I was sitting in front of floor-to-ceiling windows on the top story of the building where I worked. It wasn't a very tall building. The view was was enough. It was of a free way that I was going to sit in traffic in in a few hours. And I felt like I was succeeding at a lot of things. I had deployed Kubernetes for my organization. It was automated. I had set up all of these things that let developers use templates and deploy applications quickly. And I was a good sysadmin. I was a good systems engineer by automating those things. And even if you're familiar with Core OS at the time is what I was using, I had automatic updates, ability to have automatic updates. And most people turned those off because they were worried that things would break. I left those on and I had this cluster set up where it was a bare metal cluster. And my automatic updates would deploy updates every weekend for me. I set up a time that they could do those updates and I would come in on a Monday and the cluster was upgraded. And I was just like, wow, I have made it to this peak of Kubernetes, this peak of sysadmin where I had automated some of my job, and I didn't have to do that anymore. And I was sitting in front of these windows because I was writing a book about cloud-native infrastructure. And I was writing it with my co-author, Chris. And I was waiting for her call. And we had one last session to brainstorm because we were writing this book and it was focused on things we learned in Kubernetes and in the cloud. And we got on the call and we were going over a couple sections of the chapters that we just didn't know how to put it in words of what we learned. And she said something that I'll never forget. She said, if all of our infrastructure is APIs, then our infrastructure management should be applications. It shouldn't be a repository of automation and a repository of code. It has to be software. It has to be something that is running that manages those APIs. And everything that I thought I did right about Kubernetes was, I had just built automation. I had built the same thing I had done over and over again. And I just automated pieces of it. But she was right that we had to write software. We had to create software that controlled APIs. And software is all about just taking data, changing that data, and calling APIs. And pretty much every application and every piece of code is doing something on those lines where it's taking something in and it changes it for whatever you want in your business. And then it calls APIs. And if all of our infrastructure is APIs, then our pieces of software that should be running to manage our infrastructure had to do the same thing. We couldn't rely on infrastructure as code anymore. Infrastructure as code was just the automation piece. And it didn't scale as well, because you had to trigger it certain times. You had to make sure it worked. And from that point, I was like, what is a thing that exists that does infrastructure as software? What is something that is running software that takes in data and calls APIs and then applies that? And the very first thing that was obvious to us was a Kubernetes controller. And a Kubernetes controller just calls APIs over and over again. And it looks at the state that it wants and it looks at the state that it needs. And it makes it happen. And that was the very core piece of infrastructure as software, this idea that, oh, we should actually be managing our infrastructure with these control loops. And we found other things, once we started looking around, this pattern of infrastructure software showed up other places. If you're familiar with Netflix has chaos monkeys, chaos monkeys is like the opposite of infrastructure software, but it does the same thing. It's software that runs, that takes data in, calls APIs, but it breaks things on purpose. And these chaos monkeys would kind of degrade the state of your infrastructure on purpose with software. And it wasn't a one-time get push, it wasn't a repository, the chaos monkey had to constantly look at the state of the world and make sure, oh, is this right? Is that right? But both those things weren't GitOps. This was before GitOps was a term. But it fit really well. Once GitOps was announced and kind of solidified into these four principles that we've been hearing all day, once I heard about GitOps, I'm like, it's infrastructure as software. It's exactly the thing that we learned in Kubernetes controllers that we saw over and over again at large scales, at very high impacts in high velocity environments, you had to manage it as software. And GitOps is a core definition of that. It's an implementation of infrastructure as software. It does this control loop, it takes in that data and it calls data. And when I like to visualize things, if anyone's ever seen me talk somewhere, I like to use props. I like to use things that are visual. I'm a very visual person. And so if you've never seen these, these are AWS snowballs. They have 52 cores in them, 256 gigs of RAM. They're snowball edge devices. And I'm using them today to represent, like, standing up our infrastructure. And this is essentially, if you're using AWS, it's like a 16 extra large. It's like, that's how big one of these boxes is. And we'll shoot them to you. And you can run edge compute with them. You can put storage. I think they had 42 terabytes of storage. They're pretty nice to have at the edge. But this was what I was thinking when I had on-prem servers. I would stand these up. And what do you call two snowballs on top of each other? It's a snowman. But when I'm running infrastructure, I would deploy it. And I would run these scripts that would stand up my infrastructure. And it would get it all set so that every time I wanted to run it, I would trigger something, Jenkins, whatever, it would actually deploy this. And it was great because while I automated that piece of it, it was, I couldn't touch it. I couldn't do other things to it. And realizing time and time again that infrastructure is code again, it wasn't enough. Because someone would come along and do something to the infrastructure. Something would break. And nothing brought it back. Nothing stood those back up. Nothing brought it back to the desired state until we ran that infrastructure is code again. That repo of code that we had that stood these up, that's what actually would bring it back to a state that was like, okay, now we're good. And the main thing there of those controllers infrastructure or software is you always have something watching this state that you don't have to worry about like, oh, it's going to go. It's nope, nope. Software is constantly looking at that desired state and catching it before things fall over. Or if it does, again, it'll bring it back to that state force. We don't have to trigger our CACD. We don't have to do this stuff. I just like knocking these over now. No one tell my coworkers. They're rugged. It's fine. But I wanted to come and talk about why GitOps is really powerful in the things, the core principles of GitOps illustrates this really well. And yes, you're going to have more than one controller managing infrastructure. You're going to have Kubernetes controllers. You can probably have cloud controllers that do different things. There's all these controllers that are implementations of software. And the first thing that I wanted just to touch on again is this, what is the code piece? The thing that we always have is infrastructure is code everywhere. Everywhere infrastructure is code. And if I really like, okay, that's fine. We know running this isn't infrastructure as code, right? That's not it. Because you can't run it at your command line. You don't want people manually doing this. So what you really want is something like this. Let's echo it so it doesn't actually do something. Right? Is that infrastructure as code now? That's what we just did, right? I mean, that was essentially, I have infrastructure as code. I've made it, right? But that's like, it's just the automation of the thing, right? It's still not any better. And GitOps again is very specific in how we're implementing this. And if you didn't notice, at this point, I have code. When did it become software? Where was the piece in there that we're like, oh, now it was, oh, it's done. It's no longer software, right? It's like, software is code while it runs. It's only in that running state. It's only the code with electricity applied to it. When it goes through the processor, then it's software. And if you have a repo that is code, that is scripts, that's not software. It's static and that's fine. That's your automation pieces. But how do we break this up to say, okay, well, we need this to be applyable. We need it to be always running. We need it to be software. And again, as software, we want to separate our needs of the data we bring in, the variables we're taking in, and then the APIs we're changing things with. And for a lot of people, it's going to be, like now it's kind of software. Terraform is a controller. Terraform looks at a desired state, it looks at the current state, and it reconciles them. It's just a command line version of it. It runs locally. It's exactly what a controller inside of Kubernetes is doing. It's exactly what it fluxes and GitOps is doing. It's doing the same sort of reconciliation. And that's fine. But it's, again, it's not what GitOps is proposing. Because in this case, you could, right, like that's continuously reconciled at some level, or Terraform is going to always just do this. But that is really bad. That's going to hit limits. That's going to hit all sorts of things that doesn't scale. We need GitOps is a very specific way of doing something like this so that we can help it scale. And I really like looking at the principles because that helps us and that informs us of what other things are doing similar things. And they all kind of go along the same route. They all have similar principles and how they're going to scale and what they're going to do. So we looked at software as far as only when code has electricity applied to it. And so here's these principles that, again, you've been hearing them all day. I'm not going to repeat every single one of them. But this declarative piece is all about separating out the step-by-step from data, from the data that feeds a controller. We're declaring a state. For a long time, I falsely believe that declarative meant nothing was imperative. And that is absolutely not true. When I run Terraform apply, I declare my state in my TFRs, my Terraform manifest. But Terraform does everything declaratively. It builds this DAG that then says, okay, I need to do this first, then I need to do this first. That is exactly imperative. I just didn't have to define it. So for the longest time, I thought, oh, controllers are declarative. I'm like, wait a minute. That doesn't make sense. Something has to say this goes first, then that, then this. And the important thing is to have computers do that for us, and not in a script. Because humans have a lot more assumptions with a lot narrower scope view of the state of the world. And controllers and flux and Terraform can get a more holistic view of what's going to be applied. And that's when they can actually see what needs to happen in order. And again, it's not that declarative means nothing is imperative.  