well, look, if you wanna get the optimal dosage of all compounds, yes, then it's needed. I know that seven capsules sounds like a lot and I know it's not, I mean, it's quite an expensive supplement, I'm aware of that. But those are some individual compounds are really low. They're on the lower end because of the philosophy of near-hacker collective. So some people feel really good at the free four capsules but in terms of if you look from the scientific perspective, I honestly, I cannot say take less than seven capsules. Well, you can take five to seven, that's good enough, but yeah. But you know what? Listen to your body, listen to how it makes you how a quality of mind or whatever you're taking. Yeah, I think you said quality of mind, right? Yeah, so how it makes you feel if you need less, take less. You still get enough. If you take five to seven capsules, you're good. How do you purchase mine in India? I don't think you can purchase mine in India. Sorry about that. But yeah, it's not me signing it there. Okay, last two questions. How does landsmine mushroom work in the brain? Great question. So landsmine mushroom contains some potent compounds. They're called arenasins and heresanones. I hope I pronounced them correctly. So those are the bioactive compounds that provide the effects. And they increase the NGF, the nerve growth factor, which is an important factor for improving the communication between neurons. So this is a protein. And that's their main mechanism of action. And so you see some neuronal regrowth in the brain, strengthening connections. And that's where you get the benefits from landsmine mushroom. Okay, last two questions. That now for real. Can long-term metropics use increased IQ? No, I don't think it can, even though... I mean, if this happened, then my IQ would be like 200. I would be like two times smarter than Einstein. But I think I'm not. Or maybe, well, who knows. No coughing, but modethanol. Yeah, well, so with modethanol, I take it about once... Well, I haven't took it for like six months, seven months or eight months, something like that. But then I take it about... Well, I take it about once a year, now maybe two times a year. Actually, I used to take it way more often, but I'm thinking now, yeah, during COVID and stuff, I didn't take it that often. So when I take it, I take it during a period when we do some really extreme stuff in the company when there's a lot of work out of stress. And for example, if you're shooting videos and something happens, then we need to shoot like 10 videos in a day or whatever, something like that. Then I would take it. But yeah, it's been a long time since I took it. Then, for example, modethanol works really well for me. There are no side effects, nothing. If I drink a strong cup of coffee, I crash. So yeah, no one can explain why this happens, but that's how it is. Thoughts on phenylpyracetam, don't take it. There is not enough of research. That's the problem. Okay, cool, we extend this a bit more, but anyway, I liked it. I would talk, I could talk for hours, but let's make a few short sessions and on. I think it's better for everyone. So guys, yeah, thanks for being here. I hope you liked it. If you have any more questions, I mean, I will go live again soon, probably in about two weeks. So yeah, I'll let you know. Feel free to join me then again. If you have any more questions about brain performance in general, you know where to find me, I'm quite available and I try to reply to everyone. So yeah, thanks again. Stay safe in those crazy times. Stay healthy.  And yeah, I hope to see you soon again. Talk soon. And yeah, Mateo, I see you're saying some questions. Just send me an email, man. Just send me an email and I'll reply to you. All right? All right guys, stay well and enjoy.


Hi, this is George Cowell. So in this short video, I'm going to show you what a difference a good quality webcam makes in Making a video. So I'm going to show you what it looks like when I'm using the default webcam That comes with my laptop versus this one. So this is the default webcam on my laptop I have a MacBook Pro. This was bought in 2010 But back then it was top of the line 17 inch screen And I think they haven't really changed the webcam very much over the years even the latest MacBook has Webcam that's not not too far from this this low rather low quality and notice on this default webcam I have to like I have to adjust this have to turn the screen closer like this and To be able to see more of me and at this kind of angle It's not very comfortable for me to look at the screen if I'm looking at notes or whatever The way that I typically work is at this angle, right this angle to this comfortable looking at the screen But it's not great for the webcam. So as you can see the lighting is not so great either I don't I'm not using any artificial lighting that the lighting the natural lighting in this room is pretty good But it's not great. And so you can see it's kind of has a pinkish quality In the in the picture and so I'm going to now show you what it looks like with my external webcam that I bought This is the one that that I'm going to be showing you and with this one I'm going to show you that this can this can adjust so so this sits this sits very gently on On the top of my laptop screen like this very gently and you can adjust any of these things You can let's see. Yes, you can adjust this piece right here. You can adjust this piece So how it sits on the laptop? It's very easy to use and you can also adjust this top part So that you can you know, you can show the the angle different places. So I'm going to include the link to this webcam and the notes of this video. It's going to be a non a feeling link I don't like to be paid. I don't like getting commissions for endorsing products That's not how I make my money But I do like to endorse products because I genuinely like them and think you should consider it So in the next segment of this video, you're going to see the difference with using this instead and again I'm going to put the I'm going to put the the the laptop screen back to where it's comfortable for me to look at the screen So it's typically like this which is which is not great for the default webcam But here you go watch watch what it's like when I use and here we are back with the external webcam I have literally not changed the positioning of my laptop screen So