video where I kind of dispel you in on some things going on in my personal life with the channel and anything else that I feel like just chatting about. So let's do it. First of all I want to give, I don't know if I should be apologizing necessarily, but it turns out there are a lot of you, a significant amount of you who as it turns out hated my intro music before I changed it. And I had no idea if I would have known you guys had such strong feelings about it then I would have changed it a long time ago. I obviously liked it and all I ever heard from people was that they liked it too and that their kids would sing along to it and they would recognize it as my videos like friends and family just kind of walking by in the room would hear the little jingle me like oh she's watching sewing videos but so many of you were like thank god you changed your music I hated it before and I'm sure maybe there's a whole bunch of you out there now who hate the music that the new music that I had to to use basically what happened was that music that I was using was free and then I guess they didn't want it to be free anymore and so they hired some kind of agency and so the agency was flagging my video every single time I was posting something so easy enough to swap it out and I swapped it out for like a YouTube approved song so it will never happen again but it's good enough for now finished is better than perfect I know I'm not gonna be able to please everybody especially when it comes to something so specific as music so but I just thought it was hysterical that like as soon as I changed it so many of you were like well now I can tell her how I really feel about the song so happy to make your life a little bit better you don't have to mute it anymore you can bop along to the song now okay next up so what you may or may not know is this past Thursday was National Zipper Day fun right so all week long I have been posting little 30 second clips and videos with tips and tricks regarding zippers all of this is happening on my Instagram so if you want to go check that stuff out if you want to get like more content from me and more information it's not just like me posting pictures behind the scenes it's like also informational stuff that never makes it to the channel so head over to Instagram give me a follow there and check out all the zipper content that I've got coming for you I'm also going to be posting all of my me made may stuff there and I guess yeah I'm gonna throw this out there to you guys I'm I'm a little bit stuck on what to do for me made may most people post a picture of something that they've made they wear something that they've made every single day in May and then post a picture of that which I can absolutely do that's not a problem I've got more than a month's worth of clothes but I just feel like you guys see everything I make already and you see it on me and everything else so I'm not entirely sure that that would be interesting to you and so I've been trying to think of other ways to participate in me made may it can be anything you want there's like no strict rules so I was kind of thinking well maybe it'll be fun to like go through like each area of my closet and like kind of purge some things and show you I mean because some of those things are years and years old and maybe discuss why I'm not keeping it whether it doesn't fit anymore so my style anymore doesn't fit my lifestyle anymore would that be interesting that kind of also feels boring I don't know if either of those ideas sound like something you would want let me know in the comment section and I'll just go with whatever the majority people tell me that they would rather see I guess in a way I could incorporate both so if you're into both maybe just you can leave both in the comments I don't know I don't know what to do for me made me um today is the first day of me made me I'm filming this on Saturday and I've got my beloved Liberty knit Liberty Jersey cotton knit I think it's like seashells and starfish and stuff I don't know the names of them and what they're all called and this is a blank slate Catalina one of my favorite makes of all time this is several years old too but I still love it and still wear it so maybe that would be interesting to kind of go through my older makes maybe things that I haven't worn in a while and maybe say why maybe things that I really only wore once there are some things that fall into that category for sure um usually regarding fit is the reason why I never wore them again there's not comfortable um okay so yeah just let me know what what you'd like to see over on instagram for me made me all right so that is pretty much it that I have for like channel stuff um you guys are buying up the fast fit workbook I love that so much I would love to hear from you guys as you're using it um and let me know kind of what you think of it how if it's helping how it's helping um I would just like like some feedback from you guys whether you paid for it or got it for free um a mother's day gift guide went out that was a lot of fun if you're looking for a gift for a mom who sows I've got some of my favorite products and kind of like why they're good for moms it's not just like a random everybody likes this you know a lot of them are very specific to time saving to um like uh body positivity type of stuff as it relates to a mom to things that um make your life easier get things done faster better more accurate all that kind of stuff um yeah and I think that's it that's it for the channel I am working on the sew along that will premiere the first video