But something back there does step by step by step. So what we do is, oops. So there's my declarative state. Did I put that in here? Yeah. So I have my TFRs in there. That's my declarative state. But what is immutable? Okay. That's it, right? I'm immutable now. I can't write to it anymore. That's all we did. Like, that's all we had to do. And now we're immutable. Congratulations.  This is the fundamental piece of what we want you to do. We want you to be immutable. We want you to be versioned. And the main thing we want to do is actually, oops. See, I got permission died. I want to version this in order. I want to go to the next version. And so in here, what do we need to do? We got to, let's see, sort of first, sort that. Let's find all of them. We only want to apply latest, right? Because that's we're good. I think that'll do it. I think it's something like that. Let's see. Feet, version two. Right, there we go. We're now only pulling in the latest version. So every time, thank you, next door. Every time we're going to run our infrastructure as code, this is still just code, that becomes software, it pulls in this latest version immutable state. It's declarative, it's immutable. We're almost there. We're building our own GitOps with this infrastructure as software mindset. Pulling in GitOps is two very important things that I really like, that they put this as a core piece of GitOps.  And one is for scalability, because if you have one or two clusters or one or two servers, you're probably fine. If you have a thousand, it's going to be a little bit harder to push that out and push that everywhere. Whereas a pull states, it's a lot easier for us, we've just, technology in general, have known how to serve some files for a very long time. We can serve websites, we can serve static stuff, we can cache them. But the compute side of it is intensive, and all of the API calls that result in that action happening takes a long time. So we pull that data first. We want to make sure it's stored somewhere. Yeah, you store it in Git. Yeah, you store it in a web server wherever. But that's one important thing. And the security aspect is the other one. Because if you haven't been paying attention to any sort of breaches over the past five years, a big way in the door is CICD systems that have global admin access everywhere. And that is difficult when you can just get to one system and then you can move horizontally anywhere. And we want to prevent that. The downsides, of course, is you have to run more controllers. You have to run more software places that only have limited scope of what they can apply to. But the benefit there is if something breaks in that one limited controller, it doesn't have a large blast radius. It has a very limited for security, for downtime, for all these other things. So this pull aspect is really good for the scalability. It's just a lesson learned. Weaveworks had been doing this for a little while. They're like, hey, you should probably pull that stuff. Here's why. And it's a great principle to have. And so in our infrastructure, we want to do something like this, right? We're going to pull that data down. Let's say it stores our variables file. And once we pull it down, then we're almost set to go. Because then I can run that tear from apply. And I can assume that, bash being bash, I can assume that if that fails, my code would exit and I don't actually have a problem of half variable files applying somewhere. And this one continuous reconciliation, again, it was kind of a trip for me where I didn't know how continuous that meant. Does that just mean while true? And it doesn't. It's not about being continuous. And it's one end we can just say you run every loop. Every time you finish, you try again. On the other side is our traditional infrastructure's code, which was only when a file changed. I don't know if anyone did config management for a long time. Once I changed my puppet manifest, then I applied that manifest to servers. And that was fine as long as I kept sort of a normal cadence of changes. But in something like infrastructure, you have a low level there of say DNS network infrastructure. These things don't change very often. And if you're changing those with a while loop, you will likely break something. And that is scary. And so you wanted something beyond the like only when a file changes. Because again, if I wait for a file change, and that happens to my network, I'm going to be parsing Terraform state files. I'm going to be manually fixing Terraform state files. And if anyone has done that, that is a bad night on call. I am sorry if you've ever had to go through that. But that was a downside of infrastructure's code, right? Like I thought when I had my Terraform as code, or my Terraform manifest, and when I was on call and I got this call and I was like, how could it be down? I have infrastructure as code. Infrastructure as code solve this problem. And then my state was completely to a point where the steps Terraform was going to do, the imperative steps, I can't. I can't get from here to there because someone in the console did something that broke everything. And now you have to go fix it. I had to go in and manually adjust Terraform state or manually go to the console and figure out the knobs to turn to get it back to some place that Terraform could apply it. And that was the difference between infrastructure as code and the sort of get op style continuous reconciliation. And that's the last piece that we want here where I want to take this script and when I want to do it more than just when a file changes, I want to do it when infrastructure changes too. But we're going to simplify it for, because this is a crappy demo of what we're doing here. So let's say if you've never used iWatch, oh, and I've got to find it. There it is. Because I couldn't remember this one off the top of my head. iWatch will watch for file system changes and run a command. So we're telling iWatch as soon as a file is written in this folder, close right is the thing it's going to look for. In this folder, run my infrastructure as code script. And so over here, I can actually, let's do version 3. And now I saw like right here, it immediately applied that thing. This is still at this point, oh, I got version 2. Oh, I got a bug. See, that's a problem. Oh, I spelled it wrong. Look at that. Good catch. See, and that's what code reviews for. There we go. Now we get version 3. There it is. Thank you. See, we got that in code review. That's still in this case, obviously, looking for file changes on disk, but GitOps is doing that reconciliation. And one of the cool things about Flux and Argo and these things, they're doing that two-way sync. And it's one step beyond what a typical controller like this is going to do, where I'm only looking at my local files. And traditionally, we're only looking for a Git push. And what you actually want to do is you want to look at that full state of the infrastructure and you say, hey, I'm just going to check it. Terraform is going to go out there and Terraform plant it every once in a while and say, hey, I think something's different now. And you can hook into these sorts of signals. Amazon, we have a vent bridge. There's all these different ways that you can look at what is going on in the infrastructure and you can trigger things based on any changes. Right? I could look at my cloud watch logs or cloud trail and say, hey, whenever something happens with this scope, I want to run that controller. I want to make sure that my infrastructure is back to where it was. And that was for me just how I started growing from what did infrastructure's code look like and why was code not enough? Now to we had controllers and Kubernetes controllers and how those get applied inside the cluster was a great thing because, again, it knows the entire state of the API server, the data that stores in and it gives you a ton of different annotations and logs and events inside of Kubernetes. And then applying that in a more generic way is really what GitOps is all about. And you can apply GitOps principles, you can apply infrastructure as software principles to anything. And it's not just the Kubernetes pieces because, again, you're going to have different scopes for these controllers. And if you think that you're going to do one controller and it's going to do everything, it's like writing one Terraform main file that does everything. You don't want that scope, you don't want that blast radius for one file, for one controller. So you do want to separate these things out. You want to have that limited scope. Having pole-based limited scope inside of