This is Brett, and this 2021 Ford F-250 Crew Cab Short Box Lariat Ultimate with the Black Appearance Group is stock number 13550Z. This is also a Roush conversion package on this truck. I'm here at Summit Automotive, your new and used heavy-duty truck headquarters. This 2021 Ford F-250 has the 6.7-liter Powerstroke diesel engine, 475 horsepower, 1,050 foot-pounds of torque. It's paired up with the 10-speed automatic transmission. This truck has been fully-safetyed and inspected by our service shop has a fresh oil and filter change on the fluid tab and checked and topped off. This truck 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 vehicle possible. Star white tri-coat metallic 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.  I didn't see any dents or dings on the box. Grab and doors all look fantastic on this truck. As does the front fender, you get the special fender flares with the Rauch package. If you like the video, you can subscribe to my YouTube channel, which is youtube.com slash Summit Auto. Click the bell notifications, you'll get updates on the videos I do each and every day here at Summit Automotive, as well as one of the largest catalogs of vehicle walk-arounds on YouTube. Very nice down that side, this one comes with the special Rauch painted alloy wheels, and it has Cooper Discoverer STT Pro tires. These are 37x12.5R 20LTs, and I would say they have probably 90% of the tread left. They look very new on here. Comes with the upgraded suspension, box shocks, and springs. I'm assuming it gives it a little bit of a probably a 2-inch leveling kit on there. Frame and underbody looks good. This is a one-owner clean title history, clean car fax out of Michigan. Front bumper is in nice shape. I didn't see any major dents or dings on there. Lower valance, I didn't see any scuffs or scrapes on that. Comes with a custom Rauch grille. Those lights light up, and it does have the LED headlamps, running lamps, and fog lights. We'll turn all those on at the end of the video, so stay tuned for that. You also get this anti-glare hood decal on there, part of the Rauch package, and you get clearance lights up top. Passenger's side front fender looks really great, and the passenger's side front wheel no major scuffs or scrapes. Take a look at that white star metallic tri-coat. It's got a lot of metal flake in it. Let's see if I can get it to focus here. Really a cool color, especially in the sunlight. And that paint is in really nice condition. As you go down this side of this 2021 Ford F-250, 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 here, the truck end of confidence, and 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 salesmen know that you saw the video, that it was helpful, and that Brett sent you back wheel no scuffs or scrapes in there, and the back tires have just as much tread as the front tires. You do get fox shocks in the back as well. I'm sure that's all part of that Rauch package. Has all the remaining factory exhaust, so it hasn't been altered, and the underbody looks really good on this truck. Comes with the black painted step bars, that's part of the black appearance group on this truck. And you get the Rauch chromed exhaust tips on this truck as well, part of that Rauch package. There's no bumper, no dents or dings on that, it does have the rear bumper parking sensors, full towing package, which includes a receiver hitch four pin and seven pin wiring, and that tailgate is in fantastic condition as well. Didn't see any major dents or dings on there. Has the LED tail lamps too. Has the power drop down tailgate. Has the tailgate step assist, and a very nice spray in bed liner. You get LED bed lights back here, you also get side box tie downs. I didn't see any major damage, dents or dings back here, and that spray in bed liner goes onto the tailgate. Shuts nice and solidly. And for full disclosure, this back wheel is just as nice as the rest. Going down the rest of this side of the truck, once again, no dents or dings on the doors, it does have the driver side door code entry. We do have the code for this truck, and if you ever want to figure out what your 17 to 22 super duty door code is, in the upper right hand part of the screen, I've got a video showing you how to find that. Blind spot monitoring, built in directional signals, this one does have the 360 camera.  No rips or tears in those seats. Does have heated and cooled seats. You get the Roush stitched into the headrest. And those seats are in fantastic condition. Both sides are power. Comes with Roush, all-weather floor mats, power pedals, power drop-down tailgate, auto headlamps, and LED side lights, power tilt and telescopic steering wheel. That works nicely. Power windows, locks and mirrors, and memory driver seat. These mirrors do power fold in. I always like showing both sides so that you know that both sides are working properly. And then it does power telescope out and in and out and in. We'll take a quick look at the back seats. 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. These seats fold up. There is a collapsible storage space back here that can go down flat. Has the weather tech floor mat back here as well. There are your heated seat buttons, two USB or USB and a USB-C, and then a 110-volt, 400-watt plug-in and then a 12-volt power point. And the seats come down like so. And you get the child safety locks on the back doors. And of course, the inside and bottoms of the doors all look great on the aluminum truck here. You get these black sill plate covers. Part of that black appearance group. And as long as I'm standing right here, I'll show you the tire and loading information sticker, as well as the VIN sticker. We'll hop inside, check out the miles. Radio and everything that this truck has to offer on the interior. Get the built-forward, tough animation at the beginning there. And you can see that you get the nice digital display. This truck does have 40,752 miles on it. Instrument cluster is very nice and clean. And do note that it is a Roush instrument cluster on there. It comes with the leather-wrapped, I'm assuming this is a heated steering wheel. Let's see here. Get the Bluetooth and audio controls on the right. And cruise controls and adaptive cruise controls on the left. Information center controls. And yes, this is a heated steering wheel. There's your transmission shifter for the 10-speed automatic. You get this Roush plate up here. It's got an ID number on there, as well. And then it does have the B&L premium sound system. You get the sync 8-inch radio. There's your heated seat button or your heated steering wheel button. AM, FM, and Sirius XM radio capabilities, as well as Bluetooth. This one does have the factory navigation system on it. There's Highway 41. So that is working nicely. You get all your different apps on here, including FordPass Connect, Apple CarPlay, and Android Auto. This is also where all your cameras show up. You can see that you get the front view camera. You have the surround view camera right there. You can also check out your cargo cam. And then it also has your backup camera on here, as well. Truck's got to be running for that to work. Up here is your factory exhaust brake stability control, lane departure warning, obviously hazard lights. And you can turn those cameras off with that button.  You get two cupholders, and then you get four cupholders. That's my magic trick. You also get ambient lighting in there, as well.  I don't think it's ever been smoked in. It has the pano vista roof, power front sunroof, second row of fixed glass, it has six auxiliary up-fit switches, power sign rear window button, map lights, sunroof and sunshade controls, and your home link buttons for your garage door security systems, and lighting systems. All right, that is a mouthful. Let's start it up and take a look under the hood. Starts right up, no check engine lights or anything like that. I would personally like to thank you for checking out the video today, and hopefully from this HD video. They're your LED side lights. You've been able to verify the quality condition options and, of course, cleanliness of this truck all the way around inside and out, see all those LED lights are working fantastic, and you get them in the grill there, too. I think it adds nice little extra. Cab lights are working well, too. Under the hood, we have the 6.7-liter power stroke diesel engine, engine base very clean, runs very smooth. Once again, this truck has been fully safety and inspected by our service shop, has a fresh oil and filter change. All the fluids have been checked and topped off, and it is 100% ready to go. There is the emissions sticker, and I would highly recommend this truck from the quality condition options rarity standpoint. And to see more pictures of this truck or one of our other 550 new and used cars, trucks, SUVs, minivans, wranglers, half-tons, three-quarter-tons, one-tons, you name it. We gotta go to that website right there, summitauto.com. Full pictures and descriptions of every single vehicle all at summitauto.com. If you'd like to check out more HD videos, you can go to youtube.com. 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 to my YouTube channel, the upper left, the link to more F-250 videos that I've done in the upper right link to this vehicle on our website and lower left. And a link to one of our latest C2 videos in the lower right. Click those checks out. We're super excited to help you with this Ultra Clean 2021 Ford F-250 true cab, short box, hilarious, ultimate black appearance group with the Roush conversion package in star white metallic tri-coat. Thanks again for checking out the video. I'm the Lexus driving chair on the YouTube channel. I really appreciate it. Thanks again.