in the sew along episode one will appear on the 18th and then early early next week I'll have episode zero which is pattern fabric notions um I'm going to show you how I pick my size using the fast fit workbook and any alterations that I'm going to make to my version um so if you're interested in seeing kind of how I get to episode one um you'll want to check that video out for sure episode zero it's not technically part of the sew alongs we're technically not sewing um but I do like providing that information for you guys so that'll be there all right what I bought so I didn't actually buy a lot um I did make a trip to Joanne but I just got notions I got elastic I got some stay tape I got no I don't think I got any fabric did I get I don't think I got any patterns that trip I got bias tape all the boring stuff um I did make a trip out to hobby lobby so my hobby lobby is like 35 minutes away and the Joanne is like 15 minutes away so I just end up making it to the Joanne a lot more frequently but I did go to the hobby lobby I was unimpressed with their fashion fabric they did have the Robert Kaufman linen blend which I thought was the SSS6 linen or whatever I did think that was interesting you could get that there but it didn't feel like it wasn't cheaper or anything so I was just like well I don't know and then I did pick up some of they had like a rayon ribbonet that I thought would be a nice for like a summer tank um but I'm not like oh my god this is the best stuff since sliced bread I'm just kind of like okay we'll just give this ago and see what it is it's very thin very thin very slinky so it's going to be not necessarily like super fitted but it is going to be clingy which I'm okay with it's going to be something I probably just tuck in anyways and then I picked up every single fabric they had with a bee on it I think between Joanne and the hobby lobby I am I've got I cleared them all out of the bee fabrics I don't need very much to make honey something um so I'm just getting like a half a yard whenever I see it but like I just go to the counter and say um I have all these bee fabrics that I found myself but is there anything I'm missing and they'll be like oh yeah over there in the yellow section or over there that you know and so I'll I'll just add it all in and they're like what what's with all the beef fabrics I tell them the story they think it's really cute it's like a whole thing um so that's really all I bought sewing wise I did get some indie patterns let's see I got the pattern for Sew Together 21 in May is the Love Notions Sunday Romper they had it as their five dollar Friday a couple weeks ago so went ahead and grabbed that if you missed that sale but still want to participate and sew together there may be I mean I don't know for sure but there may be another sale coming up next week not as good as five dollar Friday but a sale nonetheless maybe I don't know and maybe you'll hear more about it in my plans video maybe I don't know anything I don't know anything um what did I get any other indie patterns I don't think so try to remember the ones that I got printed didn't get anything else so yeah that's really it kind of a light shopping month um which is honestly pretty good because Dwayne was cranking out those pattern sales and I was just oh spending like every time I go there for a pattern sale I easily spend $50 maybe a little bit more if I get some patterns and some fabric all right so let's head over to the rapid fire oh before I forget there are a few of you who are highly invested in honey content and I appreciate that so so so much so I wanted to explain how calm honey is not ending up on the channel that much one today she's not even here I take her today care and I tell you you guys it is a relief whenever I'm able to pass her off to someone else who's going to take care of her all day long and I don't have to worry about her she's just a lot she doesn't sleep very much she's very active as you have maybe have seen in like some of my instagram stuff she's always around me always on my feet you know always always around so in order for me to get anything significant done like you know mopping the floors like filming videos like if I'm doing a bunch of videos in one day it's just a lot easier for her to not be here so once a week I'm taking her to doggy day care so I can get a whole bunch of stuff done and not have to like scream at her and yell at her I just it's not a good a good symbiotic kind of balance for either one of us so she goes off and has a great time with all of her buddies and I'm able to get a whole bunch of stuff done but I did want to tell you that two things one she will be appearing on the channel more now because I am making her clothes and I'm somehow magically able to film her in these clothes without like making you guys motion sick so I'm going to be doing like dog pattern reviews dog pattern pattern reviews it's not going to take the place of me made monday but they're going to appear on the channel probably one a month so you'll get to see her um she grew out of I had to so I got the smallest harness that they had at PetSmart when I first got her she was like stepping out of it she couldn't it was like so big so I ended up having to get her a cat harness well she grew out of the cat harness this past week and now she is in that the smallest dog harness that they have not stepping out of it like it fits now so part of me is like oh man she's so big you know she's growing up um obviously she's still small but I don't know she was so little just for like the littlest bit of time I