Hi everybody it's Sam at Mixed Up Craft thank you for watching my tutorial today so I'm back after a nice week's holiday so I am rested, rejuvenated, all that lovely stuff so yeah it's been a nice week with friends and family. So first project to kick start the new week is this really lovely purse which is a gift card holder. Now I love this and I think a lot of you are going to really enjoy it so it's a double wallet so this was actually a request from my mum and it's inspired by her own purse and obviously I put my spin on it so the front here you just lift this up it's a little velcro dot you can probably you know put a magnet there if you want and this is your coin side so you can pop some money in there it will also fit a gift card as well if you'd like to and then on this side here you lift up this piece it will open up just like a wallet so here you have I haven't brought the gift cards yet so this is my boots advantage card then I have one of these nice little friendship cards that one of my close friends gave me and there are three little slots actually so you've got one there one there and this is my hobby craft club card that one that can go in there as well so my idea is is that the person that I'm going to be giving this to I'm actually going to buy her three different gift cards with a smaller denomination on so you know once they all add up it's a nice gift for three places that I know that she loves and then I'm going to pop a couple in there I'm going to get a little friendship card as well one of these you can pick these up in lots of the card shops so although I don't buy cards anymore they do have these nice little friendship cards that you can purchase for about a pound and then you've got three slots on this side as well and then also here you have an area to pop your money so there is just a 20 pound note in that one but it's a big area so it should fit most nationalities currency so I know some have got much larger notes it is a large area you can see the 20 pound note is quite big anyway but you can see how big that area is so it should fit dollars and things like that quite easily and then that closes up with a velcro now it's chunky it's got bulk to it that's nice that's what a wallet or purse is like so you can make this wider so if you want it or longer very easily and again once you see how I put it together it's dead simple so this is using the first edition gardenia papers and I finished it off with the butterfly die there from hobby base and then these are just some brads I actually cut the split pin part of it off and then just put some hot glue and just stuck them down and then I've also used a velcro dot there as well so really straightforward to make so let's crack on okay so just a few pieces here so I'm going to be doing this one using the mariposa papers and the one before was using the gardenia okay so there are lots of pieces to this I think what I will do is rather than go through all the measurements in one go they'll all be listed on my blog but I think what I'll do now is I'll just go through as and when you know we need them so first of all we will start to make this pocket side here so the coin purse side okay so you will need a piece of eight make sure that was eight so I've got my smaller scoreboard here yeah eight so this is handy to use that's why I've used these ones here because he's a eight by eight so this is a piece of eight by four and a quarter and along the eight inch side you want to score in half at four okay and then fold that in half so already now it's giving me this piece here because what will happen is the basis of this is these two sides will stick together one side becomes our coin purse and one side becomes our wallet where we've got our cards and everything okay so before we stick them together so just stick with one of them for the moment and then we want to make these side pieces so I'd already gone and prepped some pieces so these bits here so these are going to be our little side bits here so you want two pieces I've got a contrasting color here so it's all part of the same collection this is this pearlized card and I just thought that'd be nice on the sides so this is two and a half by four and a half okay and along the two and a half inch side you want to score a quarter of an inch one and a quarter and then two and a quarter okay so you'll have two quarter inch sides and then that's center score line and what you want to do is fold the two outer score lines so they are mountain folds and then that middle one will go into a valley fold so that is the shape that you want get it on an area you can see there we go and you want two pieces okay I've already gone and stuck double-sided tape on those side pieces and I've just taken a little wedge off the edges as well so we don't get anything sticking out so if I grab my pokey tool here if you do one side at a time okay so I'm going to start with this side here and I'm going to turn it over and you want to marry up the score line here with the outer side of this piece and again it should be the exact same size if it comes up slightly higher then we can trim that because for some reason again mine's come up a little bit higher can you see that it's just poking over the top but we can trim that make sure you get it right to the bottom okay so you've got that piece that then this this valley fold should be inside all right because this is going to create our side piece and then fold that piece over take off this one here now if you want you can do both of these sides so have that one stuck there now and then fold this piece over I prefer to do it actually separate and just kind of lift this open a little bit um just because I just yeah I think I just got a bit more control so I'm just going to hold that piece down and then fold this piece I use my pokey tool here if you're using wet glue then obviously yours will stick down much quicker it's just I don't want to get any of my um don't want to keep putting my fingers on it okay so now we've got our side there but because I've got a little bit coming out over the top so I'm just going to keep it flat and then I'm just going to trim come with this side actually just trim that top piece off like so okay so it's nice and neat again so if you do have that make sure you get it right to the bottom then anything that may overhang you can trim off easily okay and then again the same with this side here so I'm going to take one piece off I'm just going to open it up there and then just sit this one down let's try it up that way there we go maybe that way might be a bit easier and then you can kind of work it there we go and look that one isn't so I must have gone a little bit taller with that side because that one fits in perfectly so that one's folded over there push that piece inside like so and then take the backing off of here and then this one I'm just going to fold in push it in with my pokey tool because that's easy to pull out and then again I can just tack the bottom piece down and then work my way up and again nothing's sticking out the top there's that piece is fitted in perfectly that one I must have just cut a bit odd but now you've got your little coin purse side okay then you've got your flap okay so this is this piece here without the circle dies stuck on top this piece measures two by four and a quarter okay and along the two inch side you want to score at half an inch okay and then fold that in half then I've stuck this layer on top and this piece measures one and a quarter by four okay so I'm dropping down quarter of an inch increments each time I've popped some double-sided tape on the back kind of tab piece there so just remove the backing I've already gone and made sure it's stuck down then have a look if you've got a preference to maybe one side that's better than the other because one side is going to be completely covered okay so I know this is beautiful but you'll see it like I said once that one's down you're going to see that side of it anyway so I am going to have I think this one here it's got a few more darker purples so this piece here fold it over and you just want to lie it on the side where you haven't stuck the tape down so you can really get it right up to the score line make sure your sides are all even and then you can just fold that back piece over just with your bone folder so basically all you've made now is this nice little pocket which will open up like so okay so this is that other half there so what you need now is all these pieces here so I've got two two and two now this is optional and this is going to give us all the pockets either side okay so we've got the three pockets there and then three pockets there this back piece here is a decorative piece so this is this piece that I've got here now this piece measures at four by it's just shy it's seven and seven eighths of an inch okay now we're going to stick this a little bit differently so let me just show you so what you want to do is flip it over now I'm going to use double-sided tape I've got a thin one here you can use wet glue but I find this red tape just works really well and it's good for this project so I'm just going to go around and you want to stick it onto the two sides and one long side okay you're going to leave one long side open because that's how we're going to pop the money in okay so I've popped it on all three sides and then I'm going to take all of the backing off and then you're going to start with one side and you want to make sure you get this right up to the bottom left hand corner and then make sure it goes up nicely on that side there then you can trim away any little bits that you might have okay and you want there to be a gap okay so it's supposed to be like that and then go along it should meet up with the middle score line okay and now when you go to stick down the other half bring it up to a right angle and then stick it down okay now the reason I do say this well mine's just gone wonky there it's okay I can trim it it means that you will get this to open and close much much better okay so I'm just going to trim I've got a little bit wonky there now the reason I've done it like that is because when you open it you're not going to open it right up so it's flat you just you open it like this so it's kind of on that right angle and you get a much nicer finish whereas if you were to lie that down completely flat which