So, well, yes, Tommy say, my name is Mariana Janesa Teta, and I'm currently working in AstraZeneca as an associate principal scientist in the Advanced Drug Delivery Department in pharmaceutical sciences. And I did my PhD with Tommy, did a lot of neutrons, put him to wash a lot of troughs so I can recommend that you do that with your supervisors, but I did make sure that he got a nice dinner afterwards. Today I'm going to talk about lipid nanoparticles for mRNA delivery. So thank you very much Tommy for inviting me to this presentation, and I want to kind of summarize a bit what I would like you to learn from this presentation. So I want to have a, I want to start with a kind of high overview of what mRNA therapeutics are, especially kind of promising challenges and advances. Then I'm going to talk a bit how to make the lipid nanoparticles for RNA delivery, and then importantly how to characterize and the performance of these LMPs. And then, of course, this is a neutron core, so we're going to talk about how to use neutrons, especially a small-angle neutron scattering for the LMP development. And then I'm going to present two different cases looking at with use, with the use of sans, the effect of protein binding on the LMP structure and also how to use sans to functionalize the lipid nanoparticles or characterize functionalized lipid nanoparticles for subcutaneous administration. And I mean, if you want to interrupt me, I don't know how you do with the other presentations, just please go ahead and just unmute yourself and ask questions is fine for me. So I'm going to start with the mRNA therapeutics, promises, challenges and advances.  They're very popular at the moment. So the promises is that we can produce proteins in vivo by administrating mRNA. And this is typically the approach that we want to achieve when administrating the protein itself is not viable because the proteins degrade, because the proteins don't reach the target, because the proteins will not have the right function. So then we would prefer to administer the mRNA to produce the proteins in vivo. However, I'm a fiscum. And when I look at mRNA, mRNA is a very long charge negatively charged polyelectrolyte. So that means that it's not very easy to deliver mRNA because it needs to cross an equally negatively charged cell membrane to enter the cytoplasm and actually produce this protein. And you can imagine that that's not an easy task for a negatively charged polyelectrolyte. Additionally, our body is made to destroy foreign mRNA. So then we need to protect from enzymatic degradation before it reach the target cell. And another very big challenge for mRNA therapeutics is actually finding a biocompatible vehicle. And typically when the mRNA enters the cells, so it crosses the cell membrane through different mechanisms dependent on the delivery, enter the cytoplasm, it's read by the ribosomes and this is producing the kind of polypeptide that will become part of the protein. So when people talk about the mRNA vaccines, this is not something that was developing in gear. This is something that has 50, 60 years of development. So first of all, the discovery of mRNA was done in the early 60s. And from that we have come a long way. So from the discovery of mRNA, we have come a long way to understand how can we translate both in vitro? How can we develop delivery systems? The first were liposomes. Maybe some of you are familiar with lipofectamine. It's typically used in a lot of cell acids to transfer or to deliver the mRNAs commercially for in vitro acids, but it's not very friendly for humans. It has been tested both in vitro and typically in animal models. Already in the early 90s, there were developments of an influenza vaccine using liposomes that contain mRNA. It's also commonly used for cancer immunotherapy. There's also other applications such as cardiovascular diseases. Already companies like Moderna were reporting in 2017 clinical trials using mRNA vaccines against the Zika virus. And all of those advances allow us to have the first two mRNA vaccines that received emergency authorization against COVID last year. So I think it's pretty exciting to see this actually become a reality. But then I'm going to talk about the lipid nanoparticles for RNA delivery because we have come a long way with mRNA, but we need to understand how to deliver the mRNA. And when we talk about lipid nanoparticles for mRNA, we typically talk about this specific technology. We talk about a lipid formulation that has mainly a cationic ionizable lipid. In this case, the lipid that I'm going to talk is called D-Lin MC3DMA. It's quite used in literature, but it's also a product for S-I-R-N-A. And these kind of lipids have a head group that is immune-based typically and has what we call an apparent P-K-A. That means that the P-K-A is measured inside the LMP. And that apparent P-K-A is somewhere between 6 and 7. That means that a physiological pH around 7.4, the LMPs or the cationic lipids are typically on charge. But when the pH decreases, because this is typically the mechanism when it enters the cells in the endosomes, the cationic lipid becomes charged. This positive charge will allow it to interact with the endosomal membrane and disrupt the endosomal membrane and deliver the mRNA inside the cell. So this is an important feature because when you have a constantly charged lipid, this typically alerts the body that there is a foreign system. So it's good to have an on-charge system before it enters the cell. But then you need to have this charge to actually be able to deliver the mRNA into the cell. Additionally, we have typically helper lipids. Those helper lipids are typically acetyl or cholesterol, a phospholipid like DSPC. And then we have a polyethylene glycol lipid that helps to stabilize, sterically stabilize the LMP structure. So as I mentioned, the cationic anisolipid helps to encapsulate as well the mRNA because it forms some kind of complex cationic lipid with the negatively charged nucleotides of the mRNA. It protects it until it reaches the target cell and it also helps to release it inside the cell by disrupting the endosomal membrane. LMPs were originally developed for SIRNA. And this already in 2010 there were reports in the literature about LMPs. And there has been a lot of attempts to understand how the LMP structure looks like, but also to be able to understand if we can improve its performance. And this was a representation from molecular dynamics simulations on how we look with an SIRNA. How do we prepare LMPs? There's a different approach to prepare LMPs, but the most commonly reported literature is the use of microfluidics. So what we have is typically the lipids, which are not water soluble, we have them in an ethanol phase. And the mRNA, which is not ethanol soluble, we have in a aqua phase, which is typically a low pH. And we mix this in a microfluidic chamber. And what happens is when we have the mixture, in this case, this is a specific system called a nano sampler, which is from a company called precision nano systems. They have what they call inside a mixed herringbone structure. That helps to create this kind of flow or caotic flow to help preparing the samples and also to help to make them a bit smaller and consistently the same size. So that's why we like to prepare the LMPs in the microfluidic mixer because we typically get very reproducible results with a very high encapsulation, more than 90%. After we get the LMPs, since I said originally we have them in ethanol lipids and in acidic buffer, the mRNA, we typically dialyze them against a physiological pH buffer. And if we need them, we concentrate them. And then we do some particle characterization. That's the most standard size and encapsulation. And this is a little like spreadsheet or how we look when you're calculating your amounts for your preparation of the LMPs. So we actually make, typically mix them in a one to three ratio. That means that for each part of the ethanol volume, we have three parts of the mRNA volume. We, in this case, we have a lipid concentration of maximum typical 12.5 million molar. And the reason is because when you try to do more and more concentrated, you have a more risk that this will get stuck inside the microfluidic chip. And you don't want to make it too concentrated either. And another important thing is the N to P ratio, the N to P ratio N starts from stands for the N or cut or amine groups in the cationic lipid and P stars stands from the phosphorus of the nucleotide. So it's basically a positive to negative charge ratio. But in this case, the N is not the negative, it's the positive. So when you say three means that you have three cationic lipids per nucleotide. This is very important for the particle formation. So if you start going to lowers and to P ratio to one to one, then this can typically precipitate and not form stable particles. You typically want to have them slightly overcharged when you made them. So more cationic lipid than nucleotides in a solution. Another important is to have the composition of your LMPs. This is the most standard composition that we have is 50% of the cationic lipid around 10% of the phospholipid 38.5 of mole percent of the cholesterol and 1.5% of the peak. And this is basically, I mean, I actually have the extension that I can share with you later, but this is what we use to calculate our preparation. So we put input our volume. Another important is to have that if you have the charge density of your mRNA or DNA or, or whatever you're using to input it here. So you can have the right calculations on the weight and the solution. So as I mentioned, we use a system in our lab, which is called a nano sampler. And the nano sampler is typically what we call a bench top. So it's not necessarily what it will be called what we would use in a large scale to produce the vaccine, but it's a representative of what we can do in our lab to make an excellent piece around one millilitre to 50 millilitre. And then we typically take them out with allies in PDS into some dialysis cassettes and we can concentrate them using amicone ultra centrifugation filters. Sorry, Maria, if I may ask a question here. Yes. Yeah, so I was wondering about like what are usually the sizes of this lipid nanoparticles. So I will come into that but for this preparation that I show you is somewhere around, I will say, if you measure by DLS the intensity average distribution will be somewhere between 70 to 80 nanometers. Okay. And I was also wondering about the structure. So do you have this bilia structure that encapsulates your mRNA or it's just a sort of a micellar structure which because in the chip that you showed, you had this ethanol where lipids are dissolved and you have mRNA and then they mix together and form the nanoparticles I was wondering like looking at the cartoon. What kind of structure it is. I can say that it's not a micellar in my definition of micellar and it's not a liposome either. So it's somewhere in between so it's not it's not just a simple bilayer that is filled with water where the mRNA is it's a more complex system. Okay, so but I will show you karate and because it's always nice when you can actually see them. And of course neutrons will help us to know much better how they look. So I will go. That was a very good question because now we'll go to the characterization and performance of the LMPs. So, when we talk about physical chemical characterization of the LMPs what do we specifically we talk. So I kind of we divide them here in waves and the ways depends on the complexity because we were not going to do the same characterization for all of the preparations that we do I mean we make LMPs fairly often so we cannot do the most complex techniques for all the preparations. So typically when we have our preparation, the first thing that we measure is the size and for that we use dynamic line scattering. A simple kind of instrument in the lab a modern system. And then we measure the encapsulation with a fluorescence acid called ribograin. Ribograin is a dye that binds to mRNA.  