find out about it versus when the budget was put together? Right. Okay. Right. I should have no money. I know. Okay. But they're building, they're building their whole. Well, I'm not quite, I just, I'm, I'm trying to understand your budget is just the process. Just understanding. Well, not everybody provides this spreadsheet and set up in this way with the BAA ask and then then the budget at any rate, self for me to know in general, you don't need to answer question that I have because I don't have one. It was really of our chair. I can't speak for the administration, but maybe it would be my assumption that when the governor puts his BAA, Okay. He's going to roll up his idea into 24. So we would have to be changed. There's like, we make changes. So it's like, if it's a change that we made in the process and now was over in the Senate would not be reflected because it wasn't the governor's. Right. Peace. You see what I mean? Yeah. So like if it was a part of his. Yes. His suggestion. Sometimes you'll see, you know, it's like part of BAA written in there because they're going to make assumptions that it's going through. Yes. Does that help? Okay. But then we track not only just his stuff that we make check on, Right. But the people coming in are thinking it's in. So we go, well, you know, that was, we maybe chose differently. And then as, as the good directors point it, the Senate will make changes to even our own BAA. No way. Yes, way. Exactly. And so I'm hoping not to take your time. Maybe Wednesday afternoon or so, the Senate will pass the BAA out or give or take. Right. So when that happens, our people will come in and walk us through the changes. And then we'll go, oh, that's nice. Okay. Thank you. Is that out? Yeah. I'm sorry if I took a stand. No, no. Okay. No, it's one to me. I'm sure I was reading these things correctly. And when I, so I love the roll up because it's a nice, because you're, you're, you don't have a lot of line and you don't have a lot of money. So it's doesn't, it isn't like eight pages long. But I'm here though. All of that is on. Yes. Yeah. And I could have been more clear when I was talking about that line by saying that the ongoing, the ongoing expenses are included there. So the hundred thousand dollars for appellate officer contracts is an ongoing need. We are required by statute to contract with a hearing officer who will oversee. Appeals from final decisions of the board. So that is an ongoing expense. Okay. And then the $30,000. And I see. Expenses also an ongoing expense. That's the state vendor for processing the online fee payments. Online. Yes. The payments. So anyone who wants to pay their application fee online. We're assessed. A charge. Okay. So that is an ongoing expense. Okay. And then the $30,000. And I see. Expenses also an ongoing expense. That's the state vendor for processing the online fee payments. So that's the state vendor for processing the online fee payments. So that's the state vendor for processing the online fee payments. I see. So I'll just say a charge for that. Okay. So all of their great representative Brennan. We're going to hear your voice. Could you describe what, what an appeal might look like? Or why? Who would appeal? Would it be a. A license issue or the, you know. Yes, a final decision of the board so far looks like a denial of a license. So if the board, if we receive an application and we find that the business is not in compliance with statute or rule, we would deny that applicant for a license, that would be considered a final decision of the board for which the applicant would have a right to appeal. Also a denial of social equity status. So the board is by statute required to make determinations about applicants who have, who are considered social equity applicants. Social equity applicants are entitled to reduced fees and also payments from the Cannabis Business Development Fund, which is administered by the agency of commerce and community development. So that would also a denial of a social equity status would also be grounds for an appeal. So those are two examples. There may also in the future be appeals of enforcement actions. If the staff recommends an enforcement action, a person who's subject to that action could appeal to the board, the board would issue a final decision about that action. And then the applicant would have, or the licensee would have the right to appeal. So you expect enough, I don't know, you're talking about two positions to. It's yeah, we have two contracts now. We're likely gonna try and have a couple more. This is how OPR has hearing. I mean, many state agencies have hearing officers. I'm sure you guys are very familiar with that. So I think typically agencies keep a number of contractors sort of on, they retain them to a certain extent so they can always have a hearing officer available for an appeal that comes up. So I've got one question on the crosswalk. There's a 0.2683 with no description. And I'm not too sure what that is there. Maybe it's a mistake. It's underneath the FY23 governors recommend. I'm on the detail report. The crosswalk and green and yeah, it's always colored. Oh, I see. You see that? I think it's, I guess it's under, I think it's called 20, 20, yeah. And I don't see the description. And what's the number? I don't have that. 0.6 on page 16. Okay. Right in the middle. I've had to do it. In the special phone crosswalk. I have this weird number. You see it? I see it. It's there. Okay, yeah, that's it. Yeah, I think it's not supposed to be there. I don't think that is supposed to be there. Okay, well, I will make sure that that's not supposed to be there. I don't, do you? Hi, we think we've got something here that's not supposed to be there. Not from here, but we just want to point it out to you. All right, in the crosswalk? Yeah. Yeah. It does work, it does work, it really does. They're too, yeah, they are. 0.2, yeah. Yeah, I think it's just two different things. They are different. Yeah. Thank you. What the heck? Yeah, it doesn't make any sense. Yeah, what's? Yeah, it's supposed to be there. You're not touching. Thank you. Okay. Thank you. I'll keep plugging on. So we're at the per diem and other personal services line. So that's the line where the FY23 Governor BAA recommend the budget is 1.8, and then it drops down to 103. And that's because that was the cost of the positions which moved into the appropriate place. That remaining 103 is the last position that HR has not created a position for us just yet. So that's why that 103 appears there and not in the salaries portion. So the 103. Yes. Must be on this detail detail. Yeah. It's on the roll up. I'm looking forward to it. Okay. It's gotta be in here then. Hmm. Number's up. Where's the 103? Okay. So where does that come from? It's a position that hasn't moved up to salaries and wages yet. So you, so it's gotta be a combination of a couple lines. It will move. Yes. Yes. It's a combination of lines on the, yeah. Okay. Like 200, there's a down. It's the 1 million and seven. I haven't seen this one before, but I don't know. This one. This one's the 1, the 1, 1.7. Right. So what that is is this minus that number. That's minus. Now that's the new recommended to it's down 1.72. So it was moved up here. So this is a, so this isn't new. This is a subtraction from here. A down. Yeah. Yeah. Okay. All right. Sorry. No, I'm sorry. But yeah, that number. We're just looking at it. Yeah. That's what moved up to the salaries and wages. So I'll move on to the operating expenses, if that works. No. I'm on the rollup report, but I'm looking at. So we've got a little bit of an increase in equipment from 5,000 to 10,000. And that is just factoring in the additional positions and the equipment that they will need. I think that the IT and telecom, that the slight increase there is also reflective of the additional positions. We have that 63. So we've got a big increase in other rental for those looking at the rollup, it's line 28. So that increased from 4,500 to 63,000. Okay. Those are the fleet vehicles for compliance agents. I think I mentioned, we've got a seven member compliance team and they are all remote, home-based and they travel almost every day to go to do site visits and compliance checks. And obviously that wasn't there before because we just had our first sales. That's right. So our property and our, let's see, our property rental is just under 100,000. We are at 2889 Main Street in the city center building. We're in the old Green Mountain Hairboard space. I understand it's a very expensive space. This is where BGS has put us and that is where we are right now. I have, and then the other expenses for supplies and travel. Happy to talk about those. We typically go annually to the Cannabis, the Cannera conference, which is a national association of cannabis regulators. So regulators from every state in the country or a part of this organization get together and talk about best practices. So they typically do attend that conference every year. So those don't roll up to 291, which would be a part of the public. Yes, sir. Sorry. I have a question that's irrelevant, but I can't resist. When you go to the conference, what do you discover about the culture of your organization versus others? That's great. I could talk about it. I could give you an answer to that question that would go on for a long time. It's very different. The Vermont cannabis regulatory culture is quite different from a lot of other states. And I can just tell you briefly that it's mostly because of the enabling legislation. The way that the legislature crafted this market is quite different from every other state. I think I've said this before. We have our proportion of small cultivators is the vast majority of our licensed businesses. They are operating, they are growing on a thousand square feet of space or less. That wouldn't even qualify as a micro business in almost every other state. So the way that most other cannabis regulatory agencies operate is they've got a handful of major operators that have quite big grows that are satisfying the demands in