environments is a great idea. And then applying this whenever there's a change on the infrastructure or on files is really that last piece. And that's really all I have for why infrastructure as software and GitOps works so well together. GitOps is an implementation of infrastructure as software. And that is the main direction that all of this should be going in. And I think it's been a great progress with GitOps in general so far. So thank you. Well done. Thank you so much for that presentation. We do have a few minutes for questions. Are you available to take some questions? So questions you can raise your hand or we have a mic at the mid. You can just jump to. Any questions? I will say I am showing off running Kubernetes on these and AWS booth tomorrow with EKS Anywhere, which also implements GitOps. So if you want to see them in action with Kubernetes, come by the booth tomorrow at 10.30. Those aren't hollow? No, they are real. They are heavy. You were really just punching those over that whole time. I was like, oh, he's just got the case up there. It's not a big deal. No, they are real. All right. Well, good luck with the demo tomorrow. Yeah. Any questions? Raise your hand. Don't be shy. And I'll be around all week. So sorry. While they are thinking of one, you were mentioning that, can you guys hear me okay? Because I can't hear the mic feedback. But you were mentioning that you felt like on some level it wasn't declarative because it relied on an imperative system for operation. So would you, I mean, in that case, there is no such thing as declarative, right? Because everything always relies on an imperative operation. Like even if you built a language that was only imperative at the end of the day, it has to be, that was only declarative at the end of the day, it has to be put into bytecode, which is imperative onto the CPU. So I was wondering if you'd maybe just speak on that for another moment or two. Yeah, there's also no such thing as immutable, which was kind of mind blowing for me to think of that. It's like, no, actually everything changes over time once we run it. And those were two things that as a sys admin, I kept thinking like, oh, well, this is how it has to be. But realizing that the main benefit was to the humans. The main benefit of all of this is that you can sleep at night so that you don't get paid, so that you can get paid to your paycheck, and you can have a good balance of life. If you need to scale, if you need to automate things, like automation goes so far, but there's always has to be this trigger, and GitOps really applies that trigger. And we're still just, we're doing a lot of the same things, and we're just pushing that out. But the human interface to those imperative systems, and the human interface to those what we thought were immutable, it was okay because my view of what's immutable is like, oh, well, I want this application deployed, but then I have this other controller that comes in and says like, oh, but I need to scale it up. I'm like, well, I didn't tell it to scale up. So it's not immutable, because I told it five replicas, but something else figured out it needed 10. And at that point, I'm like, well, now it's not immutable. So is it bad? No, it's wonderful, because I didn't have to do it. It was a huge benefit of having these controllers and something to figure out that when I say I wanted five, it said, well, I need one first, and then I need two, and then I need three. And it had to do those in order, and the scheduler had to bind them, and a cubelet had to pull them down, and I had secrets, and all this stuff had to happen in order. But my view, the human interface to the rest of the system is very declarative. And I like to think of it as if I'm driving a car, I can call Uber and say like, I need to go to the grocery store. My interface to that system is I made it to the grocery store. I was there. I could also drive myself. I could jump in a car, and I could steer the wheel left and right, and step on the gas in the brakes. And that is very imperative, and that is me telling it exactly step-by-step every little thing. But I don't have to say piston one fire, piston two fire, piston three, like that's a horrible interface, right? But it's still, there's imperative things behind every declarative thing we do, and where, what stage of declarativeness that I want and I need, all depends on how much ownership and how much control I need. If I need to get there in five seconds, five minutes, and it's the 30-minute drive away, I can't request an Uber because I'm not going to get picked up. So I need some control there. I have to get there fast. I have to get to the hospital. My wife's going into labor. Let's go. I'm not going to sit there. I'm like, well, let's call a cab. Sorry, let's hope you get there. So it's all about the interface with humans. Just because I didn't see any other hands up yet. So would you say that, well, I don't know, I don't know if it's necessarily ownership, but would you say it's how much you can trust your software? Because you're describing infrastructure as software, right? Yeah. There's always bugs in software. I wrote how many bugs today in three lines of bash. There's always bugs in software and a lot of that trust only comes over time. So it's like, well, I have to trust other people. Thank you for the PR, the review. We trust the team. We trust experience. We build that trust over time, and then we can trust that software over time. And some of that trust comes from just generally other people use this thing, right? Like flux is amazing. Not because I've used it forever. It's because 1,000 other people use it and they told me some practices to do it. And I trust my car. I'm not my wheels aren't going to fall off on the way to the store because I didn't put the bolts on, but I trust the mechanic who did it a thousand times that they did it correctly. And there is always that implicit trust of just like, well, I have to trust someone in this case and being able to trust other people and trust the ecosystem and trust the tooling. Again, please don't write your own bash controller for GitOps. Use one that's fairly trusted because you're going to have those bugs. But to gain your own experience adds more trust. But a lot of that comes from knowing how the system breaks in your environment and knowing like, oh, my process didn't align with how that thing was intended to be used. And so once you have some sort of intention-based thing where you're like, oh, I think it's going to work this way, but I have five teams that are deploying to their own repos that all merged into this other thing, like that's where you kind of get into problems because you're like, oh, actually my ideal of how I was using it didn't align with how the rest of the community was, didn't align with the process inside our company. And so you have to build that trust for yourself as well. But yeah, gain that trust through these common tooling and common practices. And that's again, GitOps has those four definitions, four very good reason in the tooling implementing them for those reasons. Open for audience questions. Yep, got one over here. Hi, great talk. One question I don't know, you don't show it, but it's always in my head when I use Argo CD. If we revert in a case of GitOps, what is the best practice to revert it over the UI on Argo CD? Because you have a history and revert possibility. Or actually a single point of truth will be Git, right? You go on Git and revert it there. And then you have auto sync, you have a sync, which will, in case of issues, in case of bugs, will deploy it immediately. It's also in my head why we have this history and rollback in Argo CD. And should the developers use it or not? Should the users use it or not? Or should we go over the Git where the single point of truth lies? I don't know the very specific on Argo's implementation of that, but in general, you always want to roll forward. You always want time to progress forward. And computers sometimes have more gotchas when all of a sudden time reversed. And we said, hey, I already saw that state. I worked at Disney Plus for a while, and we were managing infrastructure. And we built our own controllers for how we were managing our clusters. And we had that same question. It was like, hey, how do we revert? We deployed something that was bad. How do we get back to a known good state? And yeah, we could go to Git. All of our state was stored in Git. We could go to Git and say, oh, I'm going to make the new head the old version. And we had so many weird things in software that didn't ever assume that latest would be something that's not the most recent. And it said, oh, no, latest is the most as far as a time stamp goes. And a lot of systems still deal with time stamps. And so what we decided for our system was always that we never went back a head version. We always did a Git revert that pushes one more head version ahead. And it says, oh, I'm going to take that thing, and I'm just going to undo this commit. But it always has a new commit, has a new time stamp. It always moves forward. And so in software and infrastructure, a lot of those systems are just easier to mentally think of when the current state is always the newest time stamp state. And so figuring out what the newest time stamp would be and the newest commit as far as history of dates, just because we live in a time synchronized worlds, it's just easier to reason about in a lot of ways for all those controllers. So I don't know if Argo has a specific way to do those reverts. But I know in other cases, it's just a lot of times more safer just to say I need to revert, meaning I go forward in time with a new check out. The Argo maintainer in me says, let's talk after this. I think we have time for one more question while they're setting up for the next talk. One more question from somebody? Big pressure. You got to get a good question. No? Okay. Well, let's give one more round of applause. Thank you so much. Thank you.