now I can't do and that's fine that you want it to kind of open up like this when you go to close it it would just all buckle it just wouldn't look very nice okay so now that's ready to pop all of these sections in so these are for our pockets either side of this piece here so it's up to you again how many you want but you're going to need two pieces that are three by four two pieces that are two and a half by four and then two pieces that are two by four okay I'll show you one side and then I will go and do the other side off camera or just on fast forward so the biggest one is the one you're going to stick down first and you're going to stick it so it lines right up with that score line there now what you want to do though is to make sure that when you pop the card in the gift card see the the deepest one here that gift card won't go down any further because I've made sure I've stuck enough down at the bottom so that they all stick out by the same amount and by doing that if you take a gift card like this one here I'm going to flip it over and you lay it down on the larger one so it's overhanging by about half an inch and then I'm going to grab my tape and I'm going to stick tape along the highest kind of area to kind of stick down like so all right now I can get rid of the gift card and then I can just pop some more tape down there again I'm not going to cover this too much because it's it's only really decorative it's not you know you don't need to go crazy and then I'm just going to pop a thin piece of tape you can use wet glue but what I would say is you want to be very sparing because when you stick this down if you're wet if you put too much on and it all loses out you won't get the gift card in there's too much of the area is going to stick down so yeah I would say just use tape if possible so I'm just going to make sure that all sticks down like so take off all the backing okay and then like I said make sure you butt it right up as close maybe leave a little bit but get it as close to that as possible it's the same width as this so it should just line up nicely there and then you can just go and stick that down okay now when I go to pop this card in just start it off a bit it won't go down any further because of where I've stuck the tape down so it's just a nice way for that person to see the gift card or the you know the friend friendship card or something that you may have given them again with the next one you don't need to do it with the smallest one but the next one do the same again making sure you've got about half an inch of the card overhanging and run some tape and I will stick some more tape down along the sides exactly the same way and then this one here you should only have to overhang it by the amount you would and then just stick some tape so I'm actually going to pop the thinner tape along that one there okay so I hope that makes sense you want to do that on both sides you may only want to have two on each side you may you know not want to do that at all you might want to put a little message or a sentiment in here so there's again there's lots and lots of ways to adapt this you may just be putting some actual money in the form of you know notes in there I think it would be lovely for someone who is going traveling um you could put the currency of the country that they're traveling to in there you could you know make a wedding style and obviously pop gift cards that they've requested or again money for maybe their honeymoon that they might have requested if somebody is going away you know you might be kind of say Disneyland and maybe surprising somebody what a nice way to surprise them you could put the tickets for the holiday in the the larger section so I think there's lots and lots of ways to you know use this for many many occasions so I've just popped the tape on there and then the next one again you're going to line it up with the very bottom of that one that you stuck down earlier and it again should all line up nicely with the other sides and then the last one again line it up the very bottom like so and now again I'm just going to test it so I've popped that one in there like so my hobby craft one and then my friendship card like so so I'm going to go and stick these three down on that side exactly the same way okay so that is now that side done and again I've got all the room there to pop the money in if I need to so next what we're going to do is we're going to stick this piece onto the back of this one here I'm probably going to stick that side there actually for some reason there we go I think I just needed to fold it better so that now will sit perfectly over the back there you can see how that sits now because I'm using this textured cardstock I'm actually going to cover the back of this with my thicker red tape because my wet glue will just not last with all of this kind of glitter so I'm not going to go right to the very bottom because by now yours is probably starting to kind of arch a little bit because obviously you are folding quite a lot of cardstock but again it all adds to the look and we will be covering it with this decorative piece in the at the bottom once we've done it all so I'm just going to cover the back of this completely okay so now I'm going to go and stick this one down so I'm going to start with the bottom first because like I said if you are slightly out you can always trim the top and I knew I would forget so luckily before I push this down too much before you stick the two together you need to add this piece which is this one here can you see it's kind of hidden between so I literally just went to stick it all down and then realised so this piece here is this is what I mean there's so many bits to it I just want to make sure I give them to you all at the right time okay so this is two and three quarters by one and a half and along the two and three quarters side you just want to score a half an inch and fold in half then I've got these little mats and layers on top again if you want to add those pieces on the next one is two by one and a quarter and then the smaller one again is one and three quarters by one all these measurements will be on my blog on the other side I'd already popped some double sided tape so see I had it all ready to go and then what you want to do so you've got all the static you wouldn't have stuck yours together yet but this piece here will go in the middle on the back side there like so and the idea is everything's concealed like that okay so now that one will come over like so so next I'm going to add a velcro dot you can use magnets if you want I just don't really use them on these kind of projects because as I always say eventually they just go in the bin and magnets are not cheap and I would rather put them on my mini albums and those things get kept so I don't mind a velcro dot being thrown because they're pretty inexpensive so I'm going to pop one on the back there and then fold the whole thing over stick the other side down and then you should be able to lift it up nicely and you know it's all lined up so that's that side there I think that looks great this side here I don't know what I want to do in terms of decorations I don't know who I'm going to give this one to this one I already know what I'm doing with it so that's why I've added the butterfly but this one here I'm not too sure so what you will do is add two circle dies if you want to use circle you might want to use square dies and pop a sentiment on stick half of it onto this piece and then put your velcro dot on the circle okay so you can see what I've done there alternatively just pop your velcro dot there and close it I'm thinking that's what I'm going to do for this one for the time being because I can still add a shape or something over the top still um and still keep the velcro in that place so take those off pop the two the pair in the middle there and then fold that over okay so it's like so and then the last piece is this piece here and this is to decorate the bottom so basically it just covers that join see you don't have to add it because all it is is those two bits that you can see which isn't a bad thing but I just thought it was a nice added extra so this is one by four and a quarter and along the one inch side you want to score a half an inch and fold that in half and then I've already popped double-sided tape on the back side here and you want to hug the outer side more than the score line okay and then again start with one side so I'm going to go in here first make sure my score line is is kind on the bottom part as possible and again it's easy to trim if you need to but that's that piece stuck down and then just curve it over and it is quite bulky on the bottom so don't worry you know too much if you've got more on one side see I've got less on this side and I've got more on that side but it really doesn't matter they both look nice and by the time I add something else there but that is it it's a really lovely it's quite chunky I don't know something I love that about you know when you add all your papers together it's like a bit like a mini album I just think it's it's just lovely really really nice so that is now ready to fill decorate a little bit further if I need to but that side obviously opens up like so and then this side you have your coin purse you could pop the little card in there you could pop a little what size pocket is this this is a four by four so you could do a three and a half by three and a half card and this could be the envelope and you can pop that in there which is actually probably now what I will do because I won't be putting loose coins in here that was more decorative than anything I was probably just going to put a little friendship card in that section but now I think I'm going to put an actual card in there and then all the gifts and everything will be on this side here so that one actually I guess they don't open upside down because it it doesn't really matter which way so if you do that one open that one first I guess and then it's up that way with the money there and then close and then open your coin that side and then this one got the coin that side flip it over and then that's upside down so on this one it's that way up and on that one it's the pocket up so it doesn't matter either way how you do it it's just like a normal purse they have it guys my gift card or gift wallets purses I'm not again too sure exactly what they are but I really like them inspired and requested by my mum so mum I hope you like it and I love to see her versions when she does them no doubt she'll share them over on mixed up crafters and I encourage any of you that do make any of the tutorials that that I share please go and share them over on the group as well so that's it for today guys hope you like it as always please give me a thumbs up if you did and subscribe to my channel so you get to see more thanks for watching bye