other states. So I don't want to say it's every state. I know that Massachusetts and California, they do have small and New York is now getting online with microprocessors and micro grows. But so far nothing really has compared to what we are doing. So it's quite different. And it was by choice. It was very much like, but very intentionally choice to make sure that we have, if I remember the conversation was that farmers in Vermont who might want to have a part of their crop be this, not be actually want to say out licensed by somebody coming in and buying, just being a giant production because there's only so much product that can be produced in a year because you have to sell in Vermont, what's grown in Vermont. So one very large place could end up being the monopoly of it. Right, so what the legislation did is it provided that everybody can only get one of each type of license. So there's no stacking of like multiple cultivation licenses or retail licenses. So there's really kind of an anti-franchising provision in the enabling legislation. So that's it. And we made some statements like through law around what a city or town can when they can say, what they can say no to, they can't because it's a different agriculture. Right, town. So it's a little bit separate for the types of. You have to be able to vote to have it sold in your town but you can't say no to being grown. Did you say that it has to be produced here? It does, yes. All cannabis has to be produced within the, it's like we've got an iron curtain. And we didn't run on the interstate commerce problems with that. No, there are, I don't know how well everybody is following what's going on in other states but in California, the cannabis regulatory agency just asked the attorney general to weigh in on whether it would be a violation of an, whether it is a violation of interstate commerce. The interstate of California, I know how that will put it. Representative Jolan, did you have a question? Yeah, I mean, interestingly enough in Vermont, in Weitzfield, we have two retailers and like a laboratory, what do you, what's the other license? Oh, a manufacturer, manufacturer, but two separate in different locations retailers which I guess in my mind, I had assumed it would be one per municipality. And you can probably see this in another. We do, yes. So I think something interesting that there is an opt-in provision, which we opted in, but I guess that one location did necessarily mean one. Yeah, the one one municipality. The one location rule applies to the licensee. So each licensee can operate out of one location, but a town was opted in can host multiple establishments. And given that opt-in provision, there are some interesting dynamics happening with how the retail operations are dispensed across the state. For example, Burlington, I think has eight licensed retailers now in South Burlington has zero because they haven't opted in. So I think, I do think there may be some sort of density or maybe density of retailers around the state given the opt-in provision. I think we have one, take one. You have one in Middlebury for Gens, we have three, I don't remember them all. I used to, I know they were just starting, they were just growing. It was the first weekend in October. One other thing that the enabling legislation did just to keep answering your question is that there's a requirement that the board prioritize people who are coming in from the illicit market. So the intent was really to make sure that the illicit market growers could join the regulated market so that we didn't keep them up. And that was another reason why I think our culture is quite different than other states. That was, again, also a choice, because they work and it would, we've incentivized for them to come to an open business. Anything else you need us to hear? I don't think so, nothing else from me unless I was happy to answer questions. So your total budget up is up about, is 1.2 million from last year, right? Right. And it comes from the cannabis fund. All right, thank you. Thank you. And thank you for helping to, I'm sorry, zero, yes. It's gonna, the eight fifties in it, I think, I think the chance that this shouldn't be about the lab work. That's not the 50th, it doesn't, that they were gonna respond to the fact that there was no, it's not to the product. If that needed to get passed, but right. So committee, you're still live and I think we can go offline and we'll see you tomorrow at 8.30 in here for,


Hey, this is Brett and this 2019 Ram 1500 quad cab six foot four inch short box is stock number 13676Z. I'm here at Summit Automotive in Fond du Lac, Wisconsin, you're new and used light duty truck and Ram headquarters. This 2019 Ram 1500 has the 5.7 liter V8 Hemi engine. It's paired up with the eight speed automatic transmission. This truck has been fully safety and inspected by our service shop per the state of Wisconsin inspection process. It has a fresh oil and filter change all the fluids have been checked and topped off and it is 100% ready to go. I'm going to go all the way around in this video inside underneath, start it up and take a look under the hood to give you the most accurate representation of the truck possible. Delmonico red pearl coat is the color and I shoot all my videos in 4k. So if you have HD capabilities, turn them on now because it is your best way to check out the quality condition and options of the truck before seeing it in person. Going down the driver side, the box looks really good. I didn't see any major dents or dings on there. The cab and doors all look really good as well. No major dents or dings or scratches looks really good. Front fenders in nice shape. If you like the video, you can subscribe to my YouTube channel which is youtube.com slash Summit Auto. Click the bell notification to get updates on the videos I do each and every day. Here at Summit Automotive as well as having access to one of the largest walk around videos of vehicles on YouTube. 20 inch factory alloy wheels with chrome covers and it has Nexon Rodeo and HTX tires. These are 275, 55 are 20s. I would say they have right around half the tread left on them and the frame and under body is in nice clean condition. Then around the front, you get the painted front bumper. Believe this is a big horn package with the sport appearance package would be my guess. Get the blacked out grille, the painted front and rear bumpers, factory fog lights, no major dents or dings on that front bumper. The hoods in pretty nice shape as well, passenger side front fender, no major dents or dings and the passenger side front wheel, no major scuffs or scrapes. As you go down this side of this 2019 Ram 1500, take note of how clean that body is, how reflective and mirror like that paint is. I take these HD videos so if you are far away or even if you're close by, you just cannot make the trip down but you're still interested in purchasing the vehicle. You can see the truck, hear the truck and have confidence in what you're looking at before you even get here. So when you do get here, there's absolutely no surprises and you can make a smart and informed buying decision from wherever you're at. If this video helps you make that buying decision, let your salesman know that you saw the video that it was helpful and that Brett sent you. Back wheels in excellent shape and the back tires have just as much tread as the front tires. Let's get framed under body, looks really good. As the nice wheel to wheel side steps and the lower rockers and cab corners are all in really nice condition as well. I didn't see any dents or dings on the passenger side box and the rear bumpers in really nice shape too. Full towing package which includes receiver hitch 4 pin and 7 pin wiring, dual rear exhaust with the chrome tips, tailgate is in really nice condition as well and it comes with a trident soft roll up tonneau cover. I didn't see any rips or tears on there and it has a very nice line X spray and bed liner. That's the bed liner I got on my truck, I love it. Goes on to the tailgate which is really nice and that shuts nice and solidly. For full disclosure this back wheel is in really nice condition as well and down the rest of this side, once again doors and cab all look really good. Has the heated power mirrors, blind spot mirrors, built in directional signals inside, the big horn package.  Get the nice cloth bolsters and wood grain trim on the doors there, back seats are just as clean as the front seats, no rips or tears back here it does have the latch child safety system for any child car seats you may have, power sliding rear window with the built in rear defrost, smells very clean inside this truck I don't think it's ever been smoked in you get a nice flat floor back here as well, child safety locks on the back doors and the inside and bottoms of the doors all look very good, we'll hop inside check out the miles the radio and everything that this truck has to offer on the interior, see this one has 22,466 miles you do get the digital speedometer and compass display comes with the leather wrap steering wheel, cruise controls and gear selector on the right, Bluetooth and information center controls on the left and audio controls on the back of the steering wheel, you get the eight speed automatic transmission, the push button four wheel drive, the push button start, the 8.4 4C radio has all your controls here AM, FM and Sirius XM radio capabilities, if we go back to the controls, get the backup camera there which is nice and crystal clear they made those HD and you can zoom in on your receiver hitch to get hooked up to your boat camper or trailer the first time every time, you also get all your different apps on here this one does have projection manager you can project your cell phone to the screen via Android Auto or Apple CarPlay so while this one doesn't have the factory navigation system you can project your phone up there with Android Auto or Apple CarPlay and if you got Waze or Google Maps it's like you have NavRide in the screen so that's a pretty sweet system we actually did demo on Android