These are what I call the wave one characterization. Wave two is to look at more complex characteristics of the LMPs. This is an acid called TNS. TNS is also a dye that allows us to measure what we call the apparent PK of the LMPs. So typically when people reports in the literature the PK of the cathodontic lipids they always report them as I mean cathodontic lipids are not soluble in water they always report them in some kind of lipid nanoparticles so it will always be influenced by the composition of your LMP. So to measure the CETA potential that can be important but measuring CETA potential of the LMP is challenging. You have to, especially sometimes when if we are used to working in systems like the Malvern CETA Sizer is very easy to get a result but we need to understand if that result is actually physically meaning. So to measure the CETA potential of the LMPs we have to try to have a low ionic strain and low concentration so it takes a bit of tweaking to actually get good measurements of the CETA potential of the LMPs. So we don't do them as a standard control. We could also look at NMR, NMR can give also in information about charge you can also give information about the structure is a bit complex to analyze but it's possible we also have a lot of NMR instruments here in AstraZeneca. Then what I call the way through is the instruments that we actually don't have in-house and we typically have to go to the university or we have to go to large scale facilities. One of them is the cryo TEM. So this is a small picture of the cryo TEM how the LMPs look and I will have more pictures afterwards. So as I mentioned that most of the particles are here and this in this kind of small circle and they are not like but some likes they are actually electron dense. So they are, you can see that they are all the way through there like dark gray. I will have a better picture afterwards. You can also do both small angle x-ray scattering and then a neutral scattering as well to look at both the structure and the LMP distribution. And I will come more to a specific sample of how they look and what does the structure means. So this is a better picture of how the LMPs look by cryo TEM and typically the standard approach when we started to work this. So I did my postdoc here in AstraZeneca with Lipid nanoparticles and when we started looking at the Lipid nanoparticles we took the example as I said from the SI RNA. What they reported in the literature is that they will have 50% of the cationic lipid, 10% of the DSPC and then they will add more peg if they wanted to reduce the LMP size and they compensate this with the cholesterol. So that means that you typically have as a base 40% cholesterol and then if you add 1% peg lipid and you will add only 39% cholesterol. And as you can see as the peg lipid percentage decreases the particle size increases. You can also see that sometimes when they're a bit bigger you can find some liposomes but in the majority of the population are basically this kind of dark spheres that are all dancing the core. And then when they're small you can also start seeing maybe a smaller that are a bit more regular. We can also compare the size from the DLS number distribution with the size from the cryo TEM distribution and find a very good correlation. These are three typical preparations so that a lot of the sizes are going to come back around the presentation so we have some around 40 this is number size number size distribution 60 and 90 so the number size is typically a slightly smaller than the intensity average distribution. So when we started to look at this this LMP so we started looking at four different sizes between 40 nanometers and 140 nanometers and then we started doing in the process and we picked two clinically relevant cells. One is human adipocytes.  So when we have administration subcontinuous administration those are the first cells that typically the LMPs are going to encounter. And then another one so we took our induced pluripotent stem cell derived hepatocytes, which are liver cells and are also very relevant because we have an IV administration of LMPs this LMPs typically will end in the liver. We label in this case the LMPs with DSPC that had 3-tune in it to be able to track how much was inside the cells. And what we found was that the uptake was more or less the same independently of the size. We didn't really see a trend on the pen if it was smaller or bigger maybe for the adipocytes the 16 nanometers was a bit more but definitely for the hepatocytes there was not a big difference. But the interesting was not the uptake. The interesting was actually what happens when they're inside the cell. And what we can see is actually we measure here a protein called adipoprotein and this protein so when we administer the mRNA this protein will be produced inside the cells and it's actually a secreted protein so we can measure it from the supernatant of the in vitro experiments. And what we can see is that the 16 nanometer LMPs had a significant higher protein expression in both cell types compared to either smaller or larger LMPs. And we couldn't understand why are these LMPs better than the other ones I mean what makes it 16 nanometer because the uptake they all look to be up to taking up more or less the same amount. And then I came that was when I was fresh back from my PhD and I said oh why don't we do a neutron experiment and I drafted a neutron proposal as I was very well coached by Richard. I think I don't know if you had had that exercise already or you will have to write a beam time proposal but I think it's a really good thing I learned from from my PhD supervisors. And I mean I guess that I mean I could probably skip this slide because you had a much better introductions to small angle scattering at the moment but what we were interested was if we could understand a bit more about the structure of the LMPs. And if these could guide us to why 16 nanometer LMPs were better than smaller or bigger. And especially I was interested in not only use a small angle x-ray scattering but to use a neutron scattering because as you have probably seen already in the course is good to be the last day. We could do selected iteration of either the leaping components or the solvent to produce different scattering profiles. And we have significant we have many components we have the cathodic lipid we have the phospholipid. We have the cholesterol and we have the peg lipid plus the mRNA. So we needed to figure out actually what can we do trade to be able to highlight so if we have for example the DSPC hydrogenous DSPC the tails are have very close matching point to the to just pure water. And the head groups are very close to do trade the cholesterol but if we have do trade the DSPC then the scattering and it is much higher. So we could really highlight the DSPC. RNA also has a scattering and density that will change depending on the content of the to because it has labelled hydrogen second being exchange. So typically the match point is around four. So so we kind of match the first time we did was okay these are all components we calculated their scattering and density we see which molecules are available to do trade and we can start playing on how to design this LMP to be able to highlight certain parts both from the deterioration of the lipid process deterioration of the soul. But you can imagine that the first thing that we did was not sense because we needed to have data before so we actually did some sex. And interesting what we can see is that when we have a mRNA inside the LMPs we can see one peak at approximately one inverse nanometer that corresponds to a correlation distance of around six nanometers. But this is only in the presence of a morning we couldn't see it in the absence of a morning. And we could see the same peak independently of the size in the same position it was a different intensity or different order but it was exactly the same peak. But I mean, I guess that you have no background in scattering so you will know that we won't pick difficult to fit a thing so it could be anything it could be in the literature has been reported this is potential and inverted micellar phase. So this could be maybe a correlation distance between these micelles. Some people says that maybe this looks like an onion so a multilambular vesicle. Could it be the mRNA some kind of work like micelles and these are kind of correlation distance between this one line micelles. So then we went and why do we care about the structure of the LMPs I mean why was it important I mean what is reported in the literature is the LMP transfection and efficacy is very low is typically single digit in less than 10%. That means that if you administer a vaccine that has I don't know 100 micrograms of mRNA perhaps less than 10 micrograms are the ones that are making their, the job. So it's very small amount and a lot of it is just being recycled. And, and we want to understand if we can modify I mean the structure of the LMP to facilitate this in the summer escape and this is not something that is new. I mean a lot of companies a company that is facing losing Camus has been doing this for many, many years, trying to understand how the kind of liquid crystalline structure can facilitate both encapsulation and the delivery of small molecules and larger molecules. And so, yeah, so I, as I said, I drafted my nice sense proposal I got some beam time in in gargling in Munich, and we went there and I didn't know what to expect I just designed a few kind of particles one was deteriorated DSPC and partially due to the cholesterol that it can be fine found in Avanti so commercially. And we look at different kind of D2 OH2 oration and we found actually was something very interesting. We found that one of the lipids that DSPC was mainly segregated at the surface so we were actually able to fit five different profiles to a core shell model, where basically we can summarize it as we had an LMP core and then monolayer which was mainly enriched by the DSPC. We had on top a peg layer with solvent in and the peg was in a mushroom conformation. And this was very exciting because what it was in the literature was not this, when we look at the first SRNA simulations we, it was believed that all of the lipids besides the peg that it was still believed that be in the surface where distributed homogeneously across the LMP. So these were really the first ones to show that the lipids were partially segregated and the LMP surface, you cannot see that from cryo TM, you cannot see that from SACS. SACS was the only technique that allows to actually see where the lipids were located. With the first experiments that we did we had very limited information about the distribution because as I said we only have a few components that we could we had deteriorated so the DSPC is commercially available, and the partially other components cholesterol is you can find a higher level but it has to be in a deterioration facility and the cationic lipid was definitely not commercially available. We could still get an estimation that we have around 20% water, 24% water inside the core. So it was mainly lipids. But finally we actually, we had continued this work and now we're collaborating with the colleagues in Malmo University with Marite Cardenas which is also a former boss of Tommy and Federica Sevastiani who is also a former boss of Richard. And then we could actually see what was the lipid distribution by not only having deteriorated DSPC but our colleagues here in AstraZeneca could synthesize deteriorated