Fellows and lutions, our country continues to battle this deadly wave of COVID-19, which from all indications continues to be fuelled by the Delta variant of the virus. Just this week, the Caribbean Public Health Agency reported another nine confirmed cases of the destructive Delta out of samples which were sent weeks earlier, confirming the suspicion of the spread of this more transmissible COVID-19 variant. It is clear that the virus continues to spread throughout our communities, affecting people of all age groups. Sadly, since the start of this wave in the middle of July 2021 to September 26, 2021, we recorded 5,703 cases at an average of 91 cases per day. Most of the cases are in the age group of 25 to 49 years. The majority female, the largest number of cases come from castries, grozile, V4 and babuno. During the period of the surge in cases, we note unfortunately 94 COVID-19 deaths, with females accounting for most of the deaths. At present, we have 1,539 active cases. We continue to prioritize the immunization program in St. Lucia and we note an increase in the uptick of vaccines over the last few weeks. A total of 45,245 persons received the first dose of the vaccines and 31,520 individuals the second dose of the vaccines. Both AstraZeneca and Pfizer vaccines have been as administered. It is encouraging to see a steady increase in the number of St. Lucia's taking the vaccine over the last few weeks. Additional sites have been established for administering the vaccines and all St. Lucia's are encouraged to take the vaccines along with their families. While our health professionals have indicated a slowing of the COVID-19 transmission rate, the number of active cases remains unacceptably high and there is still a great risk of danger to families and our communities if we continue to dismiss the seriousness of the COVID-19 pandemic and its impact on our country. We therefore must continue to be vigilant and keep restrictions in place while we prepare to allow fully vaccinated individuals to benefit from accessing specific approved services. Sisters and brothers, we must think not only of our families but of the frontline medical and other emergency personnel who put their lives on the line daily to keep law and order, to assist with the transfer of affected people, to care for the sick and to clean and sanitize the medical institutions. Workers in all sectors are also exposed and if all of us make a personal commitment to take action and to follow the recommended protocols and to follow the advice of the medical professionals, we will cause the transmission rate to continue to slow and eventually reduce transmission significantly. The new changes to the protocols are as follows. One, operation of businesses. Businesses shall cease operations on Friday the first day of October 2021 at 7 p.m. to Saturday the second day of October 2021 at 4 a.m. On Saturday the second day of October 2021, businesses will close from 7 p.m. to Tuesday the fifth day of October 2021 at 4 a.m. The curfew or no movement days will extend from Saturday the second of October from 7 p.m. all day Sunday all day Monday up to Tuesday the fifth of October at 4 a.m. From Tuesday the fifth of October to Friday the eighth of October 2021 businesses will close from 6 p.m. to 4 a.m. each day. From Saturday the 9th of October 2021 businesses will close from 3 p.m. to Monday the 11th day of October. Businesses will close from 6 p.m. to 4 a.m. each day from Monday the 11th of October to Friday the 15th of October 2021. Changes in relation to restaurants and food establishments. One, a restaurant and other food establishments approved by the Ministry of Health and the Ministry of Tourism may provide a dining service to persons who are fully vaccinated. Please note that fully vaccinated means that two weeks have passed since a person received A, a single dose vaccine such as Johnson & Johnson, B, the second dose in a two dose series of AstraZeneca, Pfizer, Biontech or Moderna, or C, another COVID-19 vaccine approved by the Minister on the advice of the Chief Medical Officer. The sale and disposal of intoxicating liquor is suspended, except for A, the sale and manufacture of hand sanitizers, B, the manufacture for sale outside of St. Lucia, C, the sale and disposal by supermarket, gas station or whole sailor if the supermarket, gas station or whole sailor does not permit a person to consume the intoxicating liquor under licensed premises. Recreational activities. Recreational activities such as boat rides and water skiing at a certified accommodation provider are suspended during the curfew hours or no movement days. They are suspended on Sunday the 3rd day of October 2021, Monday the 4th day of October 2021 and Sunday the 10th day of October 2021. Faith-based organizations. Faith-based organizations may continue to hold daily or regular religious ceremonies if the physical distancing protocols are ahead too. Special religious rites, including baptisms, weddings and funerals, are to be held using physical distancing rules with no more than 50 persons in attendance. These changes to the general protocols are intended to bring down the number of persons who may be infected with the COVID-19 virus. I urge all St. Lucia's to join with the leaders in their communities and mobilize to get the communities vaccinated. Join in our efforts to get life back to normal in St. Lucia. The protocols will be revised before the 15th of October and new protocols will be announced after the 15th of October 2021. I thank you.


preterm premature rupture of membranes, PPIOM, which is associated with vaginal dysbiosis, is responsible for up to one-third of all preterm births. Vaginal and neonatal microbiota analysis by 16S RNA gene sequencing after PPIOM may form the basis of individualized risk assessment for consecutive EONS. This article was authored by Luis Gustavo Dos Angeles Borges, Jana Pastushik, Yvonne Hyman, and others.


This tool can be rented from a library, and so can this tool. All of these tools can be rented from something called a tool library. In North Carolina in the United States, I introduce you to the Asheville Tool Library. So how do you rent a tool from a library? They're similar to book libraries, but instead of checking out novels, you check out tools from simple things like extension cords to lawn mowers to this is a wood chipper that would cost you thousands of dollars to buy. I love building and fixing things, but owning all of the tools is really expensive and most of them spend their entire life collecting dust in our garages. Did you know that the average power drill is only used for about 20 minutes ever? That's why lending libraries are so important. They help us to share resources, to cut down on waste, and to rise above consumerism.  Pretty cool, huh? For just $50 per year, members have access to about 2,000 items. So don't worry if you're like me and don't own many tools. With the Tool Library, you get to own all the tools you need without actually owning them. But if you don't have a Tool Library near you, don't worry about it. There are plenty of solutions for sharing tools and living more sustainably. You can talk to your neighbors and friends and coordinate tool sharing in your community. You can live in an intentional community where everybody shares everything. And you can go to my website and search Tool Library to see many other examples. We can all take actions to live more sustainably and to get involved in our community. So stop buying tools and start renting them at your local Tool Library. And who knows? Maybe you'll even see me at your local Tool Library.


Network, TFNN, headline news update. Welcome folks. We had the Dow Industries finish up 88, NASDAQ down three S&Ps up one and a half, and if you go over to Disney folks, Disney was the mover that got that Dow Industries up to new all-time highs. Intraday, Disney went from $136 all the way up to $148. They came out intraday saying that they, in the first 48 hours, they got 10 million subscribers and just to put that in context, 10 million subscribers took four years for HBO to get. So bottom line is that once Disney took off topside, that brought the Dow Industries also up to new all-time highs. So when we look at the marketplace, what they have is this, NASDAQ, S&P 500, NDX 100, yesterday went to all-time highs. Today you get a sideways move out there. Dow Industries today, all-time high. Small caps can't handle it at all. In fact, if you want to see how you can really get the gauge on weaker sectors and stronger sectors, weaker indices and stronger indices, all you have to do is look at the Russell 2000, gauge them against any of the other major indices, meaning the NASDAQ, the Dow, the S&P, the NDX 100, and with the transports, it doesn't matter. What you're going to see is that the small caps, the Russell 2000, they can't even get to higher swing points, never mind their high. Gold contract. Gold contract caught a bid of $11.1464, as did silver, up 22 cents, $16.91. Now, in both cases, we want to see more fall through tomorrow. We had 350,000 contracts in the gold, which is good. I still want to see a real monster today, like $450,000, $500,000. Light Sweet Crude, up 46 cents, trading $57.26 a barrel. Notes and bonds also caught a bid. They're going to need more volume, though. Ten-year up 7 ticks, $128.24, 30-year up 12, and $157.12. If we get open, we take a look at that 10-year first, what you're going to see out here is that we did do 1.3 million contracts, which is a good contract volume. We closed out at $128.25, which is inside its larger range. Now, the 30-year, we had the volume with it, but yet you're right at the—it's not in the larger range yet—$157.17 is the larger range. We closed out at $157.13. Have a great night, folks. Have a safe night. We look forward to speaking. All right, back here tomorrow morning, 9 o'clock.