Auto if you want to check that out that's in the upper right hand part of the screen down here your more tactile climate, volume and tune controls, factory brake control or stability control, tow haul mode, you do get a CD player, two USBs, two USB C's and an Augs jack, coin holders and cup holders as well as cell phone holders and keyless entry with remote start, passenger side format and seat are in really nice condition I don't know why the backrest is all the way up but still nice condition and I'm sure the backrest goes down, headlighters in great shape, map lights, power sliding, your window button and your home link buttons for your garage door security systems and lighting systems we'll start it up and take a look under the hood starts right up no check engine lights or anything like that you also get an outside temperature display I would personally like to thank you for checking out the video today and hopefully from this HD video you've been able to verify the quality condition options and cleanliness of this truck all the way around inside out all those lights are working nicely under the hood we have the 5.7 liter V8 Hemi engine engine base very clean runs very smooth once again this truck has been fully safe and inspected by your service shop as a fresh oil and filter change all the fluids have been checked and topped off for the state of Wisconsin inspection process and there's the emission sticker this truck is 100% ready to go and to see more pictures of this truck or one of our other 550 new and used cars trucks, SUVs, minivans, wranglers, half tons, two quarter tons, one tons you name it we got to go to that website right there summitauto.com full pictures and descriptions of every single vehicle all at summitauto.com and if you'd like to check out more HD videos you can go to youtube.com slash summitauto click the bell notifications you'll get updates on the videos I do each and every day here at Summit Automotive in fact in a second you'll see a link to subscribe my YouTube channel the upper left link to more Ram 1500 truck videos like this one the upright link to this vehicle on our website and lower left and a link to one of our latest YouTube videos in the lower right click those check us out we're super excited to help you with this ultra clean 2019 Ram 1500 quad cab six foot four inch short box big horn level one in Delmonico red pearl coat thanks again for checking out the video remember to like subscribe and share on the YouTube channel I really appreciate it thanks again


In recent days, markers have shown up at Six Flags Magic Mountain around the area of Tidal Wave and formerly Green Lantern First Flight. There are a couple theories going around about what could be coming for this area of the park in 2021, and I want to discuss what I would like to see come to Six Flags' Star Park in the future. First, I want to shout out Theme Park Obsession on YouTube as he made an incredible video that really goes into detail about some of the construction work that has occurred here, and even gives us a detailed view of where all of the markers have been placed. Really good stuff to check out if you want a really good idea of where this is all happening. It definitely helped me out a lot as I haven't been to the park, so I'm not familiar with the layout whatsoever. Link to that in the description. Anyway, getting into the good stuff here. Many people are suggesting that the fact Six Flags is starting construction so incredibly soon after opening West Coast Racers means we are looking at a huge addition here, and even Six Flags Magic Mountains 20th Coaster. If you weren't aware, all the way back when West Coast Racers was announced in 2018, that was intended to be Magic Mountains 20th Coaster, but they ended up having to remove Green Lantern First Flight and Intamin' Zachspin, and it is now being reconstructed at La Ronde. For all of you out there who aren't math wiz-es like me, that means West Coast Racers only makes 19 coasters for Magic Mountain. It seems like Six Flags would want to keep up with Magic Mountains title of throw capital of the world by continuing to invest in high-throw rides, and having 20 coasters would be a great way for them to cement that standing. I think the park will be receiving a new coaster sooner rather than later, especially given how long it took for West Coast Racers to finally open, so I do believe that we are going to be looking at yet another new coaster for Magic Mountain for the 2021 season. But what kind of coaster exactly? There are two very common rumored types of coasters that may be in store for Magic Mountain. Both of these models have very few iterations in existence and are both extremely different from one another, but both would make sense for Six Flags Magic Mountain, and Six Flags does have somewhat of a history with both of these models as well. The first of the two I will address is the rumor of a Mach Power Splash style ride. This could be the one seen at Six Flags over Texas for 2020, known as Aquaman Power Wave, or it could also be the new variation just unveiled by Mock Rides called the Power Loop, which actually features a huge vertical loop in the middle of the course. A lot of people are pointing to this new Power Loop model as it seems like something that would fit in very nicely at a park like Magic Mountain. I don't disagree with that, as it gets very hot during the summer in Valencia, California, and title twister has been rumored to get the axe for quite some time. If that happens, Magic Mountain could definitely probably use another water ride to help beat the heat during the summer. Correct me if I'm wrong, as I am not aware of what water rides exist or don't exist beyond title twister, I do know the old log flume was removed for full throttle, so I'm guessing Magic Mountain would need some kind of new water ride. This would be perfect to cover two different types of rides with one addition, as it would give Magic Mountain their 20th coaster and also give them a modern thrilling water ride as well. I am aware that the Power Splash going to Six Flags over Texas was originally intended for Six Flags Dubai, and once that park fell through, they just had that sent to over Texas. So maybe another one of these isn't as likely as we think. I wouldn't pick this option myself just because I don't do water rides typically, but there is another option people are mentioning that I think could be more likely that I also think would be incredible at this park. Amusement Insiders released a video explaining how the markers seen at the park are lining up with the layout of Jersey Devil, which is great adventures new for 2020 RMC Raptor single rail. I will link that video in the description as well. Based on some information and a map made by SuricatXN on Instagram, which is shown in that video, the markers appear to line up with Jersey Devil's layout, suggesting that Magic Mountain will be receiving a clone of that ride. The two Raptors that we currently have seen open already are mirrored clones of each other, and RMC has stated these were meant to be cloned rides as well, and given Six Flags history, it would be no surprise to see a clone come here. They are simply easier and cheaper to build, and I don't think there is anything wrong with cloning coasters, especially when we are talking about a thousand miles of difference and being such a well-regarded ride. Surely Jersey Devil will deliver, and could even possibly be the best new coaster of 2020, so I would not complain seeing more of these built. These kinds of rides are so much better to see put in everywhere as compared to clones we used to see at every park such as the boomerangs and SLCs. Definitely a huge step.  It seems like they are maybe trying to avoid the problems with West Coast Racers and starting really early to ensure that doesn't happen again. Which model do you think would be a better fit for the park? I'm sure most enthusiasts are going to pick the RMC Raptor clone over the power loop, but I think there is a lot going for either option at Magic Mountain. Whatever ends up being though, everything is finalized and ready to go seeing as the park is diving head first into the construction of this project. Let me know all of your thoughts about this and what it could mean for the future of the park. Thanks for watching and be sure to subscribe if you enjoyed and like my page CoasterDaddy on Facebook and follow me at CoasterDaddy official on Instagram. This is CoasterDaddy. Bye!


And we even have Outer Island Bardsmen coming in as well. And then right now we're just making sure they're done. So today we have hamburger with mushroom and swiss cheese. So I'm just sauteing the mushrooms to get a nice color on them. This kitchen is called the Baymark AT. If we are food feeding, we use this to cook all our meals out of. So we have our grill. We have our burner. We have our ovens, fridge, and our cherry ration heater. This cooks like all our UGRs. We use this a lot when we are doing exercises or we don't have an available dining facility to cook out of. It's basically all you need out of a dining facility kitchen, all your necessities. You cook your breakfast on top of this grill and we can make our burgers on here as well. We prep our meals and then we cook. Make sure we're progressive cooking so we have hot meals. And then clean up and then do it all over again. I actually really like this job. Right now we are servicing the Army Guard their exercise. And we have about over 200 soldiers to feed. And then we even have outer island guards coming in as well. And then right now we're just making sure they're not eating MREs or UGRs. They actually have good hot meals to eat. So it is important to have this mobile kitchen because not everywhere we go has a deep dining facility. If we're on a bare base, we would need this to ensure we give out hot meals and make sure everything is in temperature. Being at the field is not always the best thing. So everybody loves food. So that's the only way we can boost up the morale here. And everybody is ready for the mission.