guess I took it for granted and thought she would be that little forever and she wasn't um so yeah moving her into the big girl harness was like a moment okay so the second part of the honey thing is I do post more honey content on my instagram so if you want to see some more of her you can um you just have to go follow me on instagram that's all I have to say about that okay rapid fire what I'm reading this one is called Clara and the Sun by Sir Kazuo Ishiguro Japanese man who now lives in London he won the Nobel Prize for Literature which isn't necessarily just for a specific book but he has what three or four of his books shortlisted for the Booker Prize so this guy's pretty legit um he was writing um writing writing writing constantly and then took a huge break he was like he published a book in 82 86 89 95 2000 2005 rocking and rolling right then he took a break for 10 years didn't publish anything um came out with one book since then and I'm reading the second book since he has started publishing again it's called Clara and the Sun it is about it's like a little bit artificial intelligence a little bit it's artificial intelligence in a modern world with like a teenagery angsty kind of vibe mother daughter tension girl meets girl boy love tension so it kind of reads like young adult but in a with this underlying theme of like artificial intelligence I'm not that far through it so I don't know like how all of these characters fully developed and how the story fully develops but there's been a lot of laying of the groundwork um and I feel like things are going to start picking up here shortly I'm super excited to be introduced to this author I love that he's Japanese kind of speaking to my culture a little bit I've been kind of trying to find ways to bring Japanese culture or at least Japanese people and artists and creatives into my life um I got a new Japanese cookbook um you know little things like that so this was kind of like an effort to kind of not necessarily explore my own culture because the book is not about Japan at all um but you know reading something from a Japanese man you just I don't know you just feel more connected to it in a way so I've enjoyed doing that so that is what I'm reading and my oh what I'm watching I just started watching friends from the beginning friends is on HBO max all 10 seasons every single episode so that's what I've been putting on um I watched it when it first aired never went back and rewatched it never really watched the reruns when they come on as well I don't know I don't know why but the idea of being able to watch it with no commercials um I'm paying a lot of attention to the fashion since 90s fashion is so hot right now um the first season premiered in 1995 so what I'm seeing is a lot of crop tops a lot of t-shirts a lot of like graphic t-shirts um a lot of overalls or penifor or strappy things um a lot of like oversized blazers kind of like loose fitting trousers um a lot of that kind of stuff um so that's been really fun and then it's like oh yeah I remember that happened and oh yeah you know like you know the big overarching storylines but sometimes you forget about like the little ones um so that's what I've done I'm on season two I don't know how long it's going to take me to get through all 10 seasons but that's just what I decided to do because I couldn't find anything new I know everyone's talking about the handmaid's tale but I'm afraid I'm afraid to start watching that I'm afraid I'm not gonna like it or I'm afraid I don't know if I don't know if I'm gonna like it and then I'm gonna be like the person that doesn't like it so which is worse the person who's never seen it or the person who doesn't like who has seen it and doesn't like it like I am that way about a lot of things like uh Game of Thrones didn't like it um the British one on PBS didn't like it so like why even bother trying with Bridgerton like I don't think I'm gonna like it um why even try with Handmaid's Tale I don't think I'm gonna like it um so we'll just go with an easy breezy friends and because the episodes are so short it's literally like you know one a night kind of thing anyway so that's what I'm watching nothing exciting if you know of anything that's really really good a very well-developed series or something like that I have all the things I have all the platforms I have stars HBO max apple tv plus netflix prime regular cable tv I have it all um I just I just need something to get invested in again so um all right my latest obsession my latest obsession has to be like deep cleaning I actually rented a professional carpet cleaner and like busted it out broke a sweat whole nine yards cleaning my carpet and I am gonna tell you guys I keep up after my carpets honey and sunny don't go on the carpets like they're not allowed in those rooms the carpet was disgusting the water was so gross so gross so I'm so happy I did that all the rooms just smell so much fresher um the rooms smelled clean you know they didn't never smell bad but they didn't really I don't know it's kind of stale I guess and now it just it just feels so different so I've been doing that I have been um like reorganizing a few things you know I live a pretty organized life as it is um but I've just been like you know perfecting some things I got some new stuff for the fridge you know to make sure that is um I got uh something else for my spice cabinet so that's perfect now so just like yeah fine tuning a lot of those kind of like little drawers and little cabinets and little things um so that they are perfectly organized not just like sort