Thank you so much for coming to the session. I see a lot of familiar faces. I hope you guys like it. So we're going to talk about how to choose the best Kubernetes to deploy AI workloads at the edge. We're going to talk a little bit more about how to deploy itself. And my name is Miriam Fontanes. I am product manager with the OpenShift AI. BU in Red Hat. And I think this works, yes. I'm Jacqueline Kohler. And I'm a senior manager at Red Hat for the engineering team. So what we're going to see in this session is, first of all, we want to give you our definition of edge. We know that everyone has their favorite. But we want to kind of tell you where our mind is in that. Some use cases that we have seen with different customers on telco that can be used as a pattern for other industries. And then the challenges that we have seen when they are trying to productize AI workloads. So the challenges with serving, managing, monitoring, and observability of these workloads at the edge. We're going to see in a little bit more detail the lifecycle of the model. How do we solve some of the management challenges, the observability. And at the end, we want to give you the general picture of how we have implemented our solution. So we're going to start with what is edge. That is a very overloaded term. We decided to take the more simplistic approach to it, which is edge is everything that is outside of the data center. There are a lot of companies that have been doing edge for a very long time, and they don't call it edge. So like retail, they have had servers on the stores for a very long time, and they never thought about it as edge. But now we are seeing that it's more and more important to put powerful computing at the edge, and that's what is edge computing. And basically, the reason why we are seeing a lot more interest in edge computing lately, it's because of all of the challenges that you have when you have the data sources very far from where you want to do the computing, and all of the costs associated with moving that data between the source and whatever you want to do the analytics. And these costs can be monetary, so moving all that data, it's expensive. But also in terms of latency, when you have applications that require really low latency, like VR and video games or things that interact with end users, latency is super important. And also at the edge, we tend to see that there is a lot of high-volume data with very low value. So we basically find the challenges related to all of that. And as we talk with customers more and more, we were able to identify three main characteristics of edge. The first one is the constraint on resources. So it was not only about the bandwidth and the latency, it's also about CPU and computing resources. We think that edge computing is very complementary to cloud computing. But where in cloud computing, you have these infinite pool of resources in edge, you are very constrained by the actual physical space and the location of your servers. So that's one of the biggest challenges, connectivity. For security reasons, with a lot of our customers, we see that these environments are either air gout or intermittently connected. So always being able to access them to manage them or to get data out of it is really challenging. The second characteristic is about all of these environments tend to be data-centric. So the data gravity is really what is pulling all of this computing. It's in some space of this core edge continuum. And when it comes to data, a lot of our customers are really worried about data. Severity, data privacy, being able to do some sort of computation to move that data. So that's another of the things we're seeing over and over. And the third one is around management. What really sets apart just distributed computing from edge computing is that another resource that it's really scarce in this edge location is IT personnel. So even in a retail store, you don't see a lot of IT people there on a factory floor or on an oil rig or on all of these places you don't have really IT. So you need to be able to have operations that are fully automated, that are resilient, and that are unmanned. They have to have some sort of autonomy. And some degree of the management has to be done at the edge node and some at the core. So those are kind of like the three main things that we saw that were repeated over and over as we saw more customers. So we're going to talk now a little bit more of the challenges of these specific use cases. OK, so yes, we're going to talk on some of the challenges for the use case specifically. In our scenario, we focused more so on telco for our solution. So as you know, 5G is enabling a lot of new business models and service delivery models. And there's a lot of different products and services for multiple infrastructures and software vendors. And with that, there's, do you see here the feedback? Here, read that. Maybe this will be a little bit better. OK, so the first thing we'll cover is manageability. And with all the edge nodes that are being added, there's challenges with being able to manage them across different geographical areas and compute zones. And with 5G networks, you want to ensure that you are delivering on the promise of the bandwidth and make sure that throughput is high, that there's no low latency, and that you're using your capacity efficiently. And so some of the things in order to consider when doing this is to make sure that you have automation in place, that you can manage the edge nodes. You want to be sure that you can remote provision those through your WAN networks. You need a single painted glass somewhere, also known as a dashboard, so that your IT folks, if you have them, can watch to see if there's any alarms going off or what metrics are being sent back. You want to make sure that you can also remote upgrade those sites. And you have to ensure that all of that's going to work and be compatible between the different versions of software throughout the infrastructure. Also, you want to make sure that you have resiliency so that even if you have intermittent connectivity, that you can still run your workloads at the edge. The next thing is latency. And I think this one is easier to explain with an example. So think of driverless cars. So if you have a cloud that's milliseconds, dozens of milliseconds away, that can be a lifetime, especially if you have people in cars, big metal cars, and they're not able to react in real time. So one of the things that you can do to solve that is to put many data centers sprinkled throughout the city. Security is another big one with edge. You want to make sure that you're encrypting your communication between the core and the edge applications. What you can also do is make sure that your edge device is its own single network. You want to make sure that you can somehow ensure that the edge when it has connectivity is running the latest when it's connected and that you don't have a single source of authority. So if someone is able to compromise one of the nodes in your network, you obviously don't want to allow them to be able to compromise the rest. Value of data. So as things get distributed, where you used to be able to look at that data real time with edge, the longer that data is out there and it's not processed or anything is being done with it, then that data loses value. So you want to be able to try to be able to do some processing and some analytics on the edge. And so hopefully when you get to some of our solutions, you'll see how we're trying to solve some of these problems. The next is AI and ML models at the edge. Each edge may have a different use case. So the number of models you may need with different data and building those models starts to grow exponentially as well. To the contrary, we've touched on this just a bit. So you need to be able to do something to gather, store, preprocess, and forward those metrics before you lose the value of that data. And then network automation. So this is key too.  And so I think that goes to our next one. So we've talked a lot about the problems and the challenges. So now it's the part when we give our take on what could be a possible solution to solve these challenges with telco customers that applies to something else. So first of all, what we want to do, we are talking specifically about AI workloads. And if you see the lifecycle of a model, we have borrowed from software engineering and we divided that lifecycle into two different loops. So you have the inner loop, which is everything that happens before a model. It's ready to be deployed into a production environment. So everything from gathering the data, experimenting, evaluating the model, the data scientist, training with whatever tools they want in whatever environment they want. Once they decide that they have the optimal performance for that model, the idea is that they will store that model into some sort of repository where they will be able to push it out and hand it out to operations that will be in charge of actually putting that model into production. And that includes tasks like reviewing whatever new model is coming, building the model, deploying it, serving and monitoring it for things like model performance, things like data drift, model performance decay to be able to re-trigger, again, all of the inner loop. In application development, this is very important because you want to constantly enhance your application. But in AI models, this is crucial to be able to really have models that reflect the reality and are performing well. So we want, when we started thinking about all of these challenges at Edge and the lifecycle model, what we wanted to you give our customers was the flexibility to perform any of these tasks anywhere in that continuum from the public cloud to the Edge. So if, let's say, for any reason, your inference needs to happen in real time, you want to place the serving part of the lifecycle into maybe a camera or maybe a pole on a street where all of the cameras feed the stream of data. So that's something that we've had in mind and we decided that the best way to do that was to use container technology, Kubernetes, that it's being shrinked down and down. We just attended a talk about Kubernetes on mobile devices. We have MicroShift for very small footprints. So that's kind of like the idea. And then when we started seeing about the individual challenges about managing the lifecycle, we came to some data points where they told us that 70% of business said that management at the Edge was really the biggest challenge they had when they were deploying workloads there. And it's even more difficult because the farther away you go from the core, the more specific the environments are to a particular use case or to a particular user. The hardware, it's not homogeneous. You don't have in the cloud really nice interfaces to be able to provide you with operations. So we decided to try to find a solution for that. And if we saw what were the actual more complex challenges when managing an Edge, we saw that number one, it's the amount of Edge nodes that you have to manage. So for people that are used to cloud operations, maybe you can manage hundreds, thousands of nodes. When you go to the Edge, that could very easily multiply to tens of thousands, hundreds of thousands. So just the amount or the scalability for management capabilities. The second one is the complexity of the applications and of the environments themselves. Again, it's not the same to have hardware with GPU acceleration that can leave in a drone that having factory PC that has different computing, maybe no UI, maybe not any acceleration. So all of these heterogeneous environment need a consistent way to be managed. The next one was, again, what we saw. Disconnected operations. It's really hard to be able to manage something that you cannot see all the time. So we have to account for that on any management solution. And then compliance, how do you enforce that? What you are intending to deploy, it's really what's deployed on that far Edge location. So we decided when we were thinking about it, we came up with the approach of applying GitOps for these management challenges. It really fit well the nature of the problem that we have. First of all, because since we're using Kubernetes, it was a very natural fit. And GitOps provide us a lot of benefits to be able to manage the lifecycle of the model and deploy it. So the first one is that it's a declarative approach. So you can make sure that you declared the desired state of your inference service container in a manifest. And by automating the whole process of upgrading, you are assured that nobody at the Edge site is manually configuring something through the command line tool or through a UI. You are ensuring that you have a controller at the Edge node that it's constantly reconciliating this desired state with what is actually installed. And if somebody makes some change, this intelligent controller will make sure that it goes back to the compliance state. The next thing is that it has ingrained an approval process. So it all happens the same way that in application development, you have to go through a PR process. You can have a human in the loop approving this PR. So you can have automated processes to do the merge automatically. But what's important is that you always have these mechanisms or these gates to make sure that you don't just send anything into a production environment. Version control, it's also really important, because especially for Edge nodes, we see that right now there is not such thing as an equivalent as a cloud provider. So when you run cloud computing, you have the three big ones. And you can just ask for compute resources, and you have it. There is not such thing on Edge computing. So if you want Edge computer, either you own all of the Edge nodes at the site, or you have third parties that deliver things that are now known as Edge in a box.  