Bismillah. We have an incredible number of volunteers that have done some incredible amount of work here every night, not just in Ramadan, but every night. There are too many to list and not enough time, so we want to just recognize some of our employees here today. And I'm going to recognize, I'm going to recognize by the Mervis, by the Munir and Sister Amina, and then if President Humana wants to say a few words, feel free. Brother Mervis, everybody knows Brother Mervis. Mashallah.  We're here in the morning, we're here at night.  Then he says, I got to pray Salah first before I leave. Then in the morning he's here again. And I spoke to his wife the other day, and she says, he's always at the masjid. He's always at the masjid. He comes home, he thinks about something, he goes back to the masjid. So we have a small award for him. They're right back here. We have a small award for Brother Mervis, and I think of it as the Khadim Award, which is a beautiful, beautiful work. He's a Khadim of this masjid. His heart is in this masjid, and if you look, and in fact all three of the people over here are four of them, in terms of what they embody, the values, the feelings of MCC in their everyday behavior. So please, Mervis, please accept us on behalf of the of the community, the board, the staff, the community. And I believe there's a little, just a little, Mervis, thank you so much. Moneer, Moneer, Moneer. He runs day-to-day everything, day-to-day operations, what happens day-to-day here in the masjid. The award I think says growth. I think about another word would have been fata, opening, because he does, he talks to different communities, he builds the relationships with other communities, Muslim communities, incredible opportunity, incredible what he's done with us.  We are recognized, by the way, you should know we are recognized many, many places throughout the world, throughout the world. And two days ago, he got an invitation from the White House to attend the Ramadan dinner they have every year. Mashallah. Mashallah. And I believe you have an award here. Thank you so much Moneer. Last but not least, in any way, shape or form, sister Amina. I don't want to embarrass her. She's just done an incredible job. She is an exemplary performer in every way you can think about it. Mashallah. She does outreach to social, basically runs social activities. On the side she does some of the finance things that we have to get done also. She does it with grace. You may not know, but it's her work that led to a 50k grant where we got a Mercedes van from the Alameda County. It's her work that we got two 5k grants for refrigerators we have. It's her work that we are renting another facility where we store some of our pantry stuff. And the Alameda County pays half of that and we pay half of it. It's her work when she was in a meeting, a Zoom meeting with congressman Eric Solwell and many others. And one of the questions was, who's MCC? Who's MCC? We keep hearing about these people from Afghani refugees. Who is MCC? Right. Alhamdulillah, she's asked for a grant. I won't tell you how much it is, but we're hopeful the grant will come. And everybody recognizes her. She does a very, very hard work with talking to people that are crying on the other side. Right. That's a very, very difficult thing to do. So, Masha'Allah, we have another award for her. Bismillah. Thank you so much. Thank you so much. Do you want to say that? Assalamu alaikum, everybody. I just wanted to say that these three exemplary employees make us all look good in front of the whole Bay Area and sometimes even across the Bay Area to different states. I have learned the meaning of khidma and service and humbling myself through them. And if you leave tonight with one du'a, please make, keep them in your du'as and keep all the volunteers who run this mosque tirelessly for 30 days without sleep, sometimes without food, sometimes not even a sip of water. And on each day, they will be working from six all day, true and not spending time with their families. So, please keep sister Amina, brother Munir, brother Maroues, and all the volunteers in your du'a, especially tonight and every night. Assalamu alaikum.


Thank you very much for the introduction and thank you for the invitation to come to the meeting today and share some of the data. There we go. Am I doing this correctly? I'm pushing the green button. Oh, there we go. Oh, it caught up. Forget it. There we go. So we were initially charged with trying to come up with a question that was the basis for the presentation. We have this long standing interest in the interactions between intestinal microbes, the immune system, and inflammatory disorders. And in some cases those inflammatory disorders extend beyond the bowel into other parts of the body. And so this is the basic question that I posed was do these immune networks have the potential to address some of our public health issues? It's broken down into a number of different categories. And I'll start with a little bit of basic background. So we became quite interested in the idea, and this is almost a decade ago, of the possibility that there were microbial interactions, some of those occurring very early in life, that were going on to impact adulthood, and that those interactions involved a balance of immune cells. And everyone knows Sarkis mesmenian has this beautiful paradigm for the balance of the pro and anti-inflammatory cells. So I'm using that scale, the balance, a paradigm here. And the basic concept is that the gastrointestinal tract microbes are impacting this balance throughout the body, and that when there's homeostasis or balance between the pro and anti-inflammatory activities, that health is the outcome. And when there's an imbalance, that that's when there's a possibility for disease. And ultimately, what we would strive for is healthful longevity. So not just to live a long time, but to have those years be healthy and meaningful as healthy and meaningful as possible. And we envision that this involved this balance between the immune activities and the microbiome. So that's the background concept. So about six years ago, six or seven years ago, I met Tina Clark-Dur at a Department of Defense Era of Hope Meeting. We were both doing breast cancer research, and she oversees a cohort of women. She and Pam Horne Ross oversee a pretty large cohort of women called the California Teacher Study. And this has many subjects who are followed prospectively in interesting ways. They endeavored to do this in the early 2000s, I think. And we've incorporated a lot of Tina's data as preliminary data to support concepts of things we see in mouse models. And I hope everyone will let me take the liberty to include some of that data and to credit Tina with that. So we had this basic question, do the maternal and infant microbial ecology offer opportunities to impart good health to future generations? And many of you have presented fabulous data looking at infants, looking at pregnant women, and trying to determine whether or not there are changes in those subjects that may translate into adulthood. And we attempted to do that by proxy in a lot of Tina's California Teacher Study women. For the purposes of the discussion today, though, I'm going to present a lot of my own mouse model data. And we were interested in looking at mother-infant relationships in the mice. And for those of you who don't do a lot of work with animal models, we've certainly discussed it a lot. There are a lot of good reasons to use mouse models. And one of them is because you can create genetically uniform animals. They can have different kinds of mutations in a very controlled sort of way. You can control their microbiome, as we've discussed. And you can introduce all sorts of substances in controlled ways that are very difficult to do in human subjects. But there's another advantage, and that is that mice have very short generation time. So we use this opportunity with these short generation times to start to examine some of the things that might happen, be very difficult to capture in a human subject where it might take a very long time to have a generation, or some of that information is by proxy as with Tina's data. And we were going to use these mice to start to examine what would happen if we manipulate the microbiome in the immune system in interesting ways and then follow them for several generations. So we had a lot of difficulty getting funding for this, even though we thought this was the greatest idea ever.  Leona Samson and John Essigman agreed to fund a pilot project through NIHS. And it was on perinatal micro-exposures, and we thank them very much for the opportunity to do that. So some of the data should be credited to that grant.  And that is that when we looked generations, not just the mom and the kids, even though we have a lot of data suggesting that the children are hugely impacted by things we do to the mother's microbiome, we wanted to go one generation further. And when we did that, we discovered some absolutely shocking things. So animals that were of a wild type background, they're outbreak Swiss mice, they are animals that were not manipulated in any way other than the conventional housing that we do in the animal facilities. And they were eating standard chow, the standard rodent chow. But an enormous number of these animals are developing lesions. Some of them we had hoped to keep on the study for a longer period of time. I'm going to present a little bit of preliminary data later on showing that five-month-old, these are outbreak Swiss mice, five-month-old animals not manipulated in any way were developing 100% of them bronchial alveolar carcinoma. I mean, I just thought that this was very unusual and very interesting, and I wanted to share a little bit of that with the group. But let's do a little bit of background first. So Fiona Powery's lab has done a lot of work over the years. We got started actually working with these mouse models in around 1999 in earnest. And this is a fabulous display. I'm just going to very quickly show you this. You recognize this would be your gastrointestinal tract epithelium and your microbes, which you can manipulate in various ways. And then there's this whole cascade of events that occurs. Some of those are innate immune cells and others of those are adaptive immune cells. And