the very last segment you saw where my laptop screen was Right now comfortable for me to be reading notes or whatever as I'm recording That's the same, but I'm able to adjust the webcam angle So it looks a lot nicer The other thing I love about this webcam is that you can adjust you can download on the Mac You can download a free app from Logitech Logitech webcam C9 I think it's called Logitech camera settings is the I'll include a link to the app but the free app allows me to adjust the brightness of Of the image. So look watch this See, I can adjust however I want the brightness to be organ to make it darker darker or brighter Just very easily by my moving a slider on the app. I can also adjust the contrast easily So let me like this or like this more contrast But obviously I'm just going to put it somewhere in the middle here the color intensity is kind of Yeah, I can make it black and white easily or make it super super colorful easily very easy to do on here There's also another setting called white balance. I don't know what that is. Let's see what that does here Oh, I can't adjust that. It's it's oh, it's automated right now. It's automatic But see white balance is versus this so it's more more right or you know, so something like that So there there's a checkbox that that's put that I can put as auto for the white balance and that see now It automatically adjusts it it also Let's see. Yeah, so those are pretty much the settings and I just got a text sorry for the for the noise there and I can also adjust the focus automatically or let it be manual So right now, let me adjust the focus manually here to show you what happens See now, obviously it's out of focus versus you know in both But I'm gonna click the auto checkbox here and it snaps to focus So it's very smart and I can even adjust the zoom. So let me let me show you what the default zoom is on this So default zoom is pretty wide angle, right compared to my webcam, which is fairly smaller And then I can adjust the zoom one two three three clicks on the zoom This is kind of how I like it because then it kind of hides my my little lapel mic Which I'll include the link to the lapel mic the lapel mic makes a difference too and how it sounds it sounds much better Then then the general if I don't use the lapel mic the sound is more echoey by the way the webcam also comes with a microphone So that's kind of nice as well. The lapel mic. I think it's only $20. So include the link to that as well So anyways as you can see it makes a difference For your Skype calls for if you do webinars Google Hangouts You're you simply make videos. I'm using the photo booth app on my Mac right now Which comes with the Mac what comes with every Mac for free the photo booth app to use to make this video So I hope this helps and enjoy Getting an external webcam if you choose to do so


Welcome to MOOC course on Introduction to Proteogenomics. In last few lectures, we have heard about various ways to analyze pathways from Dr. Kerstin Krug. After understanding about how mutations affect phosphorylation, leading alterations in the signaling pathways, today Dr. Kerstin will talk about how one could use MIMP and GSEA in the hands-on session. He will talk about how to use Github to obtain the basic codes and to use them without actually coding but by manipulating codes as per your data and analysis requirement. Dr. Krug will talk about use of two different formats for GCT files, GCT 1.2 and GCT 1.3 and conditions when one could make use of this format to provide better results. So let us welcome Dr. Kerstin Krug for his last lecture and learn more about usage of MIMP and GSEA. So there will be two parts, the first part that we will try to use to predict mutation events affecting kinase substrate binding. So this relates to the first part of my talk. And here we will try something very experimental, we will try to use R and actually I already prepared an R notebook file that I will talk about more what that is, an R notebook and so on and so forth. But you will find this kind of file here hands-on MIMP.RMD which stands from R mark down in the zip file that you hopefully all downloaded. You have to open R studio and then you just load file, go to file, open file and then you open the R mark down file which is called hands-on slash MIMP.RMD. So what you are looking at is an R, it is a so called notebook. It is R code intermingled with you know just text, who of you have heard about mark down in general, ok. So it is ok, that is great. Markdown, it is a very simple text-based format to create you know structured documents or like HTML pages and stuff like that you know. And there is extensions of that that allow you to execute code in these mark in these documents. Actually there is a link here about R notebooks and if you click on this link you will get some more information here. That is a very convenient way to analyze, to document your analysis results and your code and also to share it with your collaborators. Maybe I quickly show you the result of this whole analysis. At the end of the day you will have an HTML document which looks like that which you just open in your browser and there you will have some documentation about like the different steps. So this is text that you entered so you can describe what kind, what is the goal of my analysis, what is the data input and output. But you will also have all kind of you know R code that has been executed in order to get to that analysis results. And this you can easily share with your collaborators and you can just rerun everything in order to get to these results. So but right now we just focus on that one. So as you see as you scroll down a bit you see you know this block here starts with R. So this is actually R code here in this kind of block. And you can execute this code by just clicking on this little triangle here. Please try to do that. So this might take depending on Wi-Fi connections might take some time because the first part actually sets up the entire analysis. So it downloads a couple of packages again and so on and so forth. So if you could please try to click on this little triangle here. That's the first or it's you know in line 25 that's probably easier. So line 25 if you click on there on the triangle. Which is probably not going to happen because the website is down. But so there we wanted to use some documentation for the property that