And you have cloud providers trying to also jump into this business model. But the important thing is that you need to be sure, no matter who is managing, there has to be somewhere where you can audit what is the different versions or the different configurations that you have implemented. And the last one is because GitOps reverses the management responsibility to the Edge node. It's in the Edge node where the controller is that is checking constantly. You get eventual consistency, and that allows you to scale better. There are a lot of community projects right now that are working on shrinking and shrinking also. The agents to do GitOps, like with RGocD or with OCM. So we found that that fitted really well. So basically what we had was a flow, something like this, for deploying new versions of AI workloads. You have someone, a data scientist presumably, delivering new versions of the model. They push them to a code repository. From there, you have somebody reviewing those PRs. And once they tag that as ready for production, you have a CI pipeline that you can customize. So the more basic pipeline will just download the model, download all of the dependencies, the parameters that it needs. Make sure that you are using secure supply chain to be able to graph all of those dependencies. Do some on-device testing. So that is also something very important when you want to make sure that the performance that you get of the model while you're testing is the same one that you will get on the model while it's flying out there on a drone or while it's on an old rig. And then it containerizes this inference service. Once all of this step is concluded, it will put the new inference service container image into a distribution repository and it will also publish a new version of the desired state of the model into a get repository. You have the key piece for all of this, which is the controller that has the intelligence to be constantly pulling the repository in searching for new changes. But also that controller, it's very specific to the environment where it leaves. So that controller is the one that knows that I am living on this device. So I have to do things a certain way or I have to apply the manifest through command-line tool or using a playbook or a script. So that controller is really key to our solution. And then, yes, so that controller is the one that's constantly pulling, the next one. And you know, these basic building blocks are not tied to any specific technology. So depending on your use case, you can really use all of the diversity of technologies that we have. So for example, for the pipelines, we use Tecton, but you can also use things like Jenkins or any orchestration engine that you already have. For the distribution repository, we use an OCI, which is OCI compliant because it also has a lot of utilities and tooling to do the distribution in remote locations. For the management itself or the deploying of the implementation of GitOps, we use Ocm and the pool feature that it has, it really again fits well because the communication is always from the edge node to the core, asking for new changes. And for the controller, we use a combination of Ocm and Argo CD, but again, if you are in an environment where maybe there is not even Kubernetes, you can use Ansible and deploy directly to let's say Rela and Pothman or to a virtualized environment. And if you require even more customizations, you can maybe use something like Helm or Customize. So all of these, it's again, we are just implementations of the pattern. And in Open Data Hub, we decided to build the abstraction layer for AI or for ML Ops. So we provide all of these configured out of the box so they don't have to craft it themselves. But if again, it's pluggable, it's extensible and the idea is to be flexible. Okay, so let's cover what we did for observability. You know, when you have limited storage capacity at the edge and you have intermittent or congested bandwidth, you need something that can handle that as well as being able to store and maybe do some pre-processing. So we decided to use Open Telemetry for our solution and this is how it works. So basically, we chose this because a backend doesn't matter. It can do some pre-processing of data. It can compress data. It can do intelligent data filtering, which means that if you have sensitive data at the edge that you don't want to sit back somewhere, it can actually help with that and filter on that. And it also supports all kinds of third parties like Splunk, Prometheus, Donut Trace, PagerDuty. So it just seemed like a really flexible and good fit for our use case. And so you'll see here, we have our core and we'll have our edge networks that are running Open Telemetry and it can send data back to the core or you can send it to Telemetry Gateway or you can send it to the cloud that people here at the core can work at the cloud. So it just seemed very flexible and that's why we chose it for our edge data or metric collection. And so I know we've talked a whole lot, so we wanted to put in something that showed you our deployment and how we're doing things and this is the overall picture. So we start at the data science, which we didn't cover that with our model, we're just assuming that the model's trained, it's built.  And at that time, the MLops engineer is gonna decide whether or not it's ready and they're gonna put it into production. So the MLops engineer will kick off the pipeline. That will initiate, in this case, to using Tecton to retrieve the model. It will build the model, build the inference service container image as well as test that the model is doing what it's supposed to do before it gets pushed out. If everything passes there, then the inference service container image gets pushed to some image registry. In this instance, we're using Quay, but you could bring your own, whatever you wanna put there. And also, a PR will be posted with the latest metadata in this, what we used as a Git ops repo. Now, what Miriam was saying, we can automate this or if not, the MLops person can sit there and just monitor to see if something got posted before they merge it, especially if it's going into production environment. So then, once that happens, you'll see that you have your edge node there with Argo CD running, and it's gonna keep checking back to the Git ops repo to see if there's been something pushed. It needs to pull down a new container image. So it pulls it down, it gets it deployed on the pods there, and everything just keeps working that way. In between, you'll see that the OCM spoke there. Constantly, or it checks back when it can with the OCM hub just to see if there's any updates. Should the node, should the edge node come down? Are there anything else that it needs to do? And as well as you'll see where we've fit in the data from the open telemetry. And so that is our setup and our deployment. And it's out there working in ODH if you wanna go try it. All of this is Open Data Hub. If you're interested in taking it for a spin. And that's it. Questions?


Hey, what's going on everybody my name is John Hammond and a few people asked me if I would be willing and able and I am both Of those things to share my note-taking process and some of the procedures that I did while I was taking my OSCP or offensive security certified professional so I want to put together a quick haphazard video like all my videos are and Showcase some of that stuff.  I'll quickly sign in I'm using last pass so it'll just crank through username and password super simple super easy That's good stuff, and I want to hit that new project button up on the top right I've seen this take a little bit of time to load, and I don't know why right now That makes no sense to me Okay, cool. Here we are on get lab comm slash project slash new so we can give a name to our project I'll just call mine OSCP with two E's so it's cool and clever, and that way it's not the same as one that I already have So you can give a little project description. These are my notes and resources for well I Why don't know why my mouse is like flashing is it doing that for you guys too? Sorry well I was taking OSCP cool, and we can go ahead and create that project super easy Cool, so now we have that URL and we can work with it on the command line Which is where the better stuff actually happens So what I'm gonna do is come just going to get clone that repository if you don't have git If you're on arch you should just be able to yay tack s install Don't actually use the word install, but yay tack s get or pseudo pac-man if you have any Sunday Set up any aliases for that blah blah blah or if you're on Ubuntu or some other maybe Debian based Install distribution where you just have apt as your package manager. You can apt install get so now I will cd into that directory and now we just have this as our workspace So I'm gonna use vim as my editor here. I'll just create a little OSCP notes and what I like to do when I create things like this I'll just put my name and the date was it October 6th. Yeah Some notes here. I'll be like these are all my notes And we crank through that. Okay, so I could just do a little get add get commit added initial read me Save that and let's pump that the repository. I'll just do a little git push here I don't have my ssth keys put together with git lab and I totally should I'm kind of a failure in that regard But okay now if I root or fresh that we could see my commit and my notes are in there And I could fill that out as I needed to That's super handy because that way now any pictures screenshots that you save just throw in this repository Get add get commit throw it up there and then you have a cloud backup, right? In the sense that now of your notes are in another remote location so if you happen to be working elsewhere and you've got some opportunity you've got some time you can just crank through some work and You have a backup right now. We can go back a little bit of version control see some other notes that you may have left there previously So another individuals I think it asked me for how do I actually convert my markdown into a PDF? And the way that I had done that was with OSCP Markdown to PDF. I think I shared the link in that previous video Noraj had a little template for it and this was super awesome and I shared this and wanted to showcase it This is a good Templates for creating the your exam report or lab report whatever you want to end up creating Just keep it simple keep it easy and markdown and being able to work with it so you'll need PAN doc and you need latex and This EIS Vogel or ice Vogel dot tech. I think it's a little styling there I'm not entirely too hot on some of the PAN doc