you can actually dissect them out with different kinds of mouse models, which lets you be very creative to answer scientific questions. So there you go. There's the innate immune system in solitude, and you can actually work with a mouse model. Many of you know this. It's rag deficient mouse that has these cells without mature adaptive immune response. And that's a platform that we've used quite extensively. So what we've done is we've taken this possibility of using a rag deficient mouse that doesn't have any lymphocytes of its own and substituting in these various adaptive immune cells in different sorts of creative ways. And using the Powery paradigm, which became very popular in the late 1990s, we started doing a series of experiments where we're mixing and matching different kinds of cell transfers and these different cell populations. And we found many of the things that Dr. Powery had discovered earlier. But we decided because I have the good fortune of working in one of the most wonderful places in the world, actually. And Bruce Horowitz, who is now at Harvard, was at MIT at the time. And James Fox is the director of the Division of Comparative Medicine, where I work at MIT. And the three of us started working together on these model systems. And in particular, Bruce had gotten some funding that allowed us to develop these systems. And what we did was we mixed and matched a targeted infection with helicobacteropaticus that you'll see this icon show up several times throughout the presentations. And we discovered some very, very interesting things when we started working with the rag deficient mice and helicobacteropaticus and the adaptive transfer paradigm. So I'm going to start moving pretty quickly through this material because I'm presenting it because I want you to have background to the things that we're going to do a little later, but in and of itself is not the end of the presentation. So what we found was that the uninfected animals that were maintained with their, essentially, their tachonic flora, and this in particular works most effectively on a 129 strain background, but you can recapitulate elements of it on other strain backgrounds, we found that the uninfected animals develop virtually no pathology, even if you develop, follow them for very long periods of time, whereas their counterparts that were infected with helicobacteropaticus as a targeted infection, and that was gastric gavage, ended up getting this profound inflammatory bowel disease. And the, not the rag mice, but their wild-type counterparts that were helicobacteropaticus infected didn't develop any pathology. So this led us to put together a paradigm that showed that innate immunity was sufficient for inflammatory bowel disease, and I'll talk about carcinoma in a minute, and that the cells of adaptive immunity were suppressing it. We hypothesized that this was an inflammation-associated paradigm, and that the regulatory T-cells were actually what was very important in that process. And so we set up a series of experiments probing that paradigm, and we found that as the animals went on for longer periods of time, they went on to develop cancer in the intestinal tract. So this would be typical of inflammatory bowel disease-associated carcinoma, and this is in the six to 12-week time frame where you can see these invasive lesions, the development of amusinous carcinoma. And then if you follow it out for longer periods of time, you get these, while the uninfected control has virtually no pathology at all, everybody recognizes that relatively normal colon, you get these musinous carcinomas or these poorly differentiated carcinomas. These grow out into the lumen, these grow out into the abdominal cavity, and they become quite large. They're very impressive actually. I think they're not bigger than the mouse, but they're very large lesions, very significant lesions. And what we found was that helicobacteriopaticus was actually, as a targeted infection, doing a lot to upregulate pro-inflammatory cytokines in these different settings, and these are all cytokines you recognize IL-60, NF-Alpha, and IL-17. And in particular, IL-17 appeared to be a big player, and if you modulated the immune system by looking at the paradigm of IL-10 and IL-17, what you find is this exquisite presentation. All these brown cells, and actually I've got an even higher power image here, are all expressing IL-17A. And some of these cells look very much like they might be lymphocytes and others that look like they're cells of the innate immune compartment, but there's massive, massive upregulation of IL-17 under certain circumstances in these model systems. So we went on with this hypothesis that what's happening is there's a lot of inflammation. That inflammation, if it's unchecked, is eventually leading to cancer, and I went on to test in a bunch of different settings using either blocking the inflammatory cytokines with the NT-TNF, adding exogenous IL-10 into a RAG deficient mouse, or transplanting the regulatory cells that I told you about earlier, that you could actually restore. You could take an intestine that had these lesions and restore health, and we found, in fact, that that was the case. So that allowed us to put together a paradigm like this, and it involved an interaction between IL-10 and all this stuff is published in 2003-2004 timeframe. IL-10, Tregs, pro-inflammatory cytokines, and that inhibition of the regulatory cells blocking those pro-inflammatory cytokines ends up ultimately blocking the growth of the tumors that were occurring in the intestinal tract. So as a result of a lot of this work, I was able to actually get funding, and I want to make sure that I credit individuals and funding every time I possibly can throughout the presentation. I don't know how much time I'll have at the end, but go ahead and allow me to do this if you don't mind, and I'll credit NIH grant. NCI was actually the funding source for this, and this was interrelated roles of IL-10 and TGF-Bate and colon cancer. That grant has been renewed, and it goes through 2015, and it is the basis for what allowed us to take a lot of these next steps in these model systems. So what did we say here? We were focusing on helicobacteriopaticus infections. We were focusing on the possibility of using adoptive transfers, and we were using rag-deficient mice in all kinds of mix-and-match creative ways. So the next thing we did was we moved on, and we moved on to a model system that allowed us to look at discrete polyps as an intestinal cancer model in comparison with the more overt inflammatory bowel disease cancer model, still speculating that inflammation was important in this paradigm. So for those of you who are familiar with min-mice, APC min-mice, they have a lot of immune dysregulation spontaneously. It's not entirely clear what's chicken and egg, whether it's cancer and inflammation, or inflammation and cancer, but there does seem to be an uncontrolled, massive uncontrolled inflammatory response. There's premature thymic involution, and a number of things that are very interesting in this model system. So just looking at untreated black-six wild-type mice and untreated min-mice side-by-side, you already see enormous differences in this model system, and these are stocked from Jackson Labs. So we've gone on to begin to probe this model system in a lot of interesting ways, and one of the things we wanted to do was probe the microbiome. I'm not going to present a lot of data, but I do want to credit Eric Almslab, and you may have noticed that Eric is also a collaborator on the grant with Tina and David Halfler and Tim Wong and others, and I'll show you a little bit more about that grant in a few minutes. We tried to make it as comprehensive and multi-institutional as you can on not a really large budget. Mark Burnham-Smith did a lot of this work from Eric Almslab. So we set up to use the adoptive cell transfer paradigm in the mice that I just described to you, the min-mice and the wild-type mice, and that's the same basic paradigm that we had used in the 129-RAG deficient mice only. These are immunologically intact C57 black-six min-mice, and what we found was that the regulatory cells, the same subset of cells that were purified using the power-y paradigm of purification, that same subset of t-regulatory cells was actually able to inhibit the development of tumors in the min-mice, and this fit our hypothesis of this pro-inflammatory series of events, that we had the cytokines to support that, and that IL-10, the anti-inflammatory cytokine, and its priming of the regulatory cells was important in that, and so there's two things. One of them is that this paper, it's a cancer research paper that was published in 2005, so anyone who wants to take a look at it, you're welcome to. I also wanted to thank the Shower Lab because David Shower was the senior author on this paper. We went into this together, one of his graduate students, Jane Sohn, actually had a lot of the insights that helped us develop this model further. So this is just summarizing that data, and as you can see, if you look at this panel on the left, we have the sham-treated mice. These are the min-mice that got the regulatory cells, and there were enormous differences in numbers of tumors between these animals. They were highly significant in their differences, and in fact, you could take animals that had established tumors like this, as Jane pointed out, if you caught it really early, you looked at two to three days right after we had transferred the cells, and you would actually see these involuting polyps, and the involuting polyps were packed with apoptotic figures. So the Treg cells induced regression of established adenomas, and here's that data, so we were able to show it didn't just stop the progression of it, it actually suppressed things that were already existing, and it was through an apoptosis-mediated mechanism of the epithelial cells. So we went on to take this paradigm and tweak it even further by introducing the helicobacteriopaticus. So you probably see some emerging themes here. We're using a lot of these model systems in the same way. We're introducing helicobacteriopaticus. We're using min-mice or rag-deficient min-mice on a different strain background. This is actually a C57 black strain background, not the 129 of the earlier models. And