So, I have chosen here to talk about less unknown protein protein interactions and this is of course, a little bit where you're coming from, what is less unknown. But I think that what I picked is really less unknown, but let's see. So the first slide here is also the motivational slide. I think that many of you who are here today have already have an interesting protein interactions. So if we look first here in the living cell we have actually a lot of proteins and it's a very dense environment we have up to 40% of the volume is occupied by things other than water. So there, there will be a lot of interactions between proteins and of course also other matter. And this is important in this setting also when we are talking about antibodies and solution. For example formulation of vaccines you want to have a high dose of antibodies, and there will be strong protein personal interactions. And how to describe them how to control them is very important for example the stability the viscosity of the samples and so on. And also important in, for example, food formulation where you can use proteins to control taste texture and mouse feel and so on. And lastly I'd like to to mention that as a background we have actually a very complicated matrix of ions so we have water and electrolytes, and that also plays a very important role on the protein protein interactions. I think a very good example is the pH that we often use as a handle to control protein interactions, and it's part of this background, but we'll talk a lot more about that today. So, let's think about when we say protein protein interactions. What do we actually mean. So here I have two proteins and we are obviously interested in how they interact with each other. And here we have to remember that it's not just proteins that we have to talk about, because around the protein, we actually have water. So we also need to consider the protein water interaction. Because there are now waters we also need to consider how waters interact with each other. This is a very important feature of any aqueous solution the water water interaction is very dominant on how the, the solutes they behave. We could even start to add co solutes. So this could be small molecules or salt. And they will of course also interact with the protein with themselves and with the water. And as I just described the solution we need to take into account all of these interactions we cannot skip any of them. So it's really an effect of all of them. At the same time we have to remember that often the systems we are interested in, we have a temperature in the system so they are not static they move around they rotate the diffusion so on and we need to capture that as well we need to be able to describe all of that simultaneously. So, if we consider non covalent inter atomic interactions if we really go back to basics and think about how can two atoms interact with each other, and it turns out that they're not so many ways we can think about. And basically, I would say an important part here is that all atoms they have an excluded volume and that can be some electric interactions on top of that. And by electric interactions I mean two things, I mean that I can be dispersion interactions and that can be cool on big interactions for example if we have an iron. And that's, that's about it. So we have very few interactions to, to actually take into account. And all of this, then builds up a lot of more intricate interactions we could say. So, now we have our two proteins over here and we consider the interaction between two proteins to two atoms. And we consider that we embed this into some background here of typically an equal solution. So, let's see what happens now. So, from these fairly basic interactions over here we can now describe and talk about a lot of more complicated interactions that I'm sure that many of you will have heard of before. We have hydrogen bonds hydrophobic interactions. That's very much related to for example what I mentioned earlier the water water interaction. We can have crowding, which is an entropic effect we can have screening due to ions. And sometimes if we have multivitamins we can have iron condensation over charging and so on. And now we come to three parts that I've marked in green down here and that's actually the ones that I want to talk about today. I'm going to talk about charge regression, Hofmeister effects and van der Waals interactions. I think that the, the last one van der Waals interactions I don't think this is really belongs to less unknown protein protein interactions because it's something you will find in any biophysical textbook. But I'd like to just highlight a few things that I think could be less unknown, but we'll get back to that in the in the end of the talk. That's the outline. I cannot really talk about all of this because it would be a very, very long lecture then. The idea is that these three parts they are fairly separate so I'll just try to explain what what these different effects what they are and how we can describe them. And I've tried to do this in an kind of an educational in an educational way it's not going to be much research that it's going to be a little bit about what we've done in the past. And I have a little bit of on published results but most of it is just descriptions about these mechanisms and how they could affect protein protein interactions in solution. So, when we have the possibility to describe all these effects. We can do that with, for example, computer simulations and this is what I do normally. So that's why I'm just highlighting it here. So here we have a box of proteins. And by taking into account interactions between individual particles we can then capture all of these, all of these effects that we have here. And once we describe them in simulation we can then start to calculate properties, and many of these properties that you could go out and measure in experimentally could be there's multi pressure view coefficient structure factors and so on. So, we can get information from the simulations from microscopic picture and relate that to the experimental data. So we're trying to talk, not to talk too much about simulations today but mostly about mechanisms. So, with that short introduction I think we can get started with the first topic. And that's going to be charge regulation. And I start off a little bit morbid here because these are the you see to really very highly influential physical chemists, John gamble Kirkwood and last on saga here buried next to each other and you can see here that Kirkwood was a very he made a lot of contributions including charge regulation that's why I mentioned, mentioned him here and of course last on saga a bit, a little bit more modest. It's just noble or it, and then asterisks, etc. here. And they were contemporary these two. Kirk would never got the noble price but I think it could be because he, he passed away, very relatively young I would say. And he was the, he was one of the first to describe the charge regulation mechanism and let's just try and go go through what, what that means. So, this is an effect that arises due to protein fluctuations. And what we have here is a is a molecule and it can with with some titratable sites and we have for example, an acidic acid and an amine, and what they will do that they will be in equilibrium now with the protons and the solution. So that there will be a fluctuation of their chart state. So at any given time we can come and have observed the site, and we will either find the proton or it will be free. And I think that, for example, up here the decide will will estimate between being in a being an anion or be neutral this one from neutral and being a cat ion. So, the charge distribution is not static does fluctuating. And that's the origin of the charge regulation mechanism. And to see this, we can see what happens when we take two particles, two molecules and put them next to each other. And this means that when we do that the judge distributions they can now become correlated. And that means that the will have a lowering of the free energy. For example, let's say that this side down here could be a carboxylic acid. And this one up here could be an amine. So this one can alternate between zero and plus one and this one zero and minus one. There will be a correlation between these so more frequently would find that there would be a minus and plus combination because that would lower the electrostatic energy. So all of this in total lowers the free energy. And this means that a new intermolecular interaction arises due to that. So, this was discovered a long time ago by Kirkwood and Schumacher. This paper from the early fifties, but actually turns out that the charge regulation mechanism was discovered, actually a lot earlier, 30 years earlier at my Kyle Linnaston long. The interesting thing about this is that comes from the Casper laboratories. And at that time they were very interested in describing proteins and the physical chemistry of proteins. Kyle Linnaston's boss. So he was the one that determined this that is the pH concept. So all of this in sort of an in trying to understand enzymes and proteins for production. So, if we continue a little bit, I'll try and keep the equations a little bit down in this presentation to what I have a little bit here and I think it's important to understand the charge regulation mechanism. And here we have a multiple expansion so here we have to imagine that this is one child's distribution is could be a protein and we have another protein macromolecule over here. And we can now express the electrostatic energy between these as a function of the mass sensor separation here. And this, this, this is a fairly easy to do. And if you do that you get something that looks like this multiple expansion, where we can express here the free energy of interaction as a series of terms and the first term is something that we all know quite well. And that is the iron iron or monopole monopole interaction. So what we have here is just the net charge of one of the distributions times net charge of the other one divided by the distance. So that's like a coulomb interaction, but from macroscopic bodies. This is the reason why we say that if we have to like charge proteins they will probably repel, and you could predict that using a multiple expansion like this. But there will be higher order terms and they are coming up down here. So for example, if the charge distribution is uneven there will be a contribution from the iron with a dipole with a dipole and dipole and iron quadrupole and so on we can continue this expansion out here. And you can see for the iron dipole it decays as one over R4. I should mention that the results here they are actually angularly or average so we have allowed the charge distributions to rotate as well. And you can see that the case as one over R4, this one as one over R6 and so on. Normally when we do these multiple expansions we keep the charge distribution fixed, we assume that there are no fluctuations but we've just argued that there are fluctuations if you have titratable groups, like capoxyl acid acid and amines and so on. Performing this multiple expansion and not normally if the charge distribution is constant these terms they cancel out, but if not, then we actually get extra contributions, and you see that they come in here right after the iron iron terms so we have an induced iron induced iron induced iron. And we see here that they decay as one over R squared