stuff But then you could just run this command to quickly generate your PDF So I will show you how I set that up and I'll try and showcase some of my notes here But that all all that stuff comes from this ice Vogel repository here So you need that ice Vogel dot tech file present in what you're gonna actually be working with So I will show you that In what my old setup was I'll go to PWK OSCP and there are my notes and some information that I kept track of here So I would work with PAN doc and again Yay tech capital S to install that and then I needed text live tech most and the case of arch I think I don't know if that will go ahead and install core and bin But I added those before I added most so I'm not sure which of those is particularly necessary To be able to make my quick simple generate report that I say script run quickly and easily Let me actually just go take this example Repository here, so I don't accidentally show you anything That I didn't want to previously. Let me I'll just get cloned in this current directory. So that works just fine Nice and easy now. I have that OSCP exam report markdown Director here. Okay, so I could Take one of these example markdown Files which I'll check out here and you can see nice give a little title Good little author give some information there and fill out between the paragraphs in the sections that you would want to fill out According to the machines that you broke into right according to the exercises and the questions that you wanted to showcase I'm not actually going to showcase mine because obviously that still has the actual content in there And I'm going to respect offensive securities wishes and not leak that information fingers crossed So that's how you can do the super simple super easy in markdown Just type as you need to add with the information you want These are more explanations and details blah blah blah And then we can actually go ahead and generate that output. Yeah, let me out Jim So what I'm going to do is just move my generate report script into that directory And I'll show you that in action exam report. Okay, cool Great what this generate report script is is Super small super simple just a wrapper around that pandoc command because they didn't want to keep typing that and running that over and over And over again, I just take the argument and bring it out to a PDF file And I note that in some usage up here So input dot markdown and output dot PDF and I move that ice Vogel Latex file into the directory that needs to be an if for whatever reason That's not already in place on the machine because that I had that issue when I was working on another Work machine that I wanted to get spun up on and then if that command to generate the PDF ran successfully I would simply show the PDF with evidence. So let's try that What I'm going to do is just run my generate report script. Let's use the OSCP exam report templates who is Flynn because I like that one better dot markdown And let's just say example dot PDF. So give a little bit of some time Just a quick second here and suddenly that will just pop out. Here's your penetration test. Here's your lab report Here's your exam report, whatever you wanted to put together And it's all of that same text that you've already seen now just in that beautiful easy breezy beautiful cover girl Display so you can submit that over to offensive security nice and easy, right? That's super cool let me close out of that and I'll show you some of the other stuff that I had in here because originally you do need to submit your exam report and lab report as part of a sevens that package and with your password Offensive security ID all bundled in there and I didn't want to get any of that wrong So I made a quick little script that would do that bundled it all in and it would include the files that I created as necessary That way it runs the sevens of command with all those variables and passwords and everything in place And I don't need to accidentally get that wrong or have a typo because that would be Devastating and they would not grade my not grade the exam and I would have completely failed. So that sucks trying to avoid that So that is my generate report data sage script That is the markdown PDF thing that I used that is how to create it in a simple text editor I used to use sublime now. I'm trying to get into VIM team ux and arch Linux and all that fancy stuff Now I want to showcase some of the other notes that I took because those might be handy to you One thing that I noted was I had a Windows W get that I had found online It's just a simple visual basic script that I wanted to take note of and Keep track of it in case you ever got on a Windows machine And you need to be able to download things or download some stuff from your host or other Privilege escalation scripts that you might be using etc. Etc. That's this syntax right here I'm happy to share that and you would just simply run it with C script Which you would normally be able to run and use just fine I didn't end up using this all that much because I was on low bins and That is you've probably seen a before in the GTFO bin side and you may very well have seen this one Low bass the living off the land at binaries and scripts. This one is strictly for Windows They do have a better website for this now You can check this out if you were to search for any of the commands that you're able to run on that target machine Or the victim that you're working with you might be able to do interesting things with them Like download files or in code and decode some different data streams or read files or write to files etc. Etc. So Normally I use cert util Which is pretty handy dandy for getting us a quick download They have a simple syntax here to be able to download files if you were on that Windows host Sometimes this wouldn't work for me. So another one that I found was actually extract. I think extract 32 That will still download as well Take a look at that guy Issue is that needs to be kind of hosted that file that you need to be able to download needs to be available on an SMB Share so you could spin that up with impact it if you wanted to and I can showcase that if you guys particularly need me to But it's a good quick and easy way download that file and impact it You can host it from your Cali machine or whatever attacker machine you're using so that's that But that came from my notion of this Windows W get that I tried to keep track of so Other notes that I had things that I was just trying to keep in the back of my mind as part of my preparation some Windows privilege escalation stuff a lot of these resources I found with YouTube and some information or some other people's writings and articles and blogs I tried to follow that slash our OSCP or that are slash OSCP subreddit There's a lot of good information that flows through there all the time So I monitored that a lot and some of these Repositories were pretty great for actually keeping track of that information. Let me just show you that real quick This guy had some good notes And it would honestly always boil down to just basic enumeration Basic. Hey what programs are installed? et cetera et cetera but this YouTube series was phenomenal and I always forget this guy's name and I Always fail at pronouncing it one way or the other but he has phenomenal videos quick super super short Super simple. I'm saying lots of s words tripping over my tongue here for a quick Techniques and tactics for Windows for legislation which you might want in the back of your head But a lot of times it just kind of boils down to the basic enumeration. So handy I Would showcase that as well and I took notes on like each section of this like one of these How do I do this in reality? How's what's my quick notes and reference that I can just copy and paste commands or tweak and adjust as needed And I'd do that with a lot of things Vim linux prevesc. Okay. I didn't actually have really anything worthwhile in here dirty cow rational love some simple like Colonel exploits things that you could take advantage of again. I didn't have as much mileage with that What I got to game time but for maybe the practice environment those could come in handy if you're if you're scraping It's straws. So that's handy. That's good for us And that is how I would simply generate things. I Would showcase the cover I guess of my PDF here for us OSCP OS that guy and These are the labs that I work through right but I'm not gonna show you this entire document But you can see there's 243 pages there. I had a huge lab report and my exam Page. I think that one was also Hefty. Oh, I opened that with I have no my bad I literally yelled at myself in my head for doing that earlier So and then I just had a little bit of a different display for the exam So that's that that's all that I really ended up doing use vim or sublime text or whatever text editor that you know and love Run through your stuff like jam out whatever you've been working with Because it's in a simple text editor It's nice and easy to just write everything that you're already working on document as you go and then use that simple maybe uh generate report script and Did I move that thing? I may have I may have moved that like a fool This is super duper handy and I found that really simple and easy Just wrapping around that pan doc command and being able to generate that PDF as quickly as I can So I'm happy to share this code. I hope this kind of helps showcase a little bit of my methodology And some other resources and assets that I would use and work with so Please let me know if there's more you want to see that I can realistically Show you right? I'm not I'm not trying to do anything bad here But I want to let you into my mind and my methodology for how I was able to burn through some of this stuff So mark down a PDF Use a text editor document as you go along write down everything you can take screenshots throw it in your nice cloud backup and simple repository to keep track of stuff OSCP So thank you guys for watching. I hope you enjoyed this if you did please do like comment and subscribe I'd love to see you guys in the next video. Love to see you on discord join the server There's a link in the description. Love to see you on patreon Love to see you on paypal. Just would love to see you at a conference at b-sides at defcon Thanks for watching everybody. I'll see you in the next video


I'd like to thank Leader Proctor and Owen for inviting me here and I'm very glad to be in the basic biology session because I'm going to be telling you some basic biology. I was actually hoping that David Relman would talk a bit more about babies. We should have maybe coordinated on that because I sort of expected him to. So I'll touch on microbiome, colonization and assembly in babies but then move on to a few other aspects of basic biology that we've been working on that I think you might find interesting. So there's rapidly developing data coming out about what sort of environment we actually develop in when we're in the womb. I think we've assumed for a long time that it's been sterile but studies that are in progress and coming out are indicating that it might be slightly more microbially sort of influenced that we might previously have thought but it's still a relatively protected space where we develop until we come to the outside where we then rapidly colonized by the microbiota of the biosphere which of course are very diverse and vast and so one of the first things that happens even before you take your first breath I think is that you start to be colonized by microbes and we need microbes for proper developments and this has come from years of research that goes back more than 100 years with not abiotic animals. Animals that have been raised under germ-free conditions differ quite substantially from animals that are raised normally in the normal microbial world in that they have a different kind of immune system that's not fully developed if you will because that requires microbial influence they have different physiologies they have different behaviors there's lists and lists and lists of ways in which animals that are raised under extremely artificial conditions without microbes can differ from animals that are raised under with the microbial influence. So we've done some work looking at how this colonization