we went on to show that the cells of adaptive immunity were able to suppress carcinoma and that the innate immunity was sufficient for carcinoma in the same ways that we had shown in the earlier model systems. So one of the things that we discovered serendipitously, although I have to say we weren't entirely surprised when we saw it, was that the introduction of helicobacteriopaticus into the APC min-mice actually caused mammary tumors to develop. So for those of you who are very familiar with this model, you know that when it's kept under clean health status conditions, you very rarely see mammary tumors develop, but they do have a genetic predilection to mammary tumors, and in fact, when we challenged them with the gastric gavage of helicobacteriopaticus, what we found was this very rapid development of a high number of mammary tumors. And this paper, let's see if we have it here, this paper was published in 2006, and we were able to show that there was this continuum of progression, which actually starts from around the mammary lymph nodes and turns into a carcinoma. And it was sufficient to use the innate immune response alone, so the rag deficient min-mice displayed all these characteristics. And a lot of this work was actually funded by a Department of Defense Award. I think I mentioned to you earlier, it was at the Year of Hope meeting. We had this hypothesis that anti-inflammatory regulatory cells could actually inhibit mammary tumor formation, or breast cancer potentially, by inhibiting microbial activities that were in the intestinal tract. So we were not looking at just what was happening within the intestinal tract. We had moved into an extraintestinal arena, when was this funded? In 2005, and that's where we were. All right. So we got invited to do a review paper, and that review paper is the basis for how I'm going to present the rest of the data. The individuals here deserve a lot of credit. This is Rao Verrata. He was the first author on this paper. He was a postdoc working with me at the time. This is Theophilus Putihitas, also working with me as a postdoc at the time, and of course we were working with Dr. Fox at MIT. And this was published in Cancer Research, and it displayed the ideas beautifully. So there was a review figure that basically said, we had this hypothesis that these gastrointestinal bacteria were doing these amazing things in other parts of the body, and these were our two possibilities as far as we could tell. Systemic elevation of cytokines or activated cells that were happening as a result of the GI infection, or the possibility of translocation of intestinal bacteria or antigens. And frankly, I could talk for hours on these topics, but I'm not going to do that. I'm giving you this information as a matter of background. So the U01 grant, the one I had mentioned earlier with Tina Clark, is Erdman as a PI, Kopi Aum, Kopi Tim Wang at the Columbia University. And we had set up this paradigm where we were going to probe all these different things with the helicobacteriopaticus infection. Tim was going to look at stem cells and stem cell distribution. Erica Aum looking at the microbiome. And Tina Clark, this is her initials right here, looking at this possibility of this perinatal window. And David Halfler is also working with us at Yale. Here's some of Tim's data. And again, I'm going to move through this very quickly. This was looking at the distribution of stem cells that were mobilized as a result of the helicobacter challenge in the bowel because, as everyone knows, Tim Wang's focus is looking at stem cell migration and its contributions to cancer. And so when he did some of these studies, and many of these actually occurred separately, from my own facility and my own models, he was able to recreate the same things that we had seen earlier, which is always very reassuring. And we wanted to use this adoptive cell transfer paradigm to probe this concept. And this is beginning to look pretty familiar to you at this point, with IL-10 inhibiting and Tregs inhibiting. And what we found, and this is all published in the 2006 paper, was that the bottom line was that the regulatory cells did appear to be inhibiting. This is the epithelial interactions over on this side. The regulatory cells were inhibiting, and their potency to inhibit was increased by earlier exposures to microbes. And so that is the sort of hygienic concept that we went on to follow up on. So we had this is right from that review paper, and it's talking about modern hygiene practices and in crediting all these individuals who had done all this work on the hygiene hypothesis. And we were quite sure that the anti-inflammatory cytokine IL-10 and the regulatory cells were the key to what was going on in all of this. So we've gone on to do a series of experiments where we're doing adoptive transfers, using that same paradigm in the min-mouse model, and then probing what's happening to tumor rates and changes in the microbiome. And so there were significant changes in the min-mice, the white stripe is the wild type animals. There were significant differences in the min-mice who are on the inside, and the min-mice who had received the hygienic T-cells, commensurate with these big increase in intestinal polyps, and as it turns out, mammary tumors as well. So we followed groups of animals. We followed them for extended periods of time. If we used hygienic cells versus the bacteria prime cells, we were actually able to dramatically increase the lifespan of the min-mice in this setting, as opposed to the animals that got the hygienic cells, which actually had a truncated lifespan. This all fits together in the paradigm in the way that I described. So the preliminary data that I wanted to share with you that relates to the hygiene hypothesis is the beginning of the sort of the mediest part of the discussion, and that is that Tina has taken individuals from this California teacher's cohort, taken their history by proxy, and someone had brought up earlier that it was very important you had exposure to animals, as it turns out some of the strongest protective exposures that we see against inflammatory diseases and all these types of cancer in women was actually how close they had lived to a stable or a dairy, and whether or not they had worked or mother had worked or they had worked under those conditions. So it was like a farm association. So the farm association was actually quite protective. We had put together this paradigm. It's not published, at least I don't think it's been accepted yet, but we're looking at all these cancers with the idea that this is merely an extension. These different types of carcinoma that are developing are developing with inflammatory underpinnings, and they're just an extension of all the other inflammatory conditions that we're seeing in a public health context. And so we're going on now with Eric Olm's lab at MIT to begin to look at the profiles of some of these women, unfortunately usually making associations with their early life exposures by proxy, but still looking like there's tremendous potential to begin to elucidate what the roles of early life exposures in these women may be in their later health outcomes and the establishment of their microbiome. And so the last thing that I want to present in this context is sort of dated to give you the backdrop is the interesting discoveries we had with probiotic microbes. And Jim Versalvic is actually here in the audience today, and a lot of this is to his credit that we ended up evolving the thing, learning the things that we found, evolving in the ways that we did. We became quite interested in the possibility that you wouldn't have to expose an individual to pathogens in order to get the additional priming of the regulatory cells. And we could go on about the details of that mechanism, but we thought if we could create that same phenomenon by feeding a beneficial bacteria, that the possibility existed for us to be able to use that in some kind of constructive public health setting, which is sort of the direction that we're heading in. So what we had done was we set up a series of experiments looking at simply exposing mice to yogurt. And this slide is actually the upshot, sort of at the end, the conclusion that we'll come to, but I want to make sure that I describe exactly how it unfolded. So first, we fed a bunch of mice probiotic yogurt with the idea that probiotic yogurt was actually something that human subjects would potentially be eating regularly. As it turned out, every time there was scientific criticism, and this is very legitimate, it was always based on there's calcium in there, there's vitamin D in there, there's protein in there, and on and on and on. So we also, in parallel, started running experiments using a purified organism, and that purified organism is one that Jim had shared with us. So this is the team of people. I won't spend a lot of time talking about them. They are wonderful people. They make every day fun for me, and I guess that's the way science should be, right? So one of the things we found was that the mice, the control mice, whether they were on a C57 black six background, or whether they were outbred Swiss mice, they were wild type mice, were developing this difference in their fur coat, and the difference in the fur coat was measurable in lots of different ways. I'm not going to present all the data. It's actually published in plus one in January. And one of the things you'll notice when you look at all these, the ones that were eating the probiotic have this patch of hair five days after they've been shaved and the controls don't have anything at all. So what we discovered was that there was a massive shift in hair follicle cycling commensurate with feeding these purified organisms to the animals, and it had a lot of different attributes to it. One of them is it was an antigen phase shift. You can see the hair follicle roots here. We actually went on to quantify this, and you can see the difference between a telogen hair up here and an antigen hair, and it ended up that when we plotted all this out that eating those organisms was causing hair to grow like crazy. And the hair wasn't just growing like crazy. It was really healthy. It was really shiny. And it had these interesting gender correlations that suggested that it was somehow related to reproductive fitness, which is a topic that's discussed in a moment. Paper, we call it the glow of health. So we found other things too. Some of these we set out with more intent to find than others. We were actually interested in understanding the roles of obesity and whether or not obesity was contributing to cancer and whether that might be through a regulatory cell-mediated mechanism. But what we found shocked us, and that was because we were using a number of different diets, some of them mimicking fast food and having low vitamin D and having high levels of fat and being supposed to be mimicking potato chips and french fries and bad things. We found that there was this enormous protective effect even when they're eating the fast food chow when they were also eating the purified lactobacillus roidery. And it sort of suggests, like from an evolutionary perspective, that it may have been protection against some kind of malnutrition or times of hardship.  