cationic lipid in this case MC3 and our colleagues in different deterioration facilities. I think this was provided by ANSTO, the Australian source, could provide what they call match out cholesterol so it has a much higher scat and the density, and we could actually pinpoint that all the SBCs at the surface, the cholesterol is distributed both the surface and the core but it's mainly at the surface of the LMP in the same layer as the DSPC. The MC3 is both at the surface and at the core but it's actually mainly at the core of the LMP so it's depleted from the surface. The peg lipid we already knew that was at the surface and the mRNA is at the core. So we can actually put a number of how much there is at the surface and at the core of each of the components. Yeah, in the last graph like you were talking about like, so I just missed like how did you do this analysis, like how do you differentiate between like how much, like quantify how much of this is in this part and in the core or in the shell. Yeah, so I don't have the data, I didn't put it in this presentation I should have. But we did a lot of mixtures so we started to prepare samples where we have only deuterated cholesterol, match out cholesterol, only deuterated cationic lipid, we started to mix, we have two of the cationic, you know, cationic lipid and the cholesterol. So we did prepare many different particles and start looking at the scattering at many different H2O, D2O buffer ratios and try to find what was the matching point. So we got equivalent to what we did in the first experiment but we got much more data that we could feed consistently to actually put numbers to the volume fraction of each component at the surface and at the core. So it was similar to this experiment actually I didn't put the data but it should be in a publication that came just early this year out of the preparation so it was quite a lot of bintimes to collect all that data. Okay, thank you very much. I will also, isn't this article you mentioned down in the corner. Yeah, this one here. So this one has all the preparations. I mean, I said about Federica and I will, Federica did experiments and plan a bit the mixtures, I prepared the LMP so we did around, I don't know, maybe six, eight preparations to get all of this composition. So it was very many different contrast that we tried. Okay, thank you very much. So, and so now that we knew actually how the lipids were distributed both in the core and at this at the surface of the LMPs we really wanted to understand a bit more about the core so as I said we saw from sax one peak but that one peak tells you nothing. So what we did we was, we tried to do what we call a bulk phase and what is a bulk phase. So we take, now we know that it's mainly cationic lipid and cholesterol in the core not the SPC and opaque. And we mixed this with MNA in water but this will not form particles. This will fall more of a continuous phase and it's not water soluble. But we tried to understand if we can take this phase and analyze it and have a further information on how it is just the core, not the surface. First we put the cationic lipid, cholesterol and ethanol mixed with mRNA water in a dialysis cassette. We put it in a similar conditions as LMPs will typically form which is a acidic buffer in this case citrate and ethanol in a 3 to 1 ratio similar to what we do in this nano assembly instrument. Then after we have dialyzed this for like a day we take this out and we put it against PBS because we also want to know how it looks in a physiological pH because this is how the LMPs are typically stored. And this was also really interesting so I'm going to start from the pattern look of the sex data of the core phase and the acidic condition so the citrate, ethanol, 3 to 1 phase. So this is the data. First I want to point out two peaks which are pretty large here and those peaks are typically cholesterol monohydrates and we know that these phases actually have a bit of excess of cholesterol. This was done before we did the work with Federica. So we added a similar kind of cationic lipid cholesterol ratio it will be in the LMPs but the cholesterol has a limited solubility in the cationic lipid. That might be one of the reasons why it's segregated towards the surface. But if we kind of don't look at the cholesterol crystal we know that they're there and we know that they're in excess of cholesterol what we can see is that interesting. What we can see is a main peak around the one inverse nanometer so similar to the LMPs, but we can see additional peaks. And this additional peak so if we call this the q zero we have a q one and q two they come at a square root of three and a square root of four multiples of the original peak. So this is typically an indication of a reverse hexagonal phase. And then what we believe is that at low pH the mRNA is organized in some kind of water cylinder so mRNA is a very long molecule. So these water cylinders are have an hexagonal packing and this inverse nano one inverse nanometer six nanometers is the center to center distance between this water channels in hexagonal packing. And we also did some freeze fracture micro grabs of the face and we could also see this what we can believe are the water channels and we could measure the distance between the water channels to be approximately six nanometers as well. It was interesting what happened when we actually look at the bulk phase in the pbs storage buff. It's similar to what we have measured with the LMP itself. So we can see again is that when we have no money. We stop seeing that peak that we don't have any more that that that we have a bump but we don't have any more that very highly order peak, neither the further peaks we can only see the cholesterol kind of crystal. But when we have in this case we don't use a money and it's just because I'm an extremely expensive so we use a model molecules of poly A. And we can see from the poly A is that we can still see that the main peak that we saw before at one inverse nanometer although we cannot see anymore. With the same intensity that the Q one and Q two is there got so so we believe that is this is a less order phase but somehow related to the first hexagonal phase that we saw. When we look at the first fracture of the kind of upper face so the LMP similar with the poly A in pbs. We don't see any more this nice water channels we see more of a kind of mix structure. So what we believe is we originally I think call them in the in the publication at this sort of external phase, but that sounds very on. Oh, doesn't sound very straightforward, I mean it's an external phase but it's disorder what does that really mean. So that we started calling afterwards a worm like my son so it still is this water channels where the poly A or M RNA is located. There's not any more structure in the typical hexagonal packing but there's still some kind of correlation distance because it's a highly packed system of approximately six nanometers. And interesting, if we look at the empty LMPs as we saw before and the LMPs with the M RNA, we can see quite a lot of similarities between the bulk phase and the LMP or disperse phase. So I think we're still trying to understand and model this, this, this peak. So my postdoc advisor here in AstraZeneca Lena Linford he has been working quite hard into trying to have a proper model to quantify this structure to get more information perhaps about solving is not straightforward is something that is still ongoing and if you have any ideas I'm pretty sure that Lena will be very happy to hear them. But then I said at the beginning that I was going to say I was going to talk about how the LMP structure could help us to improve actually the development because we're actually care about the performance is very nice to understand the structure but if we want this to be a product if we want this to actually help patients we need to be able to develop better LMPs. So what we found from our kind of neutron scattering experiments was that the original formulation approach that I mentioned where we will add more pectin reduce the amount of cholesterol is this one in in blue, actually was making not only the size change but also the surface of the LMP change. So when we were having larger and larger and larger LMPs were having more and more DSPC and this at the surface compared to the other components. That means that the surface area per DSPC was decreasing. That means that the almost 140 nanometer LMPs were had a similar surface area of DSPC in a gel phase bilayer. So it feels that it was mainly just DSPC and not none of the other components. And then we started to tweak. So we wanted to have different size of LMPs, but that will always have the same surface as of our best performing LMPs, which was this around 60 nanometer LMPs. So then we said, okay, we changed the, we keep the cationic lipid cholesterol ratio constant and we change the other components we change the size and we keep the same surface as as the best LMP. And if you remember the, the kind of protein expression experiments that we did with the adipocytes the fat cells. So the original formulation had the best performance around the 62 nanometer LMPs. But when we changed the size and the surface of the LMPs then we started to see that actually protein production was increasing when the LMP size was increasing. We also had bigger LMPs had more copies of mRNA in them, but originally when the surface was just DSPC. There was not enough cationally perhaps to disrupt the endosomal membrane and help this to be released into the cell cytoplasm, but it had to be actually improve the surface to be able to deliver the package, let's say. So there we see it's not only size that matters for LMPs but also the surface of the LMPs that matters. Now I'm going to talk about two cases. So if you have any questions I can take now about just the LMP structure otherwise I just talk about two examples, and I'll leave some time as well for questions at the end. I have a question about the LMP structure. Yeah. Sorry my camera is not working. That's why I'm not switching on the camera. Yeah, so I was wondering about like have you looked into the structure of the CIL phase means lipid that you are like, you know, looking into your samples like you're using in your preparation. If I remember correctly you're using DSPC, and then you are using cholesterol, peg, and this cationic lipid, right? Yeah. Yeah, so in the cationic lipid have you looked into the structure of the cationic lipid as a function of like, you know, water content, how does it behave? So this is, I mean, this is a lipid so it's fairly water insoluble but we can look at the phase behavior in water. So it's typically cationic lipids for LMPs are designed more what we call a conical shape. So they have a very small head group compared to the tails. And the reason is because it's believed that this will help to disrupt the endosomal membrane in a more efficient way. So when we look at the cationic lipid at different pH, just like a bulk phase similar to what we did with the cholesterol, you can mainly see depending on the cationic but it's typically also an hexagonal phase.  