happens this is a case study of a single individual over two and a half years which is quite a long time for current studies although more of these are coming now so that so that we can sort of compare the patterns that we saw in this particular individual and what you're looking at is bar graphs of taxa this is very simple way of looking at the data each one of these bars represents a day over two and a half years and there's certain events that associate with changes in the distribution of these taxes such as a fever here the introduction of table foods here which brought about a bloom in Bacteriades and all the while there was a very slow increase over this time period in in the diversity that was there so the richness showed a very slow incline over time increase over time and then various events bring about these big changes in the relative abundances of different taxa and the way this particular individual responded to these events is something and here's an antibiotic event for example is something that we can characterize but whether whether this these responses are typical of all individuals and whether these we can start to to go from these associations that we see in a case study to general principles is something that's going to require following many many babies and this is something for instance that David relevance group has been doing and so we can we can make some logical sense as to why things like Bacteriades increase when those table foods introduced based on what we know about their genomes and how they have the genes necessary to break down these foods and so changing the the milieu here in the gut is definitely going to bring about a bloom in the kinds of organisms that can that that use those substrates but but the kinds of microbes that are present in one individual to another is something that we're still trying to understand where they come from how much regional differences might affect the kinds of microbes are colonized with food yes we have some idea of how how food influences these things but there's still a lot of basic biology though that still needs to to be understood about this in this particular individual breaking it down and looking at the kinds of sort of consortia if you will this is again the same bar graph showing you how the phyla change but within those phyla we see discrete consortia that are members of those phyla and we see them change very gradually over time so these are the early days we see this sort of group and then later on this group and later on this group these early taxa came back after antibiotics we can see that here and then a whole new consortium replaced them afterwards and these these we know that these associations that we see like this these groups and non random but again it's going to take profiling many different types of babies over time for instance to to understand how if there's some general principles we can we can derive from these kinds of data or if or if different perturbances are acted differently in different individuals when we think about how the microbiota develop over an entire lifetime this is something that we can only piece together from looking at current studies of of individuals that have these different ages because at this time there hasn't been a single individual it's been followed over the course of a lifetime like this and so we have data on on very young people and we have data on adults most of the data is on adults and then we have some data on elderly people but but again no one person has been studied for this long and so although we we can see some sort of general themes like for instance it looks like there is differences in very early colonization based on how babies are born and it looks like stability sort of comes in maybe an early childhood and adult microbiota may be even more stable than that these are all these principles are basically derived from stitching together very different studies on very different cohorts and so going forward I think it's going to be very important to have large long-term cohorts so that we can really test whether what we think is happening based on looking at current studies is actually holds up when you when you look at people over the long term and one of the questions that the people and sort of the probiotics industry and so on are particularly interested in is whether or not what happens early on affects microbiome later in life for instance so if you have certain certain microbiota early here are they going to affect what comes later do we have distinct successional stages and do the do the what you have in these later stages depend on what comes before and that's not something I think we have the answer to at this point so what does impact the microbiome later in life or during adulthood in general this this is a very fuzzy diagram it was deliberately fuzzy that Pete Turnbaugh and colleagues wrote back in 2007 asking what kinds of influences are there on the microbiome both what's shared and what's not shared between people and you can think of this as lineages presence or the functional genes that are present and you can come up with things that you you know or think might influence this like the lifestyle and the immune system and the environment but the reason all these arrows are sort of the same size and don't intersect is because we don't have a good idea of which are the strongest how they might interact and so on and one of the areas that we have the least understanding of at this time is host genotype and I'm going to tell you now about a study that we've been doing to address how host genotype might influence the composition of the gut microbiota so there's a need for genetic studies in humans uh there've been some studies in mice using qtl mapping that reveal associations between certain taxa abundances for instance and certain loci in the genome these loci can be quite big and contain sometimes hundreds of genes and so actually getting in finer details to which genes are actually driving the associations um can is this sort of the next step for for animal studies as well um there's been studies with candidate genes in humans so for instance if you have a particular allele that's associated with a particular disease like nod 2 and crones you can select for people with different versions of that and look at microbiota and that's one example but you have to know what genes you're looking for to do this uh but there haven't been any published studies yet on genome-wide associations in humans there has been a work going back more than a decade now looking at twins and and twins are particularly sort of intriguing uh this is not actually a mirror this is a human mirror this is an improv group that got monozygotic twins and got them to line up down subway cars just to see people's reactions and so this really drives home the point that monozygotic twins are really remarkably similar phenotypically and the question is are they got microbiota is also more similar than than you might expect for say fraternal twins which might be these two although they're not and so again this goes back uh people have started to use twins uh this is an example of a paper from 2001 using DGGE fingerprinting and based on the relatedness of the people where one was the monozygotic twins they saw a trend towards more similar microbiotas in the monozygotic twins and then Pete again when he was working in uh Jeff Gordon's lab published this study so these are unifract distances this is based on 16s RNA pyro sequencing uh when the bar is smaller it means the microbiota is more similar and so now you're looking at twin pairs mz versus dz and there was a hint of greater similarities for mz's although probably because of the relatively low number of twins used here it didn't actually come out as significant so we've we've decided to look at twins and we're looking at twins that are genotypes so we can we can actually do a GWAS study as well as uh as use some of the twin based techniques to look at this and we're working with Tim Spector who runs the King's College of London uh twin registry there and it has about 15 000 twins in it that they've been working with for many years and they're part of many genome uh space studies so in this particular study we have access to about 6000 genotype twins and we're collecting stool from as many as we can so basically we're asking them to send it to us and uh as you know that's not always something that people want to do but so far we've had about a thousand send-in samples we have 250 dz twin pairs about 160 mz pairs unrelated individuals and these people are mostly middle age they're average age of 64 and they're mostly women and that's not because the twins in the cohort are mainly women it's well they are actually it's just that women seem to be more willing to be involved in this kind of study for whatever reason um there's several ways that we can we can analyze data like this so we've started with a 16s survey for and we have data for a thousand samples now and one thing that we can do is when we have abundances of different taxa we can look within twin pairs how well the abundances correlate and if you you might expect a tighter correlation for twins that are mz uh for for the abundances of taxa compared to dz twins if there's a genotype effect and we can calculate these correlation coefficients and we can do that at every single node in the phylogenetic tree and then take a look at the distribution of the coefficients the correlation coefficients and what we see when we do this is that um this is a histogram is that on average the mz twins tend to have stronger correlation coefficients than the dz twins which is shown here in the the dark compared to the light so that's telling us that there is a signal um that the mz that there is a genotype effect we can do the same kind of analysis that i showed you from pete turnbow's paper and we get the same result so if we look generally across microbiota's using the unifrack metric we don't see a difference between mz and dz's but if we look within specific families and these are the two dominant families of the fermicutes which is the dominant phylum in this in this study we do see that the mz's have a significantly more similar microbiota's compared to their dz's compared to dz's within uh within these particular families um we can also use twin based models to look at these kinds of data so this is using the ace model which is a which is a twin based sort of statistical model that partitions the variation in the data to genotype effects and environmental effects and what we're showing here is the as the effect of genotype with a heritability and we're showing it as a as a color so the strength of the heritability is on this scale with warmer colors meaning stronger and we've painted it on two parts of the phylogeny so that you can see that there's specific branches within the phylogeny that have particularly high heritability values and again they're within these two families the rumenicocaceae and the lachnospheraceae such as this part in here and there's other parts of the tree that have particularly cool colors like the bacturidides and this is telling us that bacturidides are particularly not heritable so this is allowing us to zoom in on parts of the the bacterial phylogeny that is most likely to be under host um genetic control and i just point out down here that we have methanogens and they're also coming out as um with some heritability which is kind of interesting and that corroborates what's been shown before with methanogens. We can use these techniques and look across other previously published studies so we pulled data from the Turnbaugh 2009 paper and a more recent paper from the Gordon lab that also had some twins and we do actually find some of the same patterns with these big families in the firmacutes showing us some heritability and much less heritability in the bacturidides shown there and then and i don't want you to write this down because this would probably change because as you know you know a thousand samples in a GWAS study is still a very small GWAS study and and we're in the middle of this it'll it'll expand over the next year or two but using the data that we have using the SNP data from the twins we're able to run genome-wide association tests and we we're finding things