But the quality of the fat was completely different. So the animal was in a state of health, superb health actually, and you can see this difference here. So these animals, while they're slender, that were eating the fast food chow and the roidery have fat pathology that really rivals either a very young animal or an animal that was just on control diet, even though they're getting the junk food, which was very interesting to us. It's not the topic of discussion today, but I wanted to make sure everyone understood that. So one of the things that we noticed was that the regulatory cells were increased under that setting, and I showed you that just a moment ago on the slide. And so we wanted to test, using the same paradigm we had used before, but with a bit of a twist here, this is a decade later. And so now we're using GFP-labeled FoxP3 cells as our regulatory cell marker. And there was enough background earlier for everybody to appreciate what those cells are. And they're labeled green here. We inject them into the rag deficient mice, and we were actually able to show that the GFP cells that emerged from donors that had been fed LR were able to decrease the subcutaneous fat levels and make a lot of the fat pathology changes. And so those cells alone were sufficient to do this. And so you probably see where we're going with this. We're going into the same idea that these regulatory cells are inhibiting inflammation and that there are cycles occurring within the body, and those cycles involve food, stuffs, microbes, and different kinds of bacteria, and that you can modulate them profoundly, change the cycling simply by interrupting which microbes you might happen to introduce. And it's back to the same paradigm as before, only this time it's obesity instead of cancer. And it happens to match some work that was done in 2011 that was published in the New England Journal of Medicine, and that was looking at human subjects. And in fact, the humans that had eaten yogurt were actually protected from weight gain more than any other single food item, which is really interesting. And some data from David Hafler's lab was looking at humans eating fast food in restaurants and looking at the IL-17 expression in their T-cells. And actually the whole story fit together beautifully. The animal data was supporting the human data and vice versa. So we have, as I've described, we're using a wild-type littermates, their Outbred Swiss mice. They come directly from the vendor in most cases. We did have a small in-house breeding colony for the studies of looking at the generations that I described. But we specifically were asking the vendor to supply us with wild-type littermates or we were using littermates from in-house. So, and behold, the slender animals had much higher thyroid hormone levels than the controls. And one of the things that was very interesting about this, and this is a group of females that we had followed out, was that they all start out around two months of age. We start them on the feeding of the LR. They're all within normal range, and this will be our normal range of T4 right here. And what we found was that those animals that were getting aroidari even when they reached the age of eight months or older were staying up in the very high normal range. Those animals were very active, and they were slender in comparison to these animals, which became hypothyroid as they aged. So the thing that seems to be most interesting to everyone, everyone about any of the mouse models that we've developed so far is always this. And actually it was a technician in my lab who had noticed this swagger that these mice had and we decided to take a look at what the origins of the swagger might be. And what we found was that whether you worked with yogurt or whether you worked with a purified organism, that they actually had larger testes if they had been eating lactobacillus aroidari or the probiotic yogurt. And again, this fits into our paradigm of some kind of reproductive fitness benefit that might be associated with these organisms. Makes sense because they're all about the perinatal window. And we went on to show that serum testosterone was also elevated. And so I know what everyone's going to ask. They're going to say what was different about the testes. What was different about the testes was the light excels were bigger and the seminiferous, the light excels that produce the testosterone and the seminiferous tubules were a lot thicker and there was a lot more sperm production. So those were the things that increased the size and made sense with the serum testosterone. So we were looking at reproductive fitness as part of all of this and we were looking at it because we were interested in this generational effect that I'm going to talk about in just a moment. Mother mice were taking much, much better care of their babies if they had been eating the lactobacillus reuteri. And so the effect was profound and it doesn't it's not even captured here with these numbers. I don't know if you can see this, this is about two-thirds survival and almost a hundred percent survival. It was at the first litter in particular that black six moms were not eating any of their babies. They were taking fabulous care of their babies while they're eating the lactobacillus reuteri. So like this is really interesting. But it might have something to do with plasma oxytocin levels. Okay, so we hypothesized that this might have something to do with plasma oxytocin and we call this project My Bacteria Made Me Do It. So we went on to look at everything surrounding fitness in the perinatal window. We looked at wound healing capability. We looked at maternal rearing success rates. We looked at the ability of these animals to get pregnant. And I'll present this data very, very quickly. What we found is that skin wounds extrapolate that to all the fight injuries of the males to the perinatal recovery of the mom that that process is accelerated by eating lactobacillus reuteri or yogurt. I'm just using the purified organism to be more scientifically robust. The wound closure rates are doubled and E. coli K12 does not create the same effect. And when you transplant the FoxP3 GFP cells, it recreates the effect completely, the wound healing benefit. And that's that data right there into black six mice. You can see the wound healing benefit from the mice that are reading LR. Same scenario. We went back to look at the oxytocin. We looked at oxytocin deficient animals and they had a deficiency in their ability to repair wounds after being exposed to the aurel reuteri. And so we put together a hypothesis where oxytocin in the perinatal window was improving almost every element of the fitness. And one of those manifestations was wound healing. So I told you earlier about this funding, looking at this perinatal window. We went generationally and this takes us back to where we were in the very beginning. And that was the possibility that there are perturbations in the microbes and in the immune system that are transcending generations. And with our current hypothesis, knowing that many of these phenotypes are transplantable by immune cells alone, we were hypothesizing that the first generation of progeny has a disrupted immune system and that impacts everything about the pregnancy in the second generation. And nonetheless, the rates of lymphoma were very high in these animals. And this isn't all the data. This is just a group that we had taken off at five months very recently. They were prone to liver cancer.  And just about every animal in the study had lung cancer without any extraordinary exposures at all. So there were a lot of data, but conceptually it's a very interesting concept. So here we go. Could this offer some kind of opportunity for health in future generations if we understood it? Could we restructure immune networks and would the best way to do that be through microbes? We have data showing that lactic acid bacteria in the people in the audience who are much more of an expert in this area and probably have a lot more data to support this. Is there some kind of interventions using these well characterized strains to benefit for an immediate public health remedy for some types of conditions? And maybe some of those studies are underway. Is there the possibility of setting up more prospective longitudinal studies? A lot of people have talked about this. I'm not the first one to bring this up. And we were also interested in the possibility of probing epigenetics and genetic impact realizing that if hormones like oxytocin and thyroid hormone and activities of the hypothalamus, many of which are vagus dependent, we know this because of vagotomies, perhaps we need to be quite broad in the way that we're thinking about the epigenetic changes and what the impact on the immune system might be from some of these immune hormonal interactions. And so I thank most of these people along the way, but if I didn't get an opportunity to thank everybody, there they are. I'm sorry I ran over. I'm glad to answer any questions, I guess, if there's time. Thank you, Susan, for that very nice talk. We're well over time, so we won't be able to have any questions now, but please feel free to come in at the breaks. We're going to have a short break because we're over time, so we take 12 minutes to just do the basics, get a drink, a bio break, and come back here at 10.45. Thank you.