so they are relatively long range. And we can see that they depend here on the charge on one side times what we call the capacitance C on the other proteins and so on. So, even in the case when the proteins are completely neutral, we can still have an electrostatic attraction between them as we see here so if this term with net charge is neutral. We can still have this term coming up here and that's simply just due to the fluctuations of the of the protonation states. To to use this, I think it's important that we try to describe what the capacitance is the C, and then this I'll do in the next slide here. So here we have the iron induced terms in the multiple expansion and they contribute to the protein protein interaction and you also see that it's always a negative contributions this means it's always attractive. The capacitance here, you see that this measures the fluctuation in the net charge, and that's always going to be a positive number. So the contribution to the protein protein interaction is always attractive. And it turns out that the fluctuation in the net charge here. It's actually very easy to obtain experimentally because it corresponds to the derivative of the pH titration curve. So if you have measured the protein charge as a function of pH, we can take the derivative of that function, and we get the capacitance. So with this we can obtain from both experiment but also from from theories and simulations it's quite easy to obtain this property. If we do that. Sorry for the very monochrome image here I hope we can, we can read it. So we have here we start with the pH titration curve. So charge versus pH, we take the derivative and then we end up with curves that look like this. So here we have it for NMR that's the circles, and then we have a null model an ideal model where we just consider the sequence of the protein so we can predict okay we have, we have a lysine here and we as we have pk value and we can then take the derivative of that and getting the capacitance like this. And the thirdly, fully drawn lines that from our Monte Carlo simulation. And we don't need to go into too much details about this other than know that we have here at low pH and at high pH we have peaks. And this corresponds to that we have a lot of titratable sites which are in their pH is close to their pk value. And this means that they can fluctuate a lot. And this is very typical for most proteins they will have peaks here on pH for corresponding to aspartate and glutamates and then up here in the high region we have license arguments and so on. In the middle, we typically have lower capacitance, except if you have a lot of histidines, then we can have higher capacitance up here. We see also that we can use this kind of information to judge whether the charge fluctuation attraction is at play or not. Because if we have a very low capacitance, this means that these pre factors here are the, if they're small then this is going to be a small contribution. So, we can use the concept here to determine if this is important or not. So, here I'd like to show how how the fluctuation force can influence the interaction between two proteins so here we have Lysosine interacting with calbinding so this is done using computer simulations. And we can do that with is the with static charges so it means that we just assume that we have a constant charge distribution which is, which I would say is common to do in computer simulations. And we can include a fluctuating charge. This is this we can do in Monte Carlo simulations by simply alternating the charge state. You just have to do it in the, you know, in the right way. So we keep a constant pH. So comparing these two here we have the mass and separation. And here we have the angularly averaged interaction free energy so the potential of mean force. And we can see that we start to we go from a purely repulsive system to something where we start to see a retraction. And this is due to the fluctuations of the charge. We can study this a little bit further and have a look at what happens here with the charge of the two proteins. So here again we have the mass center separation and here we have the charge of Lysosine charge of galbinding. We see that Lysosine doesn't change much. And there are two reasons for that. One is that it has this condition it has a very low capacitance. And secondly, the charge of Calbindian is fairly low so it doesn't induce as much charge in Lysosine. The other way around though this has a Lysosine has a high charge and Calbindian has a high capacitance so here we get a big response in the charge distribution so we go from 1.5 But at far separations into actually shifting a sign when we are close separations. And that's the charge is simply because we can, we can release protons as the two proteins they approach each other. And this is not just something that happens between proteins and proteins it could also be approaching a surface could be a membrane could be could be DNA or something like that. A mineral surface and you can have these effects. It could also be a small iron phosphate for example could approach protein and you could imagine that phosphate would also see fluctuations in its charge because it has PK values close to pH seven. So, that's, that's the charge regulation mechanism and just to empty section we can talk about the capacitance. So, what that is and I would say that it's an intrinsic molecular property, and it's very similar to how you would talk about the net charge and the dipole moment it enters the multiple expansion. It's a property that you can calculate for a single protein and then use that to say something about how, how proteins interact together. It measures the fluctuations in the charge. And this is in a way, how easy this to distort the proton equilibria of the touch radical groups. And it's something that's since in proteins we have a lot of touch radical groups is something that can easily occur in proteins. And it's strongly dependent on pH and the PK values of the touch, touch radical groups at pH seven we often have only small effects and that's because we have no groups tight trading there except for the histidines. So, so they can become important contributor contributors to charge regulation at pH seven. Other than that we have to go to either more acidic or more alkaline pH to see the effect. We can obtain it either experimentally or from calculations, just the derivative of the proton titration curve. And we can see that it answers the free energy between macro molecules. So, that's a little bit about the charge fluctuation mechanism. And I think this definitely belongs to the lesser known mechanisms it's a, if you if you go back in time you can see that this has come up. You know, maybe a 20 year interval so sometimes it's get some attention and then it kind of dies out again. So it's a it's a little bit. I would say in the obscure in but I wouldn't say that it's unimportant. And we can. There are now more and more methods in molecular dynamic simulations methods where you start to include fluctuating charges and there I suspect that we will start to see these mechanisms again. So let's move on to the part two, and then this is about iron specific effects, and then also sometimes known as Hofmeister effects but I would say more generally and specific effects Hofmeister series is a, is a, is an early discovery about irons. And it's a limited effect I would say. And the, the very nice picture here is done by Stephen Hansen as a former student and we are trying to use this for journal cover we don't know if they will accept it yet, but what this is about this is a cup of coffee. And what you see here in the phone. That's actually a caffeine molecule that we are investigating, we're investigating how the thermodynamics is affected by these. Let's call it ionic candy lying around here on the, on the plate so these are different ions, and they will affect the caffeine molecule and the caffeine caffeine interactions significantly. And we're trying to study that using experimental computer simulations. We're trying and go through the iron specific effects and I'll try to, I'll try to outline the sort of the major effects that's behind these observations. And this is something that has been, I would say fairly recently has been unraveled. This has been a puzzle for a very long time. But I think we know now, pretty much what's going on, but I will try and explain it. This is a series here was discovered by Franz Hofmeister. And what he did was to take different proteins and look at their solubility as you add different sorts. And what he observed was that the depending on what sold you added, you would have a different solubility is basically. And here we have a list of anions and cations and they are ranked according to how well they, they sold in or sold out the proteins. But it's important to note that when he did these discoveries, it was not at all clear that souls would dissolve into dissociate into distinct ions. This was highly controversial at the time. So this is something this is a newer formulation of his series. But back then when he did it he, he just took sorts he didn't know that they actually dissociate. And to show you what happens, I will show you some experimental data that we illustrates that this is a very strong effect. And here we have a osmotic second vehicle coefficient data measured using light scattering. And down here we have the salt concentration and here we have the video coefficient that's it's presented like this one minus the video coefficient. And then this is done in our different potassium salts. So from chloride bromide nitrate iodide and thiosynate up here and we see that side sign it here means that this induces a very strong protein protein interactions, whereas much less interaction is induced using a potassium chloride. And that, what is even more puzzling is that if we cross the isoelectric point of the protein, then these half master series they reverses. And the molecular level explanations for this, they have really puzzled us for a very long time but as I said, I think that most of it is known by now. And I would say that there are two main mechanisms that control this and I'll try and go through them. So, the mechanism behind the specific effects. One of them is that we can have ions that can bind to the macro molecule. Here we have this is this is a model we made now quite some time ago and we use molecular dynamics to do this but you can do the exact same experiment on proteins and you would see basically the same what I'm about to, to tell you now. So the green ions they are small and ions and the red ones they are big and this could be for example, fluoride and this could be iodide, let's say. And now we have macro molecule here, where we have put some cat ionic patches on the surface. So, these are plus one out here and then this part here is has no charge so it's a polar. And we embed this in explicit solvent so everything the solvent molecules are there. And we now just look where do these iron distribute on the surface of this artificial macro ion that we have constructed. And it looks something like this. And that there is a really a distinct absorption of the small lines they go to the charts patches, and then the big iodide sized ions, they sit here in between in the much more a polar regions. You could talk about that these are more they have a more a polar character these big irons and make sense because they are, they are much less solvated than the