So it would also be of course dependent on if it's charged or not so it will depend on the pH. Great also. But in your model that you have like, you know, obtained from the sax and sands experiment, sands basically, where the cationic lipids is inside the core that is inside the DSPC. So here I was wondering like how does it see the pH in this case like the change in pH because it's shielded by the peculiar and the DSPC. This is not, this is a bulk phase. So there is no, in this case, there is no DSPC or a PEC. So you take just the lipids that are in the core and you make, it's almost like a precipitate, the wide precipitate that you take. It's not an lipid and a particle anymore. So we're just trying to understand how the liquid crystalline, what's the kind of phase behavior of this mixture without the particle. So without the DSPC, without the PEC. Okay, I see that. Yeah. But it does, but it does, I mean, if you change the pH, like with the LMPs, it will still, I mean, the lipids are distributed across and there will be equilibrium of, you know, proteins in and out of the LMP. So if you do reduce the pH, it will induce a charge change inside the LMPs. So there is changes depending on the pH, on the structure. Okay. Thanks. And I mean, with that I will let maybe the future that you see that you follow Frederica and because we actually have some sense data of how the LMP looks a lot a low pH and it's completely different on how the LMP looks at high pH. But it's just still fitting the date. So I'm going to talk about the effect of the some, if you have any more questions, I can also take them now before I move to the next sample. I can also take them at the end. So the first is was the project actually that Frederica and I and Maritaz started to work together and my also my colleague Leonard effect of the protein binding on the LMP structure. And why do we care. The interesting is when once you administer and other medicines, especially through kind of intravenous administration and this is common for all kind of medicines that you administer intravenous or that is in the plasma, they will form what they call a protein corona. And I just believe that one of the main proteins in the corona of LMPs is a protein called apolipoprotein E, which is typically responsible for fat transport in our body, which makes sense because I'm leaving out a particle sleep it is typically fat. In the hepatocytes so this liver cells there are a lot of apo E receptors so that's why a lot of these particles and in the liver and hepatocytes. And since the LMPs are inside the they're taking in the in the hepatocytes via mechanism called endocytosis. Then the typically the endosome inside the pH decreases the lip cationally become charged and it's believed that this is what helps the disruption of the endosomal membrane and release of the cargo. So we wanted to understand what happens to the actually structure of the LMP when apo E is in contact with it, since we know that apo E is one of the most important structures. I don't have all the data, and maybe it's because I want you to go and click to the article, or maybe I can send you the article as well if you want. But we also did a lot of work to try to understand what happens when apo E binds to the LMP. And what we see is actually that the apo E, the apo E leads to a change in the structure of the LMP. It has been found in different studies that seems apo E selectively binds certain lipids. So it might be that the LMP can the apo E can selectively bind some of the apo E and potentially remove it. But what we can see definitely is following the binding of the apo E to the surface of the LMP, we can see that there's an enrichment of cholesterol in the surface. So this is the volume fraction in the core of the component of the volume fractions on the shell before and after apo E binding. And what we can see is that there is an increase of the cholesterol at the surface and there is a decrease of the cationic lipid at the surface and potentially an increase at the core. So there is there is a redistribution and why is this important. So as I said, we are trying to get as much of the cationic lipid to be at the surface because this will help disrupt the endosomal membrane. So if we are designing LMPs that have a certain composition then we add the product or we administer the proteins will bind to it and they will completely change the structure of the LMPs. This will affect how the LMPs perform. So we cannot, we don't need to, we need to understand not only the structure of the LMPs in our formulation before we administer, but we need to understand what happens when they're inside the body. Because we might have the perfect LMP in theory, but this might be a very different how it is inside the body. Then I'm going to end with the last example which is about the functionalization of lipid nanoparticles for subcontainers administration. So why subcontainers administration important for LMPs is because it's the most easy for patients to do in house. LMPV is more complex, I mean intramuscular is fairly, but patients for example that have diabetes or chronic diseases are very used to using auto-injectors. So subcontainers administration is a fairly convenient way for patients to self-administer. However to do therapies with mRNA for chronic administration, we know and I mean you might hear that LMPs can cause some kind of inflammation or side effects. And it's believed that this inflammatory response will be actually detrimental to be able to administer LMPs more frequently. So the idea was that if you could include an anti-inflammatory compound, typically an asteroid prodrug, this could reduce the inflammatory response. But to be able to actually reduce the inflammatory response, this prodrug had to be at the surface of the LMP to be able to allow to enzymatic cleavage before it can enter the cell. So a lot of the molecules were designed and this was also done with some of my colleagues here, Alexander who was also a boss of Tommy before. So we decided an anti-inflammatory compound that we could, that it was deuterated and we also look at how it looks using a small angle neutron scattering. So what we could find is actually find that the anti-inflammatory compound was luckily mainly on the shell. And that helped us actually to design an LMP that had anti-inflammatory properties that we could administer more than once and keep the same mRNA protein without eliciting an immune response in the body. So this was really exciting. It was also published very recently. So if you want to learn a bit more about the study details there's also this presentation here. You can also send papers if you're interested afterwards. I'm going to summarize and I'm going to leave some time I guess for questions. So I hope I convinced you and I hope maybe you didn't need to be convinced because you have seen in the news that the LMPs are the leading delivery vehicles for RNA therapy. To understand how to improve LMPs we need to understand that their transfection efficacy is not only size but surface composition dependent and this will also translate to other nanomedicines. I hope this is the last day of the science course that I don't need to convince you that science is a powerful tool for characterization and development of mRNA containing lipid nanoparticles. Also that binding of proteins such as APOE can cause changes on the lipid distribution and that's what we also need to understand not only how the LMPs look in the formulation but also how they look in contact with Plasman. And that we can actually design more complex LMPs and for example in this case we were adding an anti-inflammatory compound to reduce immunogenicity but it could also be a targeting ligand. And that can also help us to improve the LMP performance if we want to go to a specific part of the cell or if we want to get a different response in the body. And we can also use science to help us characterize these LMPs. And with that I think I am in okay time Tommy so if there is questions. Very good timing Mariana so please ask questions. Mariana I have one more question. Maybe you have mentioned this I missed it in your presentation but one thing is intriguing to me like when we are using this lipid nanoparticles as drug delivery so when you are administrating it like under the skin or it's directly into the blood stream. And how does it reach the target sort of what do you think how does it reach the target? I guess it depends on what's the kind of indication what are you trying to treat I mean if it is a vaccine which is more of a create an immune response so there is different ways of administrating LMPs typically is IV or intramuscular now for the vaccines. So you have systemic administration and you have also have local administration. Okay, so if local administration is more prefer when you know that to reach a target cell is more complex let's say I think maybe I should go back to the first of the mRNA development. So I mean mRNA although there is the most promising application is vaccines at the moment for mRNA, mRNA therapies have been developed for many different diseases because a lot I mean it's not easy to deliver proteins and diseases that are related for example in the brain in the lung. So for example, I think there is an example here from the 90s where they were looking at mRNA injection into the brain and this is an animal model of course for protein replacement I mean this is something that you would have to do of course locally because to do to cross the brain blood is very very difficult if you have to do this type of therapy if you had to do it in the lung then it might be easier to do inhale LMPs but then that will be a different way of challenge for both the kind of the development of the LMPs to be sure that things are going to work in the respiratory tract and also the LMPs are stable in whatever formulation that you have because it might not be the same type of formulation if you're going to do inhale, if you're going to do subcutaneous, if you're going to do intravenous. Typically I don't think that an oral LMP will be a product because that will be completely destroyed by the gastric acid. Yeah exactly, yeah that's what I was thinking also. Can I ask one more question? So I was wondering like if you induce any foreign particles like for example then already your immune system starts marking it as a foreign particle right and then you have this. I remember it really in terms of like you know when you are having this colloidal particles that are being put like you know that are inserted that you have this protein corona that like you know surrounds it and it practically then it cages the particle inside and then marks it for degradation. So in this case like means how does that this LMPs avoid that step of the immune system. They don't I mean. So your body will start identifying LMP so if you have like chronic or continuous administration of LMPs your body will start I mean it depends of course of everybody's immune system but they will start identifying the different components of the LMPs and they will start clearing them pretty fast your body is pretty smart. So it doesn't avoid the kind of like immune response. It does decrease it because compared to catechonic permanently charged catechonic nanoparticles. These are much easier to identify by the body than the catechonic and I saw a little bit kind of hides a bit in the kind of plasma but there are antibodies that for the peak. I think that's one of the most thing that it was hypothesized on why some of the vaccines had a potential side effects for some people like this antifelactic shock that could be related to the peak. So, so I mean, they can cause an immune response so we could think for example the first sRNA therapy that was done for a product that is approved at the moment on Patron. And this is done to basically knock down a protein production of a protein that is really unfavorable. It's a protein that is misfolding and forming amyloids. But you need to administer that product with dexamethasone, which is kind of like anti inflammatory drug otherwise your body will recognize it and will recycle it and stop the effect. That's it. Thank you. So any more questions? I see Trevor you have unmuted yourself so you want to say something? I think Trevor knows this work because he was also working with Ferik and myself. Well, thank you for inviting me. If you have any questions you can also reach out to me.