of organized and for me perfectly organized is probably something that regular people would look at and think oh my god that's a little bit over the top but that's how I like it I like to just know I like to be able you know what it is this is how I know when something's organized if I can tell Dan Dan go get a polyester zipper in six inch length and he can go do that that's when I know it's organized and he could do that that's how organized my zipper collection is you can see it on Instagram it's one of the things that that I posted for zipper week um if I can tell Dan hey go grab me the cereal from the pantry he can grab it right away it's not like which one is it this is it that but no hey Dan go grab me everything I need to make a smoothie he's got it because it's all in one little tray um go find me this spice that he can do all of that so that's how that's my beta test for or my litmus test for if something's organized or not if I say go find this and he has a question then I'm like nope I gotta find another way to organize that um okay what I am trying so I gotta say I'm not really trying a whole lot the only thing that I could possibly say is like new that I'm trying is uh with regarding planning so I used to be a kind of weekly planner with like a tinge of daily planning I am now converting to more of a daily planner where every single day six a.m eight a.m whatever I'm doing at all these times exact tasks for that day are getting put down that's something new that I am trying um I am a planner by nature I love to check things off I love to make lists so that aspect of it is not difficult what's hard for me is knowing what I can get done in a day versus okay here's everything we've got to get done for the week you know and then it comes Friday and I'm like well crap I only got half that stuff done oh well I'll just put it on next weeks now it's like every day that it's happening like oh I didn't get all that done now I just move it over to tomorrow well tomorrow's kind of already full so I really need to kind of try and get it done today it's so it's helping me stay more accountable and it's helping me I don't know just like I guess just stay accountable so I mean I guess that's something new I'm trying it's a very personal thing planning how you plan how you get things done if you make lists if you don't like so I don't expect for anybody to hear me say that and do their planning any differently if you're not planning if you're not writing out a list of what you need to get done every day every week every month whatever it is you're really missing out planning is like life life saving for me for sure I would not be able to do this job if I if I didn't have a very detailed planner very much all right so what to expect in may I think we talked about this a little little bit but we've got the so along coming up starting May 18th we've got me day may happening on social media finishing up zipper week here we've got so together 21 pattern the Sunday romper from love notions I've got two skill pop classes that's something that I haven't mentioned we're doing machine basics again also on the 18th at 5 30 p.m and we're doing a zipper pouch class so if anyone you know I don't expect for you guys to need to learn how to use a sewing machine but if anybody you know has been begging you please teach me how to sew now you can just send them to this class Lindsay will teach you once she shows you how to use your machine you make a zipper pouch come on over and then you'll have a new sewing buddy that's kind of how I envision that working we are working toward a serger class so just like sewing machine basics this will be serger basics so those of you that have a serger and it's collecting dust in some closet somewhere because you're afraid to use it afraid to change the thread I know there's some of you out there so this is for you also those of you who are kind of on the fence about whether or not you should buy one or not how difficult it is to use you could take the class it's 20 bucks see how it works see how everybody else does in the class and then use that to determine if you even want to buy one or not then if you do buy one the class is available on a recording so you could go back and watch it anytime you wanted to help you know teach you how to use the machine and stuff so that's really exciting that will be sometime in june I cannot believe we're talking about June um so yeah that's what to expect in March and there you have it that's all I have to chat about for this month I love it when you guys participate in the rapid fire so if you want to let me know what you're watching what you're breeding what you're obsessed with and something new you're trying by all means leave that information one or all of them in the comment section I absolutely love hearing what you guys are into um I know I asked you guys for a lot of other things in the comments so go ahead and leave those too and I'll just spend a lot of time in there taking in everything from you guys but that is going to do it for me today y'all thank you so much for watching I'll see you very soon bye


There's three theories on what the future of artificial intelligence can be. One is we're gonna have big, mainframe, massive computers that can do all this stuff. Second, cyborgs, like we're gonna mix, you know, like Google Glass was the beginning and all this stuff. But the third one is collective intelligence. So what if you could take all seven billion people and just like ask them any question, like what should I name my, should I name it impulsive or a little more?