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Welcome to this Reality Shifting Guided Meditation. Begin by finding a quiet and comfortable space where you can relax without any distractions. Sit or lie down in a comfortable position, ensuring that your body feels supported and at ease. Take a moment to settle into your chosen position and close your eyes. Now, bring your attention to your breath. Take a deep breath in, filling your lungs with fresh air, and slowly exhale, releasing any tension or stress you may be holding onto. Continue to breathe deeply and slowly, allowing your breath to naturally find its rhythm. As you relax, visualize a soft, gentle light surrounding your entire body. This light represents your energy, your essence. Feel the warmth and comfort of this light as it envelopes you, creating a protective and nurturing space.  This doorway symbolizes the threshold between your current reality and the reality you wish to shift into. Notice the intricate details of the door, its color, shape, and texture. Take a moment to appreciate its beauty. Take a step forward and reach out to grasp the handle of the door. Feel its coolness against your hand. As you turn the handle, the door slowly opens, revealing a magnificent landscape on the other side. This is the reality you desire to shift into. Step through the doorway and enter this new reality. Take a moment to observe your surroundings. Notice the vibrant colors, the sounds, and the sense. Allow yourself to fully immerse in this new reality, knowing that it is a reflection of your deepest desires and intentions. As you explore, you may encounter different aspects of your desired reality. Embrace them with joy and gratitude. See yourself living your dream life, experiencing the relationships, abundance, and fulfillment you have always longed for. Now, bring your attention back to your breath. Take a deep breath in, feeling the life force energy entering your body, and slowly exhale, releasing any doubts or limitations that may arise. Allow the breath to anchor you in this new reality, grounding your intentions and affirming your power to shift your experience. As you continue to breathe, remind yourself that this reality is within your reach. It is not a distant dream but a tangible possibility. Feel the excitement and anticipation growing within you as you recognize the power you hold to manifest your desires. Take a few moments to envision yourself embodying this new reality completely. See yourself expressing your true self, living authentically, and embracing the limitless possibilities that exist in this shifted reality. Now, in this new reality, find a peaceful spot where you can connect with your inner wisdom. It might be a beautiful garden, a serene beach, or a cozy cabin in the woods. Allow yourself to settle into this space and take a seat. As you sit, imagine a wise and loving guide appearing before you. This guide represents the wisdom of the universe, ready to assist you on your reality shifting journey. Take a moment to connect with this guide, feeling their unconditional love and support. Ask your guide for guidance and insights on how to navigate this new reality. Listen to their words of wisdom and allow their guidance to sink deeply into your being. Trust that they are leading you in the right direction and that you are fully supported on this journey. As you receive the guidance, notice any shifts or transformations happening within you. Feel any limiting beliefs or fears melting away, making space for new possibilities and expanded horizons. Allow yourself to fully embrace the changes and growth that are occurring within you. Now, express your gratitude to your guide for their presence and wisdom. Feel a deep sense of gratitude for the universe for providing you with the opportunity to shift your reality and manifest your desires. Take a few moments to sit in this space of gratitude and integration. Feel the energy of this new reality coursing through your body, mind, and spirit. Know that you have the power to manifest this reality and that you are deserving of all the blessings and abundance it holds. When you are ready, slowly bring your awareness back to your physical body, feeling the connection between your body and the space you are in. Wiggle your fingers and toes, allowing yourself to fully return to the present moment. Open your eyes and take a moment to ground yourself in the present moment. Carry the energy and intention of your reality shift with you as you move forward in your day, knowing that you have the ability to shape your own experience.  Trust in your ability to manifest your desires and embrace the journey of transformation. The possibilities are infinite and you have the power to create the reality of your dreams.


Alright guys, I'm Shane, the online media tutor. If you're interested in 3D modelling and animation, as well as 2D animation using applications such as Maya and Adobe Flash, then you should check out my channel. What makes my channel different is that I'm actually a teacher. I teach media production in the north of England and so this means that I'm able to put as much thought into the way I teach as into the actual skills and techniques that I'm covering. All of my tutorials are broken down into easy to follow steps and you'll be able to see your own skills develop as you work through the exercises. I also cover the theory behind the practical to make sure that your skills are built upon the principles underpinning them. What's more is that my channel is truly interactive. If you're following one of my tutorials and you get stuck, then just get in touch. Leave a comment below the video or contact me through Twitter, Facebook or email. I read and reply to all of my comments and for more complex or detailed issues, I'll create and upload a video to help you out. If that sounds good to you, then hit that subscribe button and join me for a new video every week. If you're still not sure, then check out some of my tutorial videos and read the comments left by the people that have helped so far. I look forward to helping you in the future too.


Commodity prices continue to drain the pockets of canyons. The Canyon National Bureau of Statistics unveiled on Friday the annual inflation rate in March. At 9.2%, the overall year-on-year inflation rate as measured by the Consumer Price Index, CPI remained unchanged from February. The housing, water, electricity, gas and other fuels index however increased by 0.6% between February 2023 and March 2023. The transport index also went up by 0.3% during that period. Canyons, as suffering from surging prices as well as a sharp drop in the local shilling against the US dollar and a drought that has left millions unable to properly feed themselves. During the election campaign, the president portrayed himself as champion of the downtrodden and vowed to improve the loss of ordinary canyons. Hundreds took to the streets in recent days as they wanted him to act by his words. The opposition launched a campaign of nationwide protests that kicked off on March 28th.