Welcome to Toffy TV. Everton have made a signing and it is Adritha Garnagay. He is back after three years? Three years? Three years, isn't it? Three years. Three years at Paris Saint-Germain. Finishing school, essentially. That's what it was. He's been away finishing school and he's coming back now. He's coming back on man, ready to kick on his career. Ready to kick on. Board of play with Messi and Mbappe and Neymar. Saying that was finishing school. Now it's the big leagues. The farmers leagues done with. Now it's the big leagues. And he's coming back and he's going to take this everything to the next level. I don't know what level that is, but it's the next one. Well that's good. He's back. He's back.  He's going, Messi gives the controller. Now obviously three years away, left. Obviously an integral part of Michael Silver's evidence when he left that summer. And we got John Felicobamman to the placement. John Felicobamman has gone out and Garnag's back. Now obviously he left that summer and it was a whole that Everton never fixed. Actually we said this the other week that Everton are signing Garnag to the place Garnag. Because it feels like that since he went. No one's really gone in there and took control of that midfield the way he used to. But obviously people will be rightly saying 33 soon and is he still the same player who left and all that. But in some respects if Everton have got the legs of a woby and obviously the core is in the squad. And then you've got Garnag. Then he can just do that little role of patrolling. Even though at times for PSG he's actually played further forward and scored. Which I always said he could do. We saw it in the last few months when he came in for him didn't we? He went up unbelievably till the end of the season and played a little bit further forward and scored. It'll be interesting to see how obviously how it all fits together for Frank Lampard. But he haven't been interested in him and this deal was in theory done a few weeks ago and obviously hit the buffers over. But he got it sorted and he is back at Everton. Yeah I think I can understand that people saying you know there was weird names like Mo Camare for just a little bit more money. And all these kind of things and obviously someone with a big future. But maybe right now we need a mix. And having seen more experienced players like Connor Cody and James Tarkowski helped within the leadership side of the team. And I think having someone in midfield that kind of experience. And the experience he's gained whilst being in Paris Saint-Germain winning trophies and Champions League runs there when they got to the final. And obviously they never won it.  Training every day at the level he will have been having to train at is only going to make you a better player. Of course there's going to be a question mark physically about him. But when he left I mean he was in the form of his life when he left Everton. And he hasn't played lots and lots of games and of course as you get older you're more susceptible to injuries and that kind of thing. But I think for me it is... I'm not saying a no brainer because we've been here before and there's different... For every Garth Barry there's Fabian Delph and it's where he falls. But I'm confident and I'm hoping that if he gets anywhere near that level and he's got Oana in there and a Wobie. That can really change our midfield up and if we're playing that three and give us someone who you know is busy can get round the pitch. Can sense the danger. Can allow the full backs to get forward by filling in and just understands the Premier League. And listen the Premier League I think since he's left has probably gone up a little bit because it just seems to go up all the time. But this is our fellow who understands it and this is not alien to him. This is not new to him. And then go all those things combined and him knowing the club and obviously he'll probably move back to where he lived before and all those kind of things. It's not all those things. There's no settling in period. It is a very simple transfer I think and to get someone back that the fans always liked, respected. He understood when he left going to somewhere like Paris Saint-Germain so I'm happy with this one. I am happy with this one and I think it just gives us another option in there. And it gives us an option that I don't think we've got at the moment as well. And yet you would like a younger version and I hope that's what he'll do in the next two years. It gives them time to maybe find that next version a much younger one. Again, for all the reasons you've just said, he knows the club, he knows the training ground well. Yes, a lot of the players. He knows the city. A lot of the players have left as well when he was here as well. So there is that but there'll be obviously ones he'll be playing with. She must be one of the biggest ones still here and stuff. So there is that thing coming back in and the league might have moved on but then again he's played against Manchester City squad, against Manchester City and the Champions League. So he's got that experience as well, generally a fit lad. And with all of that experience you can come in and you mentioned Gareth Barry there and Gareth Barry was a supreme footballer. And just sat in that middle of the park and dictated. Garn is a little bit different in terms of putting fires out and stuff and very energetic but he's been very energetic last season. Just looking at some of his numbers from the data from last season, he's under 97% for open play passes. Obviously his passing game was something that maybe went under the radar last time with us. It was more seen as somebody broke the play up but he is a better footballer than what we've probably given credit for. Pressure passes in 91% XG build up 97% out of obviously the top 3-4% we're talking here. For pressures on the ball than 94% for pressure adjusted, pressure 99% for interceptions and tackles 98% for defence of action regains getting the ball back. 80% pressure regains 86% 75% for interceptions. So all of that stuff which is going in and it back and winning it and getting it. All of the things where we've had midfielders over that since he's left. We've all been good at some things but no-one was ever able to do what he was able to do. We hoped that Gabbamon would do it and be a physical and more physical version of Ghana. And obviously he was a lot younger, 22 when he came at the injuries and that hasn't worked.  I've said before Alex O'Wobee's legs and getting all over the pitch and obviously O'Wobee played with them maybe four. And also having D'Corae in there as well, someone who hasn't played with but who can come in and do his job as well. That kind of thing just gives Frank Lampard real options you know. And it is like you said before, we are able to mix the make-up with the midfield up a bit now and having him do that will make a big difference. Let's have a look at some more of his numbers. So 26 games last season, three goals, three big chances created. Pass accuracy 93 and tackles per game 2.3.  But you know he's played a few games there and the pass accuracy and the tackles and stuff, you know more in his range. So I think it's a, as I said I think it's a no brainer. I know you can look at the negative side of it and I understand that and look at the other point of his age and the girls. For me as you're trying to build and get it, as you're trying to change the way you're doing things, you do need that little bit of experience. And I think you know he's got that and I think he's a brilliant player and hopefully he's got still got plenty in the tank. You know it's a talking, it's two plus one aren't they, the option of a third year taking through to 35, 36. But like you said just before it's hugely important that everything look now for what his replacement looks like. You know if we're expecting a no brainer to do that he's going to be in the team anyway. So if we've got anyone in the club we can do it at the moment so we don't know how long Yango's going to play out. He's on loan currently, he's Lewis Warrington that person, not sure. But it also means the club can look and look for the next mo camera and go there's one and we can get him. And hopefully what the club start doing is getting these players in and giving them, making them a squad player while he's still in the crossover which they've done. Obviously we've done it with Nathan Patterson which is really good. But we haven't done it enough and that's the next challenge. But going and coming in, just strengthens, Frank Lampard's my field options and that can't be a bad thing because I think a lot of people last season looked at my field and I was just like it's just not good enough. Absolutely. There you go.  Are you happy to have him back? And where do you seem fitting in with the older players? Let us know, we are happy to have him back. So let us know your thoughts. If you want to check out more videos, cruise to videos, join us over on Toffy TV. Premier the link is in the description and it will be on the screen in a moment. See you later.