Now President Mohamed Buhari has finally unveiled the redesigned Naira notes as proposed by the Central Bank of Nigeria, CBN. The unveiling marks the first time in 19 years Nigeria will be redesigning its currency notes. At the briefing after the ceremony before the federal executive council meeting, the CBN governor debunked insinuation suggesting that the early unveiling of the new notes is a means to target some Nigerians. During the ceremony which held before the council meeting, President Buhari underscored the importance of redesigning the Naira notes. There was an urgent need to take control of currency in circulation and to address the holding of Naira banknotes outside the banking system. The shortage of clean and fit banknotes in circulation and the increase in counterfeiting of high denomination Naira banknotes. It is on this basis that I gave my approval to the redesign of the 200 Naira, 500 Naira, and 1000 Naira banknotes. While this they were to be apparent to any Nigerians only four out of the 54 African countries front their currencies in their countries and Nigeria is one. Hence a majority of African countries front their currencies abroad and import them the way we import other goods. That is why it is immense pride that I announced to you that these redesign currencies are locally produced right here in Nigeria by our security printing and meeting TLC. The Naira banknotes had been fortified with security features that made it difficult to counterfeit. These new banknotes will help the central bank design and implement better monetary policy objectives. I am delighted now to fairly launch the 200 Naira, 500 Naira, and 1000 Naira banknotes. We'll speak in with newsmen after the ceremony. Governor of the central bank of Nigeria, Godwin M.A. Fili expressed the determination of the Apex bank to make Nigeria a cashless economy. According to him the amount of money that can be withdrawn across the counter will be drastically restricted. Explain it that anyone who wants to withdraw substantial cash will be tracked by security agents to determine its use. Our policy of redesign and reissue of currency is not targeted at anyone. But of course we have the laws and we will indeed be more intense in ensuring that the provisions of the law and the CBN regulations about the size and volume of currencies that people can hold or can carry or can withdraw that we would insist that this is done going forward. In other countries you will find, say in the U.S., you want to withdraw $10,000 from the counter, you will be interrogated. You will feel numerous forms. They will even track the use of that $10,000 cash you are withdrawing. Or you want to withdraw 10,000 pounds from the counter. They will refuse. And if you insist, you will feel forms. I think the problem we've had in the past is the insistence that oh, this is a cash economy. There is no economy that is imbued with the thinking that it has to be cash economy. The world has moved away from cash, predominantly cash, to cashless economy. And I think Nigeria and the Central Bank of Nigeria is prepared at this time to move towards a cashless economy. And that is the reason why following the redesign and issuance of this note, we will go forward to insisting that cashless will be nationwide. We will restrict the volume of cash that people will withdraw over the counter. If you need to draw large volumes of cash, you will feel uncountable forms. We will take your data, whether it's your BVN, your NIN, so that security agencies like EFCC or enforcement agencies like EFCC and ICPC can follow you and be sure that you are taking that money for good purpose. Hello, hope you enjoyed the news. Please do subscribe to our YouTube channel and don't forget to hit the notification button so you get notified about fresh news updates.