small fluoride ions here that would be really highly solvated they will sit the water molecules would sit very tight. And if we, if we transfer this to protein surfaces, of course gets much more complicated because the salvation, if you, if you scan the surface of the protein you will find that the salvation of the protein surface varies a lot. We can have cat ionic and an ionic groups, and then we can have polar groups we can have a polar groups, and all of that will have a special affinity to differently sized ions. So, the binding of ions is really an important. Driving force for the for the half master or I'm specific effects as a rule of thumb, we can, we can use this principle called the law of matching water affinity is not sure it here. Here we can see that ions of like sized. So like sized ions cat ions and anions they tend to pair together they form strong iron pairs, whereas similarly sized ions. They tend to stay away from each other, you can see this in electrolyte solutions in the activity coefficient you will find this kind of behavior. And we can translate that to the surface of macro molecules like proteins. And this is the reason why the small, the small ions they sit to the attracted to these blue patches here because they have similarly similar size. So that's the reason why they accumulate like this, whereas the big red anions they sit here in between they're not so so happy and and all of this is due to the salvation properties of the ions versus where they bind. So that's one mechanism behind the, the half master series. And the next one is the opposite. And that's exclusion of ions. And a typical example of this would be sulfate. So what we have here is we have for example, highly solvated. I am like sulfate. We, in order for it to approach this poorly solvated macro molecule I have over here, then they would have to let go of some of the salvation layer in order to get into here. So that's, that's an unfavorable process so in order to reduce this excluded volume that we have around this molecule and here, one way to exclude that and gain more entropy for these ions is that we can see simply push them together. So this is a sorting, sorting out effect. So the salt is pushing the particles together like this. Another way to view this is that you could say that there is an assault effect on the hydrofluoric interaction. And that effect is so specific. More weekly charged ions, like these ones we have up here, they are quite happy to enter this region we can see that up here so they can actually be absorbed on this a polar surface, especially if we have some charged patches as well. So it's really iron specific effects, both of these binding and exclusion of ions. So from a theoretical point of view, if we want to make models where we can treat, for example, hundreds of particles of macro molecules, this is really quite difficult to capture these effects because it's so it's so connected to the to the salvation of the iron and of the macro molecule. And if, for example, we have implicit solvent models, and we lose all this information simply just isn't there. So we have been working on ways to incorporate that into into our models, even in systems where we don't even have explicit ions, and I'll try and show you how that can be done now. So, one model we have and we are working on at the moment is is one where we're using solvent solvent accessible surface area approach. And here we have two proteins, they're close together and what we can do during a simulation is that we can. We can for that particular configuration we can now measure the solvent accessible surface area per residue. And this is a quite expensive calculation but it's, we can do it it's part of the we can have it as part of the Hamiltonian. So we are on every step in our simulation we calculate this sasa for each residue. And what we do is that we let this change in the in the area we let that be part of the Hamiltonian in what in what I've written here we have a parameter here that is sold specific and it's amino acid specific. And we then multiply that with the salt concentration over here. So, what this means is that, depending on the sign of this epsilon, we can either favor or penalize the creation of area in the between the proteins. So, we could, we could introduce attraction between the proteins or actually keep them apart. And if we keep them apart that would correspond to an iron binding to the surface, or if we push them together that can be an iron exclusion to the surface. I should say that we have other ways to include iron binding, but I will not talk about it today. So, this method here is mainly for treating exclusion of irons and how they can push the particles together. But it's a fairly expensive methods as of now. What we've done is we've tried to apply this on a small molecule caffeine. And this is what I've illustrated here. So, here we have a caffeine molecule and it has some distinct motifs, and we have coarse grained it like this. And now we can simulate many of these caffeine molecules together using our many body assessor Hamiltonian that has described. So by assigning. I go back once, sorry. Watch that once more. So, if we assign these epsilon values to some of these motifs, we have some idea about based on all that molecular dynamics we have some idea about how we wear the ions bind how they excluded. We can construct a model that can take into account on specific effects on caffeine. And really the reason why we're looking at caffeine is that it's, it's a proxy for a small amino acid, and we would like to extend this so that we can describe peptides and proteins as well. So, first, if we look at here we have calculated the osmotic coefficient of just caffeine in pure water as a function of caffeine concentration. So, caffeine very quickly starts to, to interact with itself and it can form, can form stacked configurations so it's, we cannot go to very high, high concentrations of caffeine because it's simply not soluble. So this is just to test our model in a in pure water and using the Hamiltonian here where we use sasa to describe the ion specific specific effects. We can then look at the change in the excess chemical potential as we now increase the salt concentration. And here we have some experimental data and we try to, to compare that with our Monte Carlo results. So, so that's, that's ongoing work I would say in trying to incorporate these effects and see how molecules are being affected by different sorts. Right. So, that's a little bit about ion specific or Hofmeister effects and then now we come to the, to the last part where I would like to talk about some a few things about fundamental interactions that I think is perhaps not so well known in the protein field. It's very well known in the colloidal field. And if some of you are from coming from there then then you will probably have heard about this before. So the image we have here is is actually painted by one of our collaborators Cliff Woodward from Australia and I just realized it looks like I put an earring on on one of us but it's actually because it's taking from a book cover. And then there are some some atoms, atoms on the side. But, nevertheless, let's continue with this topic. So the Van der Waals interaction is actually exactly a sum of different mechanisms, and we have three different mechanisms called dispersion interactions we have dipole dipole interactions between, if you have for example small polar molecules, and we can also have dipole induced dipole interactions, if you have something that's internally polarizable. Common to all of these making these interaction mechanisms is that they, they vary as one over six. So the decay, the interaction energy decays quite quickly with the distance. And because of this, it's, it's common that you will see them in models and in computer simulations they have just been lumped together to one term. So for example in a Jones potential you will have the, the Van der Waals term comes in here. And a few notes here. So, a and B in simulations and theories they are often used just in as an empirical fitting parameter. And I think this can sometimes be a little bit problematic but it's well, maybe not problematic but I think it's important to just realize that they are actually three different mechanisms. So if we talk about explicit solvent models, and then the key some part dipole dipole part is explicitly included. But only if we have polarizable simulations which I would say is not that common because they're very expensive. And then the Dubai into a Dubai part is also included, but normally it isn't. So this means that if you take a standard or Latin molecular dynamic simulation, it would be the key some and London interaction. That's, that's part of the constant up here. Right. So if we go to implicit solvent models, and then then be in principle, can be negative it includes all of these terms but it's an excess polarization that we have. And this means that it can in principle be negative I've never seen that done but I think it, maybe sometimes it could make sense it could be that it would correspond that you have something of a very high dielectric that you approach something of a very low dielectric. So that's just a comment about this and something that I think it's not talked about so much when we talk about one of us interactions in these implicit solvent models, at least not in the protein field. Finally, I would like to talk about another aspect of the Van der Waals interactions that comes from the colloidal field that I never see mentioned in the protein field. And that is that the Van der Waals or these are six one over our six potentials they decay very rapidly so they're quite short range. But I tried to illustrate this with the ants up here that when there are many of them together, they can actually reach further. And just to show you this so we have the potential here one or six, but then we have to remember that they are not alone. They sit, we have to evaluate this over all the atoms in the macro molecule. So it looks something like this and we have to, in order to calculate the total energy we have to sum up between all of these different particles here. And we can do that in computer simulations for any kind of shape we want. No problem. I think it can be interesting to do this also for more idealized shapes. So for example if we do this for two spheres. And it looks something like this, we have to integrate this one over six potential over these two volumes. And doing that, we end up with something that goes one over D, where D is the distance between the surfaces of these two spheres. So what this means is that the interaction here has gone from being one over six potential to be something that the case as one over D so much more long ranged. And this is important because this can be have very strong effects on the, on the thermodynamic properties. So a long range interaction, long especially long range attractive interactions will have a big impact when you start to calculate thermodynamic properties. Right. We have now reached the end of the talk. Yes. I would like to mention here in the end that we have, if you're interested in simulating many party protein systems we have software available for this. And we do a lot of protein simulations I would say and many of the effects we've talked about charge regulation in terms of specific effects. They are we're trying to include that in these in these models. So with that. Thank you for your teaching.