I'll move on to the next one now this is yet another example where I wanted to give you a flavor where people are using web services calls over the internet also now this is an example of a bank I think in Iceland which has enabled some of its services over the internet and we were actually working with the customer who is on the left we were maintaining their applications and here again we did a pilot this was not a production use maybe now it has gone but this about a year ago where we worked where we wanted to show them or they also wanted to be convinced that we could make a web service call and more important was there were a lot of financial data this is about transferring credit card information between a credit card processing company and a bank so there is a lot of sensitive financial information which is getting transferred and we showed web service security again you all have come across so we kind of implemented web service security for them the bank hosted a secure web service but then there is also a lot of work to call a secure web service so your application also has to have the appropriate security setup and then you have to utilize the web service security standards and the APIs there and then the exchange of digital certificates and so on which is in the picture which was you know kind of executed the idea here is that to put up this slide is today people are also kind of using it for transferring financial information sometimes you know you get the picture that web services are called more to get you know non-mission critical informational kind of you know services only but that is not the case things are changing on that front as well okay this is a case where you know where one of the insurance companies they have used the ESB more to integrate services so the lower layer where you see there are different kinds of sources of information which they have over time they have their customer information in the CRM system the policy and the claims related information in the legacy application which is shown in the middle box and some other information in their databases and now when they you know as a part of the initiative to become more customer centric they have you know self-service kind of portals and things on their website so how do you do that so they have used the ESB approach where they have service enabled their applications and then these are available on the service bus so to speak so that's what is illustrated by the middle layer and that can be consumed by you know the search insurance policy search customer kind of modules in their web application so they have started off this way okay and then later on they they want to at least the direction is they want to do some kind of process orchestration as well okay but as far as I know the first two things that is the search customer and the search insurance policy kind of applications were done using the ESB approach to call the different services so here if you see it is not a if you compare it with the first case there we have used direct calls to the to the service okay whereas in this application there is a ESB in between of course there is no protocol conversion kind of things which are there but then this is more done with the future in mind so that when more applications would be consuming web services they have done some kind of upfront investment okay yes primary needs a web application but then the after the web service enablement which we have done these are all dotnet thick clients windows clients yes this is just an example okay you could have a web application also consume web services sorry you have created okay okay it's a simple windows based form application yes yes so the question is this is more about the windows application and how would you distribute okay so yes so there will be a exe which you would have executable which you would create and distribute it to different users internally when it executes it would be making a web service call to your I'll try to clarify it using diagram and maybe then I'll take your question when you move on now I'm describing the system how it would look in this new way of doing it so you would have essentially a client server kind of environment this server would be exposing some web services okay and then the rich client or the thick client which I have would be the in web services terms the web service clients so they would be calling the web services number one and the question you asked earlier so this client exe will have to be installed on multiple desktops right that was your question right earlier okay sure yes see one of the benefits here is in this environment it was all windows application so it doesn't you know kind of bring benefits of web services on the table but imagine that this was a just to kind of give a different flavor this was a java application you could still consume it using a dotnet client which otherwise is very hard or next to impossible kind of thing that you wouldn't try to do that okay so you know different kinds of clients so once you have a web service the interoperability mechanism you can put to use by using a thick client then I told showed you the the mobile kind of applications also they could use the same service okay and of course the web applications also yes right so the question is now you when you want to service enable you have to look for support for web service development and deployment so as far as I know vb6 doesn't have that okay so what you would have to do and we have done the same thing here is you may have to put a layer of dotnet in a get it into picture because dotnet natively supports you know web services development idea is much more easier okay so it will have to coexist along with the or vb that's the most easiest way I can think of right now okay the number is what is the process orchestration orchestration okay I'll go back to the explanation I had in bpm I'll paint the big picture first and then I will talk about the process orchestration part we have four essential phases so to speak one is model so you model the process execute what bpm says is you first model the process create a executable process so wherein we may have to use tools we know which will generate a bpel so there is certain tasks which the role we say the integration developer will do this you will actually tie it to the application services you will use decide on the services which you are going to use and build the whole executable model okay at you deploy in a orchestration server this is where the orchestration server will come in the picture this is where you will create your bpel after you execute you realize that maybe the process requires tuning or there is a external customer demand which is necessitating some change okay many things are there which can be kind of uncovered by monitoring okay and this monitoring is not at the level of technical monitoring of what is the web service what is the data which has gone but more at a business level where you say okay my purchase order process which I talked of it is taking there is a bottleneck somewhere and you know it is taking larger time in terms of hours which my customers are not getting their acknowledgments so what do I do but then before that you have to measure it or you have to have measurement now it is taking seven hours on an average at least that number should be available to you so with monitoring and that is more of business activity monitoring is another word these vendors have been using so you get to know that and once you have the monitoring information you have enough information to change your model so you rework on your model optimize your model you know these are things people talk of and then the whole cycle again we runs so now to answer your question the bpel and the orchestration engine come in this half of the bpm cycle yes now another interesting thing is many people also because the bpm implementation requires lot other tools for modeling as well as the monitoring part so there is a in-between kind of solution that there are tools to develop the bpel okay you must have seen so you just do the deploy and the execute part you don't do the full bpm that is also being done okay the the mindset is not to have the entire bpel sorry not the entire bpm so as to be more model and monitor also so you remove that part you just have process orchestration as the objective and do it right any other questions right right yeah today unfortunately we have not yet matured to such things yet at least not to my knowledge where you know you put in some figures and you are able to have a calculator kind of of thing which which which you will show you yeah so that's what even this the model or the diagram which I showed you earlier so we have we have to use it to you know get at a effort estimate for ourselves also okay so we have to break down the problem okay there is no entire you know first of all what should I say formula or a calculator I said so you'll break down the problem number one second is as I said the SOA adoption is a journey it all doesn't happen in one shot so you would you would find that you may want to build some applications and that in the design you would propose some services so in our kind of environment this would get budgeted in the project cost okay there are those certain customers who go upfront and say okay I want help in my SOA adoption there what we do is we kind of have some you know kind of metrics where but it's more on gut feel I would say where based on our experience we'll say okay these are the service we'll of course go talk to the users business users understand the requirements and then evolve what kind of services they would be requiring look at what their existing applications are okay then we get an idea what we are going to you know use the which of the legacy which we will use to convert to services sometimes we may have to develop new applications with service interfaces as well then we would go on to say which applications will consume them so that their entire kind of solution requirement is satisfied then when doing that we will say I mean this again see it's a investment mindset which the customer also needs to have right but then you know when we look at technology we will be talking in this way but when it's a customer what they're going to say is why should I do this I'm going to in my next slide there's a business case yeah so you require actually with SOA what I saw is that you require a little bit of investment mindset then many things get very easy service creation level I think our traditional uh you know methods still work but when you actually move on to a solution which will which will involve say ESB kind of things so there what happens is nowadays lot of uh you know requirement is of configuring those ESBs properly so you have services to be developed and when I look at the whole solution perspective my solution is not complete until I configure the service and build the application so the building of application and building of services your normal software development life cycle still largely goes on as usual but then the configuration part actually you know over time I believe again we have not done many of these installations such but that should become a routine or we may have some thumb rules for doing that we have not exactly yeah you may have to develop them you have to taste them and then people are new to this concept as well okay yes yes that's a very good very good question you know when we make a service usually it happens that we have some application in mind and from that we are driven okay normally at least I have not seen where people are gracious enough to say we will develop one service without any application in mind and then we will you know later date we will use okay having said that so one is we are there is a motivation for making that service because that's the application in hand which we are developing and we get kind of driven by the requirements of that application but then the whole point gets defeated if it is we are not able to reuse it okay because the whole underlying you know notion is we have to be able to reuse so in that sense we say the service has to be generic this becomes a little conflicting requirement so you have to keep in mind that so the testers have to be kind of have that broad mindset that this service will be a generic service and test with that way so you have to do additional test cases also so that this becomes generic only thing problem is there are no clear definitions of what will when will it become very generic okay so that's the experience part so this is the last slide and more to appraise you or make you aware of what are the difficulties which we have you know encountered in the field and let me before I move talk about this the earlier case studies also which I talked these are some of the illustrative case studies there are vendor examples who have done much more than this they were implemented bpm so what I'm trying to say is there are a whole lot of work which different companies do so what I'm going to now share is what has been our experience so one thing you know in the web services world last three four days you must have seen it's all