like this where for example u-bacterium shown here is has a correlation with the genotype of a particular gene so this particular SNP is coming up in association with you with a particular species of u-bacterium here and it there is actually a risk allele for crones in in this gene but but again it's early days it's particularly underpowered at this point and so it will update we'll be able to update this going forward but in terms of gaps we still don't have an understanding of how the host genotype determines the microbiome and we don't know at this point how the microbiome interacts with host genotype to determine risk susceptibilities but i think incorporating microbiome into GWAS studies for particular diseases is going to be very fruitful we already know for instance that different kinds of helicobacter pylori pose particular risks to particular kinds of to people with particular genotypes and so it it would just makes intuitive sense that this is also going to be the case with microbiome and at this point it's it's going to be interesting to find out how much variation in any kind of host trait could be explained by microbiome component alone or in combination with the genotype and and i i see this as as i really look forward to seeing microbiome incorporated into all kinds of GWAS studies in the future especially those that have some kind of inflammation basis that the microbiome might be driving so now this kid wants his own personalized microbes or at least he he wants to be able to determine you know the the abundance of certain kinds of things that you might get colonized by and what does he get well there's some thought that he might be getting his mother's microbes at the very beginning and and these are data that i think Maria Dominguez-Bello might talk about later on that very early on we know that that it looks like microbiota come from the mother and then other sources come in later but i'm going to use this to segue into pregnancy because and this this nicely shows it a pregnant woman who's just given birth is still not exactly like a pregnant woman a non-pregnant woman and you can see this like Kate for example would not have been looking like this a year ago so pregnant there's profound changes that happen during pregnancy and and persists to a certain degree after pregnancy and we were interested in those and i'm going to talk about some work we've done on pregnancy now and the microbiota that are associated with pregnancy we know that when women go from the first to the third trimester of pregnancy several things happen they gain adiposity their blood glucose levels are higher and their insulin sensitivity is decreased and these are thought to help grow a baby by keeping the blood glucose levels higher and then the increased adiposity is is thought to help prepare for breastfeeding later but these these these actually these things fat mass blood glucose insulin sensitivity have also been shown to be regulated by microbes in non-pregnant animal models so we were curious if microbiota had any role in this so we worked with Erika Ausolari and Seppo Salmonen from the University of Turku who had ongoing studies with pregnant women and they provided us with stool samples and diet data and clinical data and and baby stool samples from 91 women in Finland and first and third trimester and what we saw was the first view of the data they got was this so here are the same women sampled first and third trimester and the first trimester samples are very similar to one another and this again this is based on 16th but the diversity increases expands quite dramatically in the third trimester and the purple dots are postpartum one month so this point we didn't know what was normal the tight clustering or the not tight clustering so we put it in the context of the human microbiome male and female obviously non-pregnant people here and although there's a shift along this axis that might have to do with geography because these are Finnish people what what we see is that this big expansion of beta diversity is really not really not normal so we tried to associate the pattern with different things we had obesity and overweight data for prior to pregnancy but that didn't explain the patterns and neither did gestational diabetes status because some of these women develop gestational diabetes but but this didn't seem to map on to the expansion at all what did however seem to be driving these patterns were the the gradients of how much of the bacteriides and proteobacteria were in these samples so cool colors mean less and warm colors mean more so on this axis we have samples separating according to the bacteriides abundances and proteobacteria here indicating that the third trimester had many of the samples had more proteobacteria present and that was true for about 60 percent of the samples so if we look a little deeper and in use machine learning techniques to find the ot use that can discriminate the two trimesters we find that there's more short chain fatty acid producers in the first trimester which is kind of the normal looking state and the third trimester we saw things that reminded us of opportunistic pathogens things like proteobacteria which seem kind of odd and they're the kinds of types of bacteria that are typically associated with inflammation we also noticed a drop-off in the alpha diversity so the richness is reduced in the third trimester so in other words everybody has a reduction in the diversity but all in their own way because they become quite different from one another and this persists one month postpartum which is shown here so this is another way of showing the clustering tight clustering is a shorter bar and then you see the expansion of between subject diversity it stays one month postpartum and then when we looked in the kids you see that higher beta diversity in the children and then by four years of age they look like first trimester or normal people and I just note on the side here that we couldn't actually match children to their mothers at any time points even though the greater similarities between kids and their own mothers was for the four-year-olds in the first trimester but we couldn't actually match the one month or the six months old to their own mothers they weren't more similar to their own mothers than to anybody else's mother so we see this increase in proteobacteria for instance and other kinds of bacteria that don't look like they should be sort of that dominant in a normal setting and so we thought is there inflammation we'll look for it and we measured greater levels of inflammatory cytokines in the third trimester stool shown here some of these like IL-6 TNF alpha and IFN gamma so then we thought well what happens are the microbiota driving these these phenotypes at all so we took microbiota from the first and third trimester and and transplanted them to germ-free animals and what we found and now these are data for the recipient mice is that we after two weeks in the mice you could see a difference between first and third trimester microbiota and now looking at mouse cytokines we saw that the third trimester microbiota induced a higher cytokine load in the so so there was a sort of sign of inflammation in the third trimester recipients we saw higher blood glucose in the third trimester recipient mice and finally we got fatter mice um those that received a third trimester microbiota had greater adiposity gains after two weeks and these were swiss websters germ-free sort of same genotype that we used for both obviously for both inocular here so what we what we think is happening from this study is that the first trimester has a relatively normal microbiota and then as you go to third trimester there's an altered microbiota and what might be driving this transition is something that we still have to determine it might have to do with changes in immunity uh that that change mucosal immunity and when we transplant these microbiotas into germ-free animals it's enough to see some of these phenotypic differences recapitulated in in the uh in the recipients here so that we have a fatter mice with insulin desensitization which is actually what we see in the third trimester host when she's pregnant so these metabolic changes that are induced by by microbiota it's something that we've seen that's been talked about in the context of a metabolic syndrome in human beings and it's possible that they have that this kind of host microbiota interaction has maybe evolved in the context of pregnancy where it's really much more adaptive and maybe microbiota kind of used as a link in the chain here so that when there's a change in hormones or immune state that could alter the microbiota and then they altered microbiota then has an effect on the host but in this context it's actually beneficial as opposed to this other context where maybe it's less beneficial and so one of the things we're doing now is trying to compare you know do we see the same kind of host microbial interactions underlying these um these metabolisms in the metabolic inflammation that might be happening here and it's intriguing to think of whether these kinds of host microbial interactions might have actually come out of the context of reproduction from an evolutionary standpoint so I'm gonna this is my last slide I'm gonna finish with uh what I think some of the big knowns and unknowns are and um where I think there's there's some areas that that might be be very interesting to go into and that's thinking about what the extent to what extent do microbiota affect host phenotype so we know from these germ free studies for instance with transplantation that have been done in various labs and pioneered by the gordon lab that microbiota can affect metabolism immunity there's there's work coming out showing that microbiota can affect behavior but there's many many other things that we haven't actually looked at yet such as aspects of fertility uh aspects of longevity how much activity animals or people engage in um basic physiology uh there's a lot more to look at the extent to which microbes can do this how they do this on what kinds of microbes do this and and and how much can you push it so and this is a picture that's stolen from garland's lab at UCR where they've been selecting for my say that enjoy running a lot but I think that we could do these kinds of studies where we select for microbes that confer these kinds of behaviors and it's going to be very interesting to see going forward just how much microbes influence phenotype and then I think we can use that as a basis for therapeutics later so I'd like to finish up by thanking the people in my group specifically uh Armie Coran who worked on the pregnancy study and Julia Goodrich who's been working on the twins UK study um I have a number of collaborators just as just some of them um including Tim Spector and Andy Clark um who work with me on the uh on the GWAS um on the GWAS study and Erica and Seppo who worked with us we're very generous with the uh pregnancy study that I mentioned and we have some other ongoing things but finally I'd like to thank uh from MIH the new innovator award which has really allowed me to do creative things and not worry about it um this is a fantastic program and then Bob Carpenter IDDK for funding our twins UK study and thank you okay if somebody we have time for one quick question especially if you're standing near a microphone and also as uh the questionnaire is going there remember especially people online that the email address is hnvisionatmail.nih.gov so with respect to one of your earlier slides saying that nobody's been followed from birth to old age what am I wrong no no no okay no but I mean uh cesarean versus natural birth is something that everybody remembers and it's a relatively easy thing you could correlate at later age has anybody done those studies well that's interesting with our twins we've we've been uh asking them were you born by c-section and were you born uh vaginally and funnily enough not everybody remembers or or knows but we're trying to use use this cohort because because we can't ask them all sorts of things that's something that we're we're trying to do okay let's thank Ruth again let's thank Ruth again thank you okay our next talk is uh Jacques Revelle um microbiome dynamics in adults and here's Jacques