I really can't describe to you what it's like to be an alcoholic. It's miserable. So anxiety riddled. Every morning I would wake up and wonder how I got to that point again. How can I hide it? The first thing I would think of when I'm helping my career, how would you be able to help me? How much can I make for you? It was running my life. Yeah. Probably better than I could. So Michael and I met in seventh grade. Mr. Koss is math class. He actually sat behind me. We had a cute kiss together at around that age and then never really spoke much after that. We just like remained friends and life kind of parted ways. And then when Michael came home from Iraq, I was out celebrating my birthday with a few of my friends and he happened to be at the same place and I like came down the stairs and he out of everybody in the room stood up and was like, you look so beautiful and the whole night we just talked and it was instant connection. We've been together ever since then. Well to be honest, I knew that he had a problem drinking but I don't think I could have even imagined how bad it was. My drinking really started running out of control and binge drinking when I came back from Iraq. My career was demanding. I felt out of control in every aspect of my life. I felt overwhelmed. We would go out to dinner and I'd have a glass of wine and leave it at that. He would order a drink and then have another drink and have another drink. Like God, I don't understand. I'm praying for my husband to like find you to find salvation for him to want to go to church with me and yet we're having these same arguments so it's frustrating. This alcoholism was this insidious onset. It was the first thing I would think of when I wake up. How am I going to drink today? How much am I going to get? How can I keep my career? How can I keep my family but manage this drinking problem that was running me? The more I drank the more my faith was lost and it led to this incomprehensible demoralization for me. It's like one day I looked behind me and all of a sudden there's this line and I had crossed it and I had no idea how I had gotten to that point or how long I had been across that line. My life was just completely out of control. I got a phone call from the ambulance. You need to head to Bronson. I had our son with us who at the time was almost three and we were allowed to see him in the hospital room and when we went in there the first thing he said to me is I just want to get my life right with God. Laying there in a hospital bed this overwhelming peace came across me and I remember very clearly God meeting me in that moment and the Holy Spirit said Michael give this to me I can do this and as long as that took to get to that point where my life had just become unmanageable it was resolved within a moment. I felt loved. I felt welcomed. I felt like I was myself again. I felt just I felt an overwhelming calming peaceful presence in my life and I haven't had a compulsion to drink it's doing for me what I could not do for myself. For it to come in this way is not the way anybody would have wanted it but finally I was just like thank you God for answering this prayer. It was a miracle. I feel like God has put it on my heart to share my story to let other people know that there is hope and there's good news out there in the love of Jesus. Before I came to Christ looking to stop drinking I wasn't expecting to become a better father a better husband I got saved and my heart had been changed forever.


Hello everyone, I want to welcome you all in this presentation. My name is Suzan Marjan and I'm working as a senior watershed management officer in Basin Management Center, Pokhara, Nepal. In this presentation, I will briefly explain about Fivalek and its watershed area with information about its degraded condition decades before and the restoration activities implemented by the government of Nepal and the results that we achieved and the lesson we learned from the implementation of restoration programs. Fivalek is located at mid-western part of Nepal in Pokhara valley. The watershed area of Fivalek extends from 742 meter altitude to 2480 meter above sea level. Pokhara valley received the highest amount of rainfall in Nepal. It is estimated the annual average rainfall of the Pokhara valley is 4062 millimeter with average rainy days of 128 to 164. The estimated mean annual maximum temperature of the valley is 29 degree centigrade and the minimum mean annual temperature is 5.3 degree centigrade. Among many rivers, Harpun, Antheri, Betany, Belondi and Firke rivers are the major rivers that draining into the Fivalek. The people living around the Fivalek mostly involve in tourism related activities such as hotels, restaurants, trekking, bar, boating and certain percent of people also involved in agricultural activities and this farming also. Fivalek is one of the 10 ramshar sites of Nepal. This lake was included in ramshar sites in 2016. Fivalek is the second largest freshwater lake in Nepal after Rara lake. Decades before the landscape of Fivalek was very disturbed, denuded and degraded due to different anthropogenic disturbances such as open and overgrazing deforestation and forest degradation that caused high siltation in lake Fivalek. Realizing the extreme environmental degradation of the area and the immediate necessity of the restoration activities, government of Nepal has designed and implemented different kinds of restoration projects with the help of technical and financial support from different donor agencies in this Fivalek landscape area. In all level of restoration activities from planning to implementation and monitoring, the involvement of local communities was given high priority. Among many restoration activities implemented in this area, community best first management is one of the most important and successful interventions. In 1970s, government of Nepal has passed a provision that allows to hand over certain area of forest area to the nearby communities to manage as community forest and at present there are 160 number of forest user groups managing about 3745 hectares of forest lands and the areas are in good conditions. Deforestation, deforestation and agroforestry activities were also extensively promoted at that time by establishing different number of permanent and temporary nurseries in statistical locations to produce important tree, father and grand species seedlings. Shoe conservation activities such as torrent control, gully treatment, riverside treatment, check dams, terrace improvement and trial