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Sometimes they have us together Sometimes they don't hopefully do today because my business partner is in Go getting busy. She says you're gonna be doing like 10 minutes.   It was I think the guy was new so he kind of didn't know what he was doing But it may everything take a little longer, but it's all good, baby Everybody gotta learn it sometime I said leave it to do a second word we're gonna do Right there Step back in those photos Let's go my business partner went in there and got busy So I had to put all the stuff in the car. I had to go to jump off that Wendy's order That went kind of bad, but we back to the bag, baby.  You know what I'm talking about? Sometimes it gets critical But it the way it goes we gonna get go and I don't think we're going to be late to this one. So it should be all right Sometimes you got to do that type of stuff. They don't they only see what we show them on the camera, baby Sometimes we it be critical. I'll be zoned. I'll be asking the zoom. I'll be at the zoom It's wrong bad and I zoom right by it in the curl roll a crispy cream on the tires What y'all know about that cover below a lady over say that He don't say that but he say something like that. You know what I'm Um, put this right here This stuff is like kind of crammed in here, but I'll be okay Put that right there make sure it's right make sure it's right to baby and they got two coca-cola with the yellow the mini coca-cola This right here Lift that up coat that John. Wham time to scram. Let's go Jump up this order this Go Put it in the arms ski.  Come on. Let me know It's slid off the bag. Where's that bag at? This bag I don't know how I slid out. Oh, it's so thin. Maybe I gotta lean to the side like lean to the back like that Joe lean to the side, baby Put it on my arms ski again for the second time.  I'm doing great in yourself. Great. Thank you. You can sit it right there and I'll just give it to you This is a beautiful house sir, by the way You stay here for a long time. I just bought it February.  Love it everything's so close. Yeah You got the food everything the stores. Yeah Okay Okay, how do you guys like the ev6? Yeah, we've been looking out we've been looking at if we were thinking about buying one would you would you recommend it? Okay, you said in kanosa Palm and kea. Okay. I'll check it out. Thank you guys. You guys have a good one Who says they behave but they wrong over the pace bus we in a rush starbucks 3.9 miles for 6 97 We steal they can go out for our blessings. We get his money when he tries to let's go How you doing? You're welcome. Thank you I'm I have an order for steve. I just want to make sure this is everything. Yes, everything. All right. Thank you You have a good one. Are you steven? No, okay. Thank you See we had to go on this drone to see if they got a Where they get I had to text him because they said Texting when we got here they're going to be in the lobby I don't know where I'm supposed to go. I've got to wait here. I guess I don't know Yeah No, steven pc. I don't know. I don't know the the number or anything. It's not on here. It's a real number I think so. I don't know Steven pc. It just says steven pc Check the front desk They said that they're deaf. So they told me the text one on there. They all back in their room back there playing Going to David back there. Oh Okay All right, thank you. What's up? What y'all doing here? Oh, yeah, what's going on? I'm jumping off this order right quick. What's up? What's happened? I'm what you what you don't go pro on or something? Yes, man. I got the youtube y'all youtube right now What's going on, bro? Yeah, let's see youtube Steven Or the sientian ultra we about to pull on right now again though Jewel isle five out of 4.3 miles of red car order for 1150 rp nipsey order ultra is mariano's 23 items 1.5 miles for 2789 Wait, somebody tell me shy. There's the plan stand. We about to get these beans man We dropped my business partner off at mariano's and we gonna deliver the jewel I scroll and we gonna keep on getting this though. I hope you guys are enjoying this video Subscribe and give me a thumbs up and do not skip those ads. We're right right now to get to this bag. Let's go Baby, we literally just came from this house like a few like few orders ago, baby We about to get this money and they gave us a 20 dollar tip. We got us together We're trying to let's to get to this cheddar. You know, I'll see my god had a little sand last Let's get this money Let's go get busy, baby Step back and don't fall Of this mariano, baby the thing about delivering to something that you are delivered to guess what we remember how to get here because This that one that got the uh, the crazy apartments that we have to go to a different Dang this joint heavy. Oh, he probably got the same exact thing he had last time. I remember this before from before Yeah, he had these monsters. I remember all this Yeah, any stirrer Put on the left hand so we get these bands and the stairs are weird like to set up all the stairs are weird. So This joint extra heavy baby ain't gonna lie to you and that is everything so you have a great one. Enjoy Yes, it's just like that. It is the end of the video. I hope you guys join the content today. We made 150 a 16 We can't even got that green baby and nothing in between we got done 1131 We can't even get it done and we got ourselves some of that money, baby Everything was going great today and then guess what? Well, the orders have stopped coming So don't turn a great day into a terrible day guys.  We came and did our thing. I hope you guys enjoy the video. Make sure you lock it tomorrow Cause going back down where you playing around to just to instill bad season We don't keep on getting maries. I'll see you guys the next one and before we go Y'all we know we gotta go. Let's go.  I hope you guys have a great day Go out there and get to that cake. I'll see you guys on next one We go on before you already know the ddk and i'm on my way


So thank you everyone for staying so late. It's the last. I wouldn't be promising like I wouldn't be taking a lot of time here. And thank you for Ramya covering most of the testing strategies, so I would be just skipping it out. Thank you Ramya. So my name is Rohit Vyas and I'm a suite quality engineering manager at Red Hat. And mostly involved with the automation part of the CI off of a test suite. So this talk is mostly about revamping the QE automation pipeline, QE automation testing and tech on pipeline. This talk is mostly about my journey towards like how we integrated our integration testing from end to end point of view. If you look around the QE CI process and if you want to think about the CI and CD process, it's like building your software from building, deployment, building, testing and deployment. But what I'm talking about is just the CI process of the testing side. And this CI process of testing side includes from trigger identify at what stage your test suite would get triggered. Then it from what repository you are using the code to be checked or it could be GitHub, it could be GitLab. You need to also link your code. You need to ensure that you're coding like JS. If you're using JavaScript, you need to use JS slant, it can be using pilot, Yamalint, depends on the code which you are using for your integration testing. Then you have a provisioning environment where your SUD will be run. What is your provisioning environment? Whether it be a cloud, it would be a bare metal. What kind of provisioner tool you're gonna use it in integration testing some might be using Ansible as a provisioner, some might be using Terraform. It totally depends on the set of tests which your test engineers have built. The SUD setup can be in AWS, GCP. It could be a standalone bare metal machines using Vagrant or Liveware or something like this. Maybe it's running on the podman containers. So that's the SUD part. The test execution constitute of your Ansible test, your Python test, your Jmeter for the performance, your API test would be in SOAP or it would be running on the headless mode with using the postman. Or it could be a go-kingo for the go framework. The last part, not the last, but it's a test logging because that's the most important part where you're a stakeholder keeping an eye on. The logging part and the analysis part. That's also a very interesting part because what everyone care about is what's the percentage of a test case is passing and what's the product readiness for your entire workflow. Last but not the least, the important part for getting the notification. Your notifications should be very productive at any point of time where your test execution get failed or there's an identification of any deviation from your workflow and you can have your test notification integration with your Google chat Slack or even getting a PDF reports on the Gmail. So this part is a small criteria in the CI CD workflow where QECI scenarios and the workflow fits in. There are so many CI tools available. It's pretty hard to identify which one suits your requirement. There's like Jenkins where you can write your Jenkins files. You can grow up into multiple stages, build up a pipeline of these steps. You have GitHub actions, which is good to integrate with your Git repos where you have to run some kind of action files for your integration test. You have Ergo workflows where you can run your execution pipeline on multiple clusters by integrating to it and then you have Tecton, which I am trying to target today.  This is one of the use cases which I was working for my last project. We were certifying an operator certification use case where we need to certify different vendors, operators, and also we need to certify different OpenStack plugin and have like tons of different test scenarios that we need to cover on. If you see that there are like proper triggers, it could be triggering our test suite twice in a day or probably on every Git check-in. Then we have to check out the workspace that definitely a part of our CI part, provisioning and building a test data, provisioning on multiple environments like OpenStack, then configuring, setting up an OpenShift cluster. It could be using a mini cube, code ready containers over a bare metal machine, or it could be using just kind cluster. Deploying and configuring the operator side of it, triggering the GitHub actions pipeline, which is on the upstream side, pulling the events notification from the GitHub comments just to start and initiate our integration test suite. Integration test suits were in many different formats from UI test which were in Cypress, the backend test which was on Python, the API test on Go, then we have Go Catalog test, we have tests which certify different Docker images of the container images, then we have backend DB test and this has to be integrated on together. It requires all configuration, so we have bundled all the tests in a container format, container image, and we keep those container image in a registry just to ensure that we are well versed with the latest images for our test suite as well as it has a vulnerability scan, we are always updated over it. A vulnerability scan on clearance with clear services, so it's like quite giving us the updates of the health of our images. The last part would be the logging