about standards good part is it is a made out of consensus and then you know many good things also come in the standard but the problem is the standards are ever evolving it doesn't stop okay so for us for developers like us who are developing applications we have to be you know constantly having an eye as to where the standard is moving number one because if we want to have the new ones we have to be ready second is related to this you know second point the tools have to be available to us which will implement the standard for an example if I have to give you are using they were using jboss or net beans so you have a net beans environment which will generate soap with some standard which will say 1.2 and then give you with wsdl 2.0 and things like that now if you go on to add web service security to it okay and then BPL and things that all that has to kind of standards have to be kind of what should I say supported or compliant by whatever are the requirements of the whole infrastructure and that is where the difficulty comes because you start off with one tool you have developed your service you move on there is some you know time delay then you are going to build your orchestration at that time you will find that the orchestration engine demands a higher version to be supported on the service so then you have to break your service and redo it kind of you know I'm little bit exaggerating but these are some of the situations we have landed up in so web service security which was there it took a long time for the standard to come this about two years ago story of two years ago but then it was very hard to find tools which would implement the web service security number one second is you may have situations where you may have to use tools of different vendors okay because you have the service framework may be a java kind of environment so you would use net beans and you know some kind of software to implement it but then the user on the user interface side or the consuming application is dotnet now to be again to make soap calls you may have to use the dotnet say 1.1 framework and then they have some other enhancements also because the standards are changing there are a lot of enhancements also which come into the picture okay and then they don't interoperate okay now again I don't want to say that you know that problem this problem has been recognized it has been solved also to certain extent but I want you to be sensitive about it that this can happen today there is a organization called web service interoperability organization whose core job is to work out these problems of interoperability but then in certain areas where you might venture you may still get into some problems of evolving standards and the tools not being mature enough so you may be working with the older tool so that's where you may get down really get stuck okay third thing is about the interoperability which I talked of when you are making you know at a higher level we say okay it's a soap call we can even look at the soap message and you know thing should be all fine is what we feel and nowadays there are all tools which allow you to not take the wsdl create proxies and then you just create a client and get done but then that is not really the case when you have to build real applications you have a lot of complex objects for an example you know the data structure itself is complex what we have seen is sometimes you have issues with serialization of those objects or you have to take special care to serialize that kind of objects normally whatever we try you know while learning phase is all simple data types and we you know they move back and forth much more easily but when you have complex objects and out of different systems because so when you say you could have a cobalt system you could have a dot net system you have a java system so in all this whatever data types you are going to use across for the application have to be supported so that's when this interoperability kind of situation becomes more critical so this is another aspect you have to think of and you know as a project this is a kind of risk which you have to identify and that's where the proof of concept kind of things helps you if you are kind of trying to do something for the first time in the sense suppose you're going to have an array of objects which you are going to transfer I'm just giving an example over a web service for some reason then if you have not tried it out please ensure that within the environments which you are going to use make a small case try it out and be sure that it works okay and then you can build on onto your in your entire project okay so adoption in India unfortunately we don't have much customers who have done in our customer base but what I gather because we also in part of center of excellence we talk to a lot of vendors and there is a lot of movement in companies because one of the advantages which we have in India is you know we didn't have too much of computerization earlier so whatever is happening is happening now or lately and with new generation of tools and these new generation of tools actually help you you know doing SOA on these kind of tools is much more easier so for an example at least what I know of in the segments of say insurance and telecom and things like that and very you know a lot of multinationals have come in and the kind of applications they are building they're all talking of you know having them as SOA based and utilizing the benefits of SOA because one of the things if you see now we look at our ticket booking we have internet sites banks which are giving us you know self-service kind of things even insurance companies giving you self-service kind of things when you want to you know be all this customer centric and give facilities to the customer behind the scene you have to have these kind of systems otherwise it becomes difficult it is not that you can't do it so to answer I mean though I have not been involved myself but I hear and that is what I'm saying that there is a SOA adoption in the new kind of applications which are getting developed now people are aware and you know adoption is happening fourth point that's the developing a business case now this is one of a big problems and this is not a technical problem though it's more about budgets for the SOA project see the the if you look at it closely what happens is you develop a service okay we say we develop a service it has to be a reusable asset and to be used by multiple applications but then as I said it doesn't get created that way it is done as part of some project now imagine I am doing a project I will make the service I will host it then I have to have the mindset to be able to share it with all of you okay and when it is departments there's a lot of cost consciousness of between the departments also okay so in organizations where they don't have these mechanisms of cost transfers or you know sharing of shared service centers kind of things it becomes very difficult so as I say it's not a technical problem but that's the reality because you see why should I build that the question is like this make that extra effort to create a service for the for everybody is good you know so funding becomes a problem and there are organizations grappling with this problem because how how do you fund because this is if you take it in the project cost that's going to increase the project cost for the project executor okay so what we are also seeing is now many companies recognize this problem and then they are having some kind of mechanism for corporate funding okay then it becomes easy so the corporate funds and then apportions the cost to a lot of them but that mechanism is not there there's a lot of problems for taking SOA projects of the ground and you know you may have similar that's why I brought it up here you may have similar situations in your organizations also where you know how do you get approvals for this lastly creating a SOA roadmap that's again is a you know you have to have a very long term view of what you are going to do because as I said it's a SOA journey it is not one project where we are going to build some application which will consume a service and finish it is going to be there for a long time to come the benefits to accrue the return on investment that word somebody used to really get that you should have at least two applications using your service when the second application is going to come up what state I mean what state our service will be you know in what is the infrastructure which will be needed are we going to have the service integration or a ESB kind of requirement people's mindset to use services share services all that is there you know these are problems when you want to create a roadmap what all do you think about so you know we kind of have now started getting into engagements where we also sensitize people about these aspects and work with them okay these are your department different departments get people there start with the basic education okay saying that how it is important you know what benefits it will bring in it's an investment but then over the long term it will pay it for you and things like that then have a buy-in and then you know depending on the landscape which they have what kind of systems service enabling prioritizing which systems to service enable first what is the infrastructure which they have to put the cost aspect which you are asking me you know so some ballpark figure we can't give them very precise estimates so we kind of give a plan and that is very very kind of essential requirement is what we feel so that they are able to budget it over years yeah so these are but very difficult tasks still yeah so these are few things I thought might also be you know relevant in your organizations and it would be good to know so that's all I had to share yeah the service interface changes so if you modify the services still you have to question what is it which is going to change you know one of the things probably they must have talked is in a web service the contract if it remains intact nothing is going to get affected if the implementation is going to change no problem okay changes yes yes so interface changes yes your application will break down because the whole the soap call will not be processed on this side because it is expecting in some form but if I modify just say some part of the services yes which part is the question right there are two parts there is an abstract part right and there are the for example if you create the part on which the service is running it won't work the board has changed right if you change the abstract part your interface has changed so it has been compiled so it depends on that back and implementation changes doesn't matter so as I said like that when you are making any services specifically to some application in mind now later on you need to design that service so the interface would change so most likely you will be compiled in that case there will be a versioning issue yes to the whole point of reuse everybody breaks so how does SA handle that versioning issue yes now I would say the simplest answer I would say yes it will break so there is a option of versioning which you have but then you can make little intelligent clients because there is a concept of discovery which is available you could figure out if there is a new version and then try to use it but then it is not easy okay so you have to have mechanism so you will have to rely on some other mechanism to be able to detect that there is a version that is one way second is there are tools today available which work at the soap level at the infrastructure level which will allow you in certain ways to deal with two different services so that mediation example which I told you okay but then that's not an automatic process you will have to do some intervention there but you could still not change the client and still make it work but these are you know some non-standard ways or not a very neat ways of tackling the problem that doesn't have any mechanism to indicate that this is the version of this something like that and a mapping between the two versions in rmi if you remember it's not part of the standard at least not it's not part of the right it can it's at least to my perspective and so you can you know add to that it's certain tools may give you that certain render tools may give you that but that's an convenience but just because a server side versioning doesn't mean that your clients will continue to function non-interrupted right there is still has to be some way of changing a call from a 1.2 to 1.1 point over the whole point is we need a mechanism and in the standard as sir said it's not there we will need a mechanism at the client side to detect this change first of all and when you write clients do you really worry right now see the other point is we are also kind of taking for granted that this service interface and we are writing clients that way so if you want to really if you know future proof your application you will have to get all these aspects also into the programming of your client yeah but I have not frankly seen any examples right in this direction but this is just my thoughts.