improvement were also intensively implemented to reduce the soil erosion and to treat the landslides and ultimately to reduce the siltation. Similarly, the local people and the technical manpower involving any restoration activities wells also provided different kinds of trainings to enhance their capacity to improve their quality of work in restoration activities and most importantly the establishment of payment for ecosystem services board which was established to resolute the conflicting situation between upstream and downstream communities regarding to the continuation of conservation activities in upstream sites and the sustenance of the ecosystem services for downstream communities such as volume of water, quality of water and reduction in siltation etc. With the implementation of restoration activities for long period of time we were able to achieve numerous good results among them the increment of people's awareness level in natural conservation and their participation is very important one. Similarly, the large area of degraded lands are also reclaimed in these two pictures you can see these changes that we achieved through implementation of restoration activities for a long period of time. The first picture was taken in 1970s where you can see a big landslide and large amount of sediment deposited at base and in second picture taken recently of the same site which where you can see a good forest covered and the settlements you can see there developed after the restoration of the area. The green coverage area of the pheabolic watershed area is also increased by many fall at present there is 55% of land covered by forest area. Similarly, the flora and fauna diversity of the area is also increased and most importantly there is sharp decline in siltation which was 30 metric ton per hectare per year now estimated around 15 metric ton per hectare per year. With the successful implementation of restoration activities we learned certain very important aspects that could contribute a lot in conservation and restoration activities in other areas also for example the community involvement in restoration activities is very important without their involvement or their participation in restoration activities we cannot achieve the targets of restoration. Similarly, the coordination among the stakeholders is also very important for effective and successful implementation of the restoration activities. There must be a good connection between conservation activities and livelihoods of the local communities. The conservation activities must help or must contribute in improvement of local livelihoods otherwise the people participation may not be there. Similarly, the capacity of the community people and technical staffs must be enhanced time to time and there must be a mechanism that will monitor all field level activities and provide the instant feedback to the working groups that will help to improve the quality of the works. Based on experience that we gained from the implementation of restoration activities here I have listed down certain aspects that must be considered by policy makers and project developers before designing and implementing the new policy and projects for the similar areas like Phewa Lake Landscapes. The projects must adopt the multi stakeholder approach with interdisciplinary actions. There must be a provisional stable governing system that will look after the project activities for long period of time. There must be a good link between development work and environment conservation. There must be a mechanism that will assist to develop a harmonious relationship between development works and environment conservation. The restoration projects must be provided technical and financial support for long period of time and all projects must well articulate the upstream and downstream link cases. Thank you very much.


So my name is Huamin Chen. I come from Red Hat's Office of CTO, Emerging Technologies. I'm working on the sustainability at the moment. If you have met me before, I used to work on the Kubernetes 6 storage, which is so exciting that I want to also create some physics here, not to create the new functions, but EMCQ, resource record for the visions of sustainability as Red Hat's emerging technologies. For the M, we are using the metrics, specifically that's what we're going to talk in this project, Kepler. So when you have the metrics about how much energy consumed by your workloads, that is very powerful information that you can use in your configurations, in your scheduleings, and in your reporting. So once that information becomes available, the metrics information becomes available, we want to use it in the workload scheduling. So one of the theories that's when you're using your servers as the certain capacity and a certain utilization, the performance per watts is the optimal. Meaning that if you want to wrap up your Kubernetes clusters to 70%, for example, the CPU utilization, you probably get the best performance per watts curve. And we can use that information in your scheduler to make a smart scheduling. And we are trying to use in this project, it called PIX, to investigate such potentials. The project's going back and forth, but we finally settled to using the existing Kubernetes plugins called Tamron, so that we can configure the servers to certain capacity and using Kepler metrics to advise the scheduler to make the scheduling such that the overall aggregates across the utilization, power utilization, will be at the most optimal level. The next project is called Correction, which we presented as the last year's cube come. It's as we're using the Kubernetes of Vertical Part Autoscaler, the VPA, and then you incorporate with Kepler metrics, the power consumption metrics from workloads. And then we dynamically tune the CPU frequency such that this also has a background. The higher the CPU frequency, the higher energy it consumes. The lower the frequency and the less power. But in order to maintain the same level of quality of service, we set up the objectives. The objectives using the clever is called instruction per cycle. So the amounts of CPU cycles that is to run your workloads were deterministically determined the quality of service that you will receive. In the same manner, if we are giving you more resources, then potentially you are using more to power your application, the less resources then you are going to have more constraints to run your application. So as the higher CPU frequency, if you are maintaining the same level of IPC, we give you