part. We push the entire test results into our centralized data logger, which is report portal. We are using report portal for pushing all of our test artifacts, test reports, the UI test logs, everything. And then after that we have notification section where we push all the notifications to our Google chats, Slack, and Gmail. Just to bring a light introduction about Tecton CI, so Tecton CI is a Kubernetes resource which help us to build up our pipeline and by pipeline we have multiple areas. The smallest one or the shortest one would be the steps where you execute your commands and your test scenarios by bypassing the arguments. Then we have a task which bundles the steps. After this task you can imagine as different stages of a pipeline. Pipeline is also a Kubernetes customer resource which is used to bundle and execute your task whether you can run it sequentially or you can run it parallely. Pipeline runs with a pipeline run customer source which is then been triggered by a trigger template. You can bundle it with the trigger template. Trigger template is something which listens for the trigger event and you can listen those trigger events with the event listener services from the Tecton which helps you to listen different triggers like from webhooks or you can just directly use chronicles. Why we shifted to Tecton? Because most of the workload which we were running on was run by container side because the test bundled container it was easy to use the same format where we can just execute our container test scenarios. Also applications can be deployed on the same cluster so that was easy for us. Then Tecton provides a good mechanism of executing using CLI and GUI. So Tecton has a CLI client which is called TKN. You can use it to run CLI. It's easier to integrate and it also provide a GUI dashboard from where you can just manually run the test on demand. Then we have a huge benefit of using Tecton was that it's catalog source where you have a predefined task which you can just directly integrate with your pipeline. It has a huge community and the best part is like it's task are reusable so you need to write your task once and you can reuse it in multiple pipeline scenarios. Also Tecton tasks and pipelines are just YAML. You can write your YAML over here defining what steps you need to execute the images on which your task would run and each task run as a pod and each pod executes as a single instance for the process. So you define your task in a simple YAML format which is easy to understand by Q engineers or it's easily maintainable because any changes on the test side will reflect on the image. So you can just update your image it's quite easy to integrate in the entire format. Then you can integrate those tasks on the pipeline which defines whether your task would be securing parallely or sequentially on a specific time. Then you have a pipeline which defines how your pipeline, sorry, you have a pipeline defines how your pipeline would trigger you can pass all the arguments as the input and the output resources. You have a trigger template and event listener for like if you want to create an event listener for Git events, you can have it as a Git event with respect to the push hook or something like this and also you can integrate it for a different like you can use it as a web hook like I think in GitHub Actions there's a STTP response dispatch workflow so you can also trigger your event listener directly from any STTP request. All right, I think this is a small video of Tech Turn catalog where you can just directly search for your task and let's say you want to create a task for a pie test one example, you can just directly go in the pie test download the task you can download the task, go in the CLIs you can install the task, give a reference give a reference of your task to your pipeline can directly install the task in a cluster you can give a reference once you have a task in your it directly installs the task in your Kubernetes cluster and once it's available you can use it as a reference. So I just downloaded the task of pie test and it's in my Kubernetes cluster I can reference it and use it in my pipeline. Okay, all right. So this is one of the screenshots for my operator pipeline where we have a set of environment setup then we have a provisioning area where we provision multiple bare metal machines and deploy a Kubernetes cluster on it using CRC and kind of the Git event which we trigger on the GitHub side we wait for the polling agent to get the information from the GitHub side and then we execute multiple tests in parallel which is from the UI back and DB and the API side. Finally, we submit the test run and after that we can get the notification of completion. Well, this is the demo for how you can execute a pipeline from the CLI as well as from the UI. So this is a simple example of running your CI pipeline using the Tecton CLI. You can use overriding your default parameters as well as if you want to use the existing default parameters you can use set perms default. Once you run it from there it's pretty easy that it gets executed on the UI side. You can also redirect these locks from the terminal as well as you can see on the OpenShift side under the task locks. So that was an example of how we trigger the test from the CLI. Also you can directly trigger test from the UI side. It depends on what's the demand for execution of your test frequency. So you can directly go and override the variables from there and you can execute the test. All right. For test logging and notification we are using report portal where we are pushing all over test results into the launches. It supports the launches format where you have all the test results from multiple source. You can also see and configure your customized dashboards based on which you can understand what is the failure analysis for your entire test suite and for that program. The notification part consists of custom messages that we are sending on the Google chat side which gives the information from the tech logs as well as from the test results. So we customize our notification format. Well this is an example like how does the report portal analysis looks like. You have your test results over here. You can see the test logs published. It also has a capability of predicting the failure. So it saves a lot of our time in identifying like what was the reason of failure. So let's say if there's a system related issue. If it's a product related issue or it's an automation bug. So we can just identify from this test log analysis and it's automatically reflects with all these kinds of defective. So rather than investing times. Okay. I got another event coming. I know time to do. Sorry. Okay. All right I'll just hurry up. All right so you can see the dashboard over here which has the predictions of the test run. And also you can see the Git chat notifications which we have customized has information of the tech logs. You can directly navigate TechTown dashboard as well as the board portal log instance from here. All right. I think I won't be taking much of the time. These are the resources and the reference which you can refer for for TechTown OpenShift Pipelines. We have our project operator pipelines over here in GitHub from where you can take a reference of it. And also there are different tutorials on the Jenkins and other CI tools like GitHub actions. You can just refer it. All right. Thank you. Thank you everyone for being so patient. I thought I covered the talk on time. Let's thank the speaker. So you are completely on time. I'm not sure what happened here. So don't worry about this. But I mean, there's a minute over. Okay. Well then thanks everyone for joining. Let's give our speaker a round of applause. Thank you everyone.


At Audiology Associates of Prestonsburg, you can live your life the way you want and find the freedom of better hearing. With 40 years of being in the hearing care industry, you'll experience patient care that is specific to you with exceptional follow-up care that ensures your hearing and balance needs are being met. Audiology Associates at 1428 Northlake Drive in Prestonsburg. November is National Diabetes Month, which works to boost awareness about the risk factors, symptoms and types of diabetes. We spoke to Public Health Director Tammy Riley about how you can prevent and manage diabetes. Currently, Kentucky is fifth in the nation for prevalence for diabetes. It's very important to Pied County residents to have the information that they need. Currently, Pied County has 13.8 percent, according to county health rankings, prevalence of diabetes. That is one out of six Pied County residents have documented diabetes diagnosis. Millions of Americans, Kentuckians and Pied County residents are pre-diabetic. Unfortunately, around 80 percent do not know that they're pre-diabetic. The big message is if you can catch the diabetes when it's in the pre-diabetic stage or prevent it entirely, it is manageable, reversible. So if you fall in the pre-diabetic stage, your diagnosis is reversible. One in four Americans have diabetes and don't know it. Symptoms of diabetes include frequent urination, excessive thirst, sudden weight loss, blurred vision, dry skin, and tingling hands and feet. So pre-diabetes is reversible or preventable. So we have a CDC-approved program called the Diabetes Prevention Program, DPP, and it's managed by a nurse who's on staff, Jessica Anderson, and she meets weekly with the individuals. They have weight loss goals, a lot of management, food intake, education. It's a wonderful program and the good news is it's free. It's free to Pied County residents to participate in our DPP program. If you've already received that diabetes diagnosis, then we assist individuals with the referral to what's called DSMES program and that's a program to help individuals manage their diabetes so that they can decrease their risk factors for a poor outcome. For more information, go to Pied County Health Department on Facebook. Reporting for Mountain Top News, I'm Brianna Robinson.


Professionalism. This is something that is important to every nurse and midwife working in the UK. And it is for this reason that the UK chief nursing officers decided to commission a review and to create a report that describes the practical application of the nursing and midwifery code into professional practice. We have now launched a report today which sets out a way of working and understanding what it is to be a practicing nurse and midwife. I think you'll find it useful and I would encourage all of you to read the report and engage with the conversation about what it is to be a professional nurse. What does it mean to be a professional to me? Well I actually think being a nurse or a midwife is a very privileged position. We engage with people in every walk of life through all stages of their life course. We're there at birth, we're there at death, we are there when people are at the most trying times of their lives. That's a privilege and it comes with a responsibility. To be a professional nurse and midwife means that we have to understand our responsibility to put the person at the centre of care. And I'm hoping that the report when you see it will help you make those day to day decisions that make you a wonderful professional practicing nurse and midwife.