the CGA sampled. We wanted to try to show you how you would avoid this tool. So yes you can click here on search, search mutations. Then you can upload your VCF file. So you know the VCF file you've learned about. I mean the VCF file is called a very common format. And depending on your pipeline that you use to call your variance you look at a VCF file. It's a pretty standard data format for a very common. So it's not about that you have to create a VCF file, it's more about you should have gotten it from somewhere. If you have shipped your sacros to sequencing center they do the sequencing to get a bank out of that. So running past Q is for the genome, which is back. Then typically you would have some pipeline running in your lab or maybe you're collaborating with someone or it's becoming spiked last and greats. They would take these back parts and you know the phone invocation card. The result of that are VCF files for example. I mean it's not a very complicated format, but I can do it here. So when we install all of the technologies we need to specify more data. And here I have chosen a mutation file and a muscle size file and the cross only database file for one of the TCGA pages again. In order to make it more convenient for you I put everything on GitHub. So it's automatically fetched to the GitHub. So you don't have to go anywhere to download if it will happen automatically. And here you actually have a link to GitHub. So you can also just copy these things.  So one is called mutation.txt, the other is tsize.txt, and the other is support.txt. And again it's basically the format. So can everybody do that? So this is very specific to the software that we are using here, right? So this is now linked, and the mutation file is divided into two columns. The first column is the gene name, and the second column is the non-SEM. You can ask the substitution that this mutation calls, easy as that. So this link will be there also after the workshop, so we can always come back. So the installation succeeded. The installation succeeded because it's called on par with the data sheet. And then we will try to render this markdown document into an HTML report. Let me go and look at the HTML report, because it's just easier to do. I don't know why, but this is now a problem that I can see here. Again, so the link to HTML. So actually before we go, these are the last calculated patterns. However, if you first want to go and look at the HTML document, and if successful, this HTML document should be in the same order. And here we have the entire report as HTML, which includes some description about the project, and also documents all different steps that we've done here. So first we install all of the packages. You also see the output that was generated by the different code chunks here. Here you now have the direct link to GitHub. If you just click on that, you end up on my GitHub page and so on and so forth. Meaning we specify the input data, what a script then does, it downloads the input files, and then what a script does, it imports all of these files and just shows the first couple of entries. You all have that in your HTML file here. So this is the R code that creates that table. Does that make sense? So again, we're looking at the mutation file. We have two columns, gene mutation, gene mutation. The phosphosite file is similar. Again, so this is the code that generates this table. And what happens then is it will open up this result page. So this is what just happened when I ran MIMP. This is also one example that I was showing in earlier this morning. With this result page, here we see there's three mutations that effect in total like 22 possible phospholation events. We see that most of them are losses. So meaning a motif got lost. So the phosphosite is most likely not phospholated anymore by the kinase it used to be. But we also observed two gains. So meaning now we suddenly see like an increase of or potential, a predicted increase of that phosphosite to be occupied. I just go to the last page. So these are the two events where we predicted a gain. And here I just was showing or show you two examples of a phospholation gain. So basically that's the wild type. That's the mutated version. So we see this aspartic acid here, which is now being recognized by like a novel kinase. So this motif fits to that particular kinase. So we predict or we assume that this phosphosite is more likely to be phospholated now. So that's the type of data that we get out of here. The objective or the goal of this hands-on session is to explore breast cancer subtype specific pathways. We're going to use the breast cancer data set that we have used. But we're only looking at two subtypes just to make it a bit easier. We're just going to look at basal and luminary. And the tables that I already created only contain these two subtypes. So we're going to do, so this first exercise is optional. So this would involve morphers. And I'm not sure how well this works here again. So we can skip that. I can just demonstrate how we use morphers or how you could use morphers to create or to convert your protein centric tables or your phosphosite centric tables into gene centric tables. So this is always what you have to do if you want to do path analysis. So again, this is optional. So you have already the correct tables to move on. Let me do two different ways of pathway of GSEA analysis. One is like the classical, so to say, GSEA using this Java application that everybody was able to download or most of you. In the second approach, we're going to use the same data set. So we will use single sample GSEA to project our protein matrix into pathways. And then I actually plan to use morphers again to perform some cluster analysis and marker selection on the pathways. So again, I cannot guarantee that this is going to work due to internet connectivity, but we will at least try to do that. And again, so I tried to make the slides as comprehensive as possible. So you should be able, in theory, to just go home. You have all of the data. You go through the slides and you can repeat these exercises on your own. Just a quick recap. So that's the data set we've heard about this data set a couple of times already. And now we only want to look at basal versus lumen and A. And just by eyeball and protein space or in this proteogenomic space, you clearly see differences. Now we're interested in what are these pathways that are differentiating basal and lumino. And in this case, we're only looking at the cancer hallmark pathways. So it's a very small compact and well annotated curated pathway database. So it's basically just what I said here. So we have two different data formats. And I got a lot of questions about GCT and how to create GCT files and how to open GCT files. These files are simple text files. You can open these files in any text editor. And I would highly recommend to install or to use a text editor on your PC. I would highly recommend Notepad++. This is what I use on Windows systems. And again, if you just Google Notepad++, it will bring you to the right page and you just download that. That's like a general recommendation from my side. So in this hands-on, we are going to use two different versions of GCT. One is called 1.2. That's the older version of GCT, which has been around for 10 years now, I suppose. And since a couple of years, we rise to format and came up with a new one, which is called 1.3. And the only difference is that in 1.2, we only store the data. So we just store the data and we have two annotation columns, two annotation columns to describe the data. So somebody made that hard-coded, so you cannot change that. You always have to have two annotation columns and then you have your samples and your data. GCT 1.3, you can store metadata, which describes your experiment, for example. So for example, so this is like a snapshot of this data that we are going to use. And here's the data in this corner. And on top of that, we have all the metadata that describes the sample. So this is one sample. Here you have the TCGAID and then we have all kinds of information, so which team teacher has been used to quantify that, which subtype it is, HER2, ER2, PR2, PR status and so on and so forth. So the advantage of this format, although it might not be very intuitive in the beginning, but if you are getting used to that, it's very convenient because you store all of your metadata together with your data.  What is the GCT file format? That's what you want to know. And how this file format is organized. I mean here, if you spend 10 minutes or so, you will better understand what GCT means and how to create one. We have to use both versions because the Java application does only support GCT 1.2. So this tab is now optional. Let's try to make it work. Let's try to go to Morpheus. And here you have different ways to import your data. You can just browse your computer. If you have it on your Dropbox or you can provide an URL, or you can just simply drag and drop it into this window here. So now what we're going to do, we go to... So that's the zip file. I'm going to quickly extract that here. So that's the one that you downloaded. So now if I now go into this file, you see two folders, GSA and single-sample GSA. We are going to focus on the GSA one. Everybody with me? And here you have two GCT files. So one, you know, proteome-basin-luminal-A 1.2. And the other one says proteome-gene. So this is gene-centric. This is proteome-centric. So in case we are not able to use Morpheus, we already have the gene-centric matrix. That's what I'm going to... This is what I want to say here. But right now we try to... Just as an exercise, I just want to show you how you would do that. And for that you just drag and drop this file, the one without genes, into Morpheus. So drag and drop means you do this. Does that work for everyone? And here you already see, you know, that you have genes that appear multiple times here, right? So these are different isoforms. And, you know, these types basically tell you that we cannot really resolve these isoforms. They have very similar expression patterns because they have many peptides that are shared between those. And for pathway analysis, we need to have a single row for each gene. Does that make sense? If you would open it in Excel, this is what you would get, right? Just Excel, that's the same file. You see the number of genes here, or like 30 inns and a number of sample columns, like 42. Then you have the gene ID, you have some description, and then you have the data matrix. That's 1.2 format. Easy as that. That's the same file, just we look at this in Morpheus. You can easily, I mean, you know, I don't want to go into too much detail, but you can easily... I think in the later slides, I show how to change the annotations. For example, you could go and say, okay, I also want to look at the description, right? So this is highly customizable.  But what we want to do, we want to create gene-centric tables. So you click on Tools, that's the first step. Then you click on Collapse. Then you should see there's been no popping up here. I'm going to do the same in parallel here. Tools, Collapse. So then you have to pick the field that I want to use to collapse. And in your case, it's the ID column. So this is the first column here shown, which contains the gene IDs, right? And here you can also specify whether you want to collapse rows or columns. It really depends. We want to combine or collapse different rows. And here you can choose how you want to collapse them, median or mean. Again, it's very, there's no clear answer what, you know, what would work best. So median is usually more robust against any, like, outliers. So we're going to do that. Then I just click OK. Then you will see that you will get a new data tab here. Now you can also go back and forth, right? If I click here, that's the protein-centric matrix. If you click here, that's the gene-centric matrix. So you see that each gene symbol now is listed only once. So it's, you know, very convenient to create these tables. And if you want to download that result table, you can just do so by clicking on File, Save Dataset. You can, you know, pick a GCT version. You can give it a name and so on and so forth. And then you can just click OK. So now you're ready to do GSEA. And that's what we're going to do now, okay? So now I want you, and again, so here is a step-by-step manual how to do all of this, right? So you should be able to do that at home. So now it's GSEA time. And I saw for a large fraction of you guys, you got it to one. So please try to open GSEA, the Java application. So you should be able to see this kind of screenshot here. Or like this kind of window. I tried to do the same on my PC. So if this JNLP file is not automatically associated with Java, like here on my PC, you can just right-click on it. Then you should be able to see Java Web Start Launcher. Launcher. So then you should be able to open the app. And once this is finished, we should be able to see the GSEA window. So now it asks me whether I want to run this application. And I just say yes, run. All right. So it's a little bit small on my screen here, so because of my resolution. Java also or GSEA also comes with a very extensive documentation.  Because here on this start page already, they actually describe all the different steps that you have to do. Steps in GSEA. So this is exactly what we're going to do now. So what do we need for GSEA? We need the expression data. So this is our GCT 1.2 file that we just created. We need phenotype annotation. So because we don't have the metadata about our samples in our GCT file because it's 1.2, we need an extra file in order to tell the software what is luminal samples and what are basal samples, right. And that's why GCT is so convenient, because you don't have to worry about any other files where everything is in your file. But in order to make this work, we have to create a phenotype label file. And I'm going to show you how we do that. And we have to just pick a gene set database. And again, so here you can upload your own gene sets. You can download different databases which you can upload here. Or it also, you know, directly links to the MCDB page. So you are sure that you always get the latest version. The phenotype labels are stored in so-called CLS format. Here if you follow that link, you will get again more information about that format. It's again something broad specific that has been used for a while, but now because we have GCT 1.3, you know, that's not really required anymore. But however, for this particular application it still is. And I mean, what is important here is the third line. So the third line contains the same number of, you know, columns here than your GCT file has samples. So in this case, we have, now with CLS file, we will have 42, which is in the same order than the columns in your GCT file. And then you can say, okay, first column is basal, second column is luminal, and so on and so forth. And I already prepared that file, and you can find that in your GSA folder. So which is called phenotypelabels.cls. So if I open that in WordPad, so the first line tells you how many samples do you have, 42. The second line specifies, the second cell here specifies how many groups do you have in your file. So it's two, luminal and basal. And the third one has to be always one. Don't ask me why, but this is what it says on the webpage. The second line always starts with the hashtag here. And then it lists both labels, like a unique, you know, representation of your class labels. And then the third line is the important one, where it just, you know, defined for each sample. And again, this has to be in the same order than your GCT file. And you say, okay, this is the first sample is basal, second sample is basal, third sample is luminal A. Okay? Okay, now let's try to import the data first. So we're going to go through these steps, and this is also the order which is shown here on the left. So the first step is to load data. So you will end up on this page here. So again, you have different options how to import data. And now just make sure that you select the genes version this time. So that's the gene-centric version. And I just drag and drop it here. So now the file is here. And I do the same with the phenotype labels. So we have both files here.  So there will be this pop-up window which tells you, okay, I uploaded two files. These are the names of the files. And files loaded successfully, two out of two, which is promising, and there were no errors. So now just going to hide this window, I just click OK. That's it. And again, if you go back to my PowerPoint presentation, you will see all these steps that we just have done here. Okay, now we are going to the next page, which is called 1GSEA. So here we are going to define parameters that we're going to use during our pathway analysis. So please click on 1GSEA. I'm going to do the same on my PC here. 1GSEA. So here on the first set of parameters, so these are required fields, so you have to define those. So which is, you know, this is an expression data set, the gene set database, number of permutations. So why do we need to do permutations? Say it again? Almost. So we are doing permutations. I mean, if we would just calculate it once, we would get an enrichment score, which wouldn't tell us much because we don't know, you know, what does this score tell me. So we are doing permutations, we are permuting the class labels or samples, repeating this entire analysis 1,000 times in this case. So we will get a distribution of enrichment scores. Then we can go back and actually calculate the probability that our actual enrichment score that we got isn't a tail of the distribution or not, which tells us, okay, this one is significant or it's not. We can use that to calculate p-values, right. So that's the main purpose. So we are generating a background distribution, you know, of false positives enrichment scores, because we randomly shuffle these last labels, so it shouldn't make any sense, right. We get a negative distribution. Then we look where this distribution does our actual enrichment score fall into. It is at a number of permutations, and then we have to specify the phenotype labels. So that's how about we just do it. We just start at the top here. If you click on that, there will be only one data set loaded, right. So we specify that one. Gene set database. So this might take a second or two because now it's connecting to the broad servers. Okay, here we go. So now I'm going to click on here. So it says gene matrix local GMT. Okay, okay. So you have to import this database first, like we imported the GCT file and the class label file. So please go back to load data, and then please go to the single server GSEA folder. So right now we were here in the GSEA. Now I go one folder up, and there will be another one called single sample GSEA. And in that folder you will find this file, H.or version 6.1. So that's the hallmark database. And you can just again simply drag and drop it into GSEA. So now if you go back to one GSEA, we should be able to see the database. Once we get the error message again. Okay, now I'm able to see this file here, right? Okay, so number of limitations we just discussed. So now we load the phenotype labels. So here you can select in the first panel here you can select the source file. So there's only one, right? So you could also have multiple phenotype labels and you can play around with different ones. But here we only have one. But here now it actually lets you select what kind of comparisons do you want to do. Do you want to do basal versus luminal? Or do you want to do luminal versus basal? Right, you can just, you know, pick one. And I just leave it at basal versus luminal. And I click OK. Okay, now we're almost done. So the next option here is actually very important. So collapse dataset to gene symbols. So that's what we have done already in Morpheus. So just keep in mind that this software has been developed in 2005. I mean, not this particular software itself, but like the principle of gene set enrichment analysis. So there was no proteomics and no RNAseq, no, I mean, you know, there was no RNAseq and no proteomics to the extent that we know now. So this has been developed for micro areas, right? And the software comes with the option to collapse micro area probes to genes. So this is what this option is for. So we are not able to use that here. So that's why we have to deselect that. And just say false. Use dataset as is. So that's what we want to do. The permutation type, you can either select whether you want to do permutations on your phenotypes, meaning on your sample columns, or you can do the permutations in your gene sets. So why would you do, why would you have to choose or change this option? So can you think of a scenario where you cannot do your permutations on your phenotype labels on your sample columns? What would that be? One is based on a phenotype and the other is based on a gene set. So in option one, we would permute the sample columns and the phenotype labels. In option two, we would permute the gene sets. We would randomly generate gene sets, nonsense gene sets to create our background distribution. Exactly. If you have a sufficient number of samples and I would already consider 40 or so as, you know, sufficient, you can do the permutations across your samples. If you just have two biological replicates, you cannot do permutations on two replicates, right? So then you would choose gene set. But in this case, we have like 40 samples in total. It's totally fine to do our permutations there. Okay, so now we actually filled out all required fields. So now we can expand the basic fields and we're just going to do some small adaptions here. So first of all, we can just give it an analysis name. Hands-on IIT workshop. What is probably the most important option here? I mean, you know, in principle, you don't have to worry about these kind of parameters. What you would have to worry about probably in some scenarios is how you do your ranking. If you remember, so GSEA works on a ranking of your genes. So it would rank, in our case, luminal versus basal. So it has to do some sort of marker selection or some sort of ranking that differentiates luminal from basal. So the default option is signal to noise, which is basically, if I'm correct, it's basically the average between luminal and basal. The difference in average is divided by the product of the standard deviations of both groups. That gives you a measure. It's basically the full change divided or the full change between luminal and basal scaled by the standard deviation. If you have a high full change, but a high standard deviation as well, you would end up with a lower full change. Whereas if you really have a high difference, a high full change, and a very low standard deviation, your denominator would be very small and you would still get a high full change. You could also do, I think this requires at least, they say on a web page, you should have at least three or so samples in each phenotype if you want to do that. You could also do, for example, t-test. That's probably the second that I would recommend. Or other metrics like Euclidean distance between luminal and basal or correlation and things like that. There's different metrics, how to rank your genes. In general, I would recommend to just leave it as signal to noise as long as you have sufficient number of samples. So, we're going to leave that here. And, you know, so this might be interesting for you because that's the folder where you can find the results afterwards. So, this is where GSA stores its results. And you can also change that folder, but this is like per default, you will find the results here. And these other filters here, you can exclude gene sets that have fewer than 15 members and more than 500 samples. I mean, these are pretty good default parameters, so you don't have to worry about them too much. Okay, I think this should be everything that we need to actually perform the GSA analysis. And in order to run that, you just have to click this little one button here. Okay, now it shows success too for me, so let's take a look at the results. And you can just click on success and then there should be a page, it should pop up like an HTML report which summarizes your results. That is false detection rate, right? FDR is a false discovery rate. False discovery rate. Exactly. So, that has to be less. Yes. This is basically the fraction of, let's say we have like 100 pathways, right? And if it says you have an FDR of 5%, this tells you that 5 pathways are actually false positives in your list of significant pathways. Yes, I was actually genuine. Yes, so here the default parameter in GSA is 25, which is very loose, right? But you can also adjust that parameter. So, we are looking at FDR, so false discovery rate smaller than 25%. So, 25 is actually pretty high, right? So, that's the default setting here. And you know, there's probably nothing that you would put in a paper, right?  You will have all of these results in an Excel sheet as well where you have the FDRs and then you can just filter or only look at the pathways at a certain FDR. So, this is basically, I think this parameter has been used in your Wichita study back in 2005 and it still made it until the latest version here. So, it's basically a summary of, you know, very high level summary of your results. So, you have like these two blocks here. The first block tells you this is the enrichment and phenotype basal. Everybody with me? So, we see we have 19 samples in basal. The second block is enrichment and phenotype, Blumenet A, where we have 23 samples. Then you can get some, you know, high level summary here. So, for example, 34. So, I told you that the Hallmark database has 50 gene sets. So, that's why here it says 34 out of 50 gene sets are upregulated. They have a positive enrichment score. So, they are more enriched in basal compared to luminal. In luminal we only have 16, right, and this should add up to 50. This does not mean that they are significant or whatsoever, right. This just tells you the direction. So, here at FDR 25 percent and again this is very high, I know, but again that's a summary and it tells you it's 5 gene sets that are below 25 percent for basal. And there is 2 gene sets that are below 25 percent for luminal A. And all of these, you know, this entire page is again, these are different hyperlinks that will forward you to the actual results. So, here you have a summary about your data sets. You are looking at 11,000 genes and so on and so forth. And here is the summary about your gene set database. And also, you have very detailed and very extensive documentation about GSA because it is such an old software, old approach, very well developed, very well maintained and curated. You have a lot of documentation and tutorials online. And here already you find the direct link how to interpret the results. You can just click on that and you will find all the information you need in order to make sense out of this result page. So, what I am going to show you and here you also have a direct link to the Excel sheet, as you can see, right. If you click on that, it should be an Excel sheet. So, that is now the path values for Bazel, right. And here you also have the FDR value where you can just focus on the first two which are below 5 percent and then below 10 percent, right. Okay, let us look at an example here. So, what I did now, I clicked on enrichment and phenotype Bazel. I clicked on detailed enrichment results in HTML format. So, this is kind of a similar table that you got in Excel format, but now it is in HTML and you have these different hyperlinks here. So, the most significant or most differential pathway is apparently G2M checkpoint, so some cell cycle. So, now for each of these gene sets or pathways, you can click on G as details. And you will actually get these enrichment plots, right. So, here we are looking at the G2M checkpoint signature. We have the P value and the FDR value, which is associated with this pathway. And we also have this enrichment plot here. So, in the x-axis, we have the genes which are ranked ordered according to the differential expression between Bazel which is shown on the left and Luminal A which is shown on the right. So, these are all the genes here in this area. All the genes that are more abundant in Bazel, here are the genes that are more abundant in Luminal A. And all of these vertical bars in this case are members of that particular pathway. And again, just by eyeballing, you see, you know, this cluster of members here, right, which are, which basically do cluster among genes that are very abundant in Bazel subtype, right. And if you calculate the enrichment score. So, if you now look at another example for Luminal A, we can also look at a snapshot of enrichment in the results. This should be like a summary about all of the pathways. So, what are the, so this is the most significant ones. This is the second most significant one. And we see both of them, both of them are estrogen-related, estrogen response early, estrogen response late. Does that make sense? So, we are comparing Luminal A with Bazel. Now, we are looking at Luminal specific pathways. And many of these Luminal tumors are positive. And this is actually the Horma cancer pathways that we are seeing here in this set. So, what is shown here is the signal to noise that we have calculated. It also says that here. And here, according to that, this is the ranking of my genes in my entire data set. So, we had like 11207 genes in this data set. And this is the ranking according to the signal to noise statistic comparing Bazel and Luminal A subtypes. So, again, so what these genes here are more abundant in Bazel subtype. These genes on that side here of the ranking are more abundant in Luminal A subtype. And again, so here we see a clear enrichment. So, these vertical bars, again, are members of this particular gene set. The estrogen response early. And we see a clear cluster of these members here in genes that are more abundant in Luminal A subtype. So, I think the most difficult part is to get the data into the right format. And I gave you some hints to use Morpheus and so on and so forth. In today's lecture, I hope you learned how to use R-scripts and incorporate data in MIMP and GSEA-2s for understanding and visualization of the data. Use of Morpheus to convert your data set, which is protein centric or phosphocentric to gene centric, which can be used as an input for GSEA. The next session is going to be, again, hands-on session in which Dr. Bing Zhang will talk about how one could use linked omic tools. Thank you.


By far, the easiest way to get started with raw is to simply go to the web page raw.densitydesign.org. And that's where we are right now. It brings you to this interface. And I'm going to go full screen just to give a little more space here. And really, you've got one thing you need to do, and that is to click use it now. If you click on that, it takes you to a page and gives you an option of inserting data or choosing a sample. Now one thing I want to make really clear here is you may notice there's no place to log in. That's because you can't create an account on raw. Now that works in part because everything is done in the browser, everything is done locally. And they don't save or process any of your data. It also means that there's a very unusual feature here. You can't save your work. You can save the output. You can export the final product, but you would have to recreate it from scratch. And so that's an important limitation of raw, but also it makes it really sort of quick and easy to get started with. And so you simply open up the browser, click get started, and then you can start using the sample data or inserting your own data. And I'll show you how to do both of those.


Was there ever a time where you felt a spark with someone you were talking to? Maybe it was a blind date or someone you've met in a bar, but it felt like there was a connection. Or maybe you felt a spark while reading a romance novel, and so you're curious about in-person sexual tension. Sexual tension is when we feel sexual attraction or chemistry with someone, but don't have sex, either right away or ever. Our brains can often judge how we feel about someone quickly. A 2015 University of Pennsylvania study of over 10,000 participants found that most people make their decisions about attractiveness within the first three seconds of meeting someone. And considering our bodies respond to the signals our brains send out, we might notice some signs of attraction and sexual tension between us and our person of interest.  What you might think of as awkward but fun flirting might be someone else's I'm feeling uncomfortable. So go slowly with these things and be careful about reading the signs. Here are nine signs you might be experiencing sexual tension. Number one, attraction. The first and very obvious sign of sexual tension is attraction.  So a big difference between a friendly connection and a romantic one is the attraction part. So your first question to tell if what you're feeling is sexual tension is to ask yourself, am I attracted to this person? Do you find yourself checking them out? Remember, our brains know these things sometimes before we realize it. Take a moment to think about how you feel about your new interest. It's a good way to get in touch with your own thoughts and feelings before you go further. You might not be able to tell if you're special someone feels the same, but maybe with some of these other signs you can get a better idea. Two, awkwardness. This isn't always present, but sometimes if we're attracted to someone and still figuring out our own thoughts and feelings about them, we might feel awkward. We might be too busy thinking about the right thing to say or we just keep quiet to make sure we don't say the wrong thing. This can lead to a bit of awkwardness until we're a little more sure of ourselves than the other person. But it's important to remember that awkwardness can also be a sign of other things, such as shyness. So while awkward conversations can be a sign of sexual tension, it shouldn't be the only way to judge it. Number three, immediate connection. On the complete opposite side of awkwardness, you might both feel an intense and instant connection to one another. You've probably heard people describe the feeling of having known this person for a long time despite having just met. You might feel that same sense of connection as you talk to them. This chemistry, the way you two just seem to click, could indicate a level of attraction or sexual tension. Number four, similarities. You might have heard that opposites attract, but in some cases of attraction, like attracts like. So in addition to an immediate connection, you might also notice that you and them share a lot of similar features. This could include shared interests, hobbies, or goals. But it might also include having similar physical features as well, such as the same hair or eye color. Scientists call this assortive mating, where we bond or connect with people based on similarities we can observe. So it's possible that those similarities you're recognizing might be a piece of attraction. And it could be contributing to that immediate connection you're hopefully both feeling. Number five, speedy heart and sweaty palms. We usually tell kids that butterflies in the stomach mean we like someone. That's not far off. Do you notice your heart starts racing more around that special someone or do you notice your palms start to get a little sweaty? These are usually signs of attraction. But in a 2022 study that set men and women up on blind dates, it found that those who were mutually attracted to one another experienced heart rates and sweaty palms that increased and decreased at the same time. Why does this happen? Well, it could be because attraction is often caused by hormones like adrenaline and norepinephrine getting ramped up, which leads us to getting excited, hence the racing hearts and sweaty palms. So if you and your partner are both noticing these signs, you might be feeling a touch of sexual tension. Number six, touch that excites both of you. Are you both feeling a little touchy?  This could be the brush of a hand or a light touch on the arm or even leaning closer to one another when talking. If you're hugging, you might notice that you both hang on a little longer than you would with others. When we find someone attractive, we want to get closer to them. And one of the ways we do that is through touch. But the touches are usually ones that affect both of you. You're both feeling some level of excitement from them.  Number seven, you enjoy one another's company. Do you both find that you can't get enough of one another? You can talk for hours and hours and still not be tired of each other. Maybe you realize you feel immediately comfortable with each other or you both feel secure in each other's company. And you might even be a little worried that when you both separate for the evening, you'll miss the conversation or feel like you even missed an opportunity. Sure, some of these signs can also mean you're experiencing a friendly connection, but when you combine it with the attraction part, it's usually a good indicator that sexual tension is occurring. Eight, your conversations are very flirty.   Does complimenting each other come naturally? And are you finding you're both bouncing off each other in the same playful way? If so, this may be a good sign you're both feeling sexual tension.   And number nine, you continue thinking about them after you've separated.  Are you constantly thinking about your conversations or how good they looked in their outfit? Or maybe you're trying to figure out what your next conversation will be. Perhaps you're also thinking about when you'll see them next. If you continue to think about them no matter how much time you've spent together, you might be experiencing some attraction. If you find out the other person is feeling the same, that's probably a good indication you're both feeling sexual tension. Navigating the world of dating and sex can sometimes be a challenging thing. What we think is a sign of attraction can be someone else's shyness or even discomfort. It's important to remember that even using these signs, it needs to be felt on both ends. And while we can't always tell what someone else is feeling, it's good to understand our own feelings about someone else. Sexual tension can be a fun and exciting start to something more meaningful. But only if for sure, both people are feeling that same pull.  Let us know in the comments below. Thanks for watching and don't forget to like and subscribe for more.


Bemidji wrestling hosting a pair of top 10 teams tonight, taking on Anoka first at 120. Seth Newby gets the takedown. He wins 15 to 7 for the first win for the Jacks. At 126, Thade Osborne against Caleb Brandenburg. A low-scoring affair, those were the only points. And at 138, Darren Roth working on a pin finally gets it. Bemidji was down in the match, but they win the last four contests to win by just one point. However, in the second match of the day, Wilmer would beat them 42 to 19. If you enjoyed this segment of Lakeland News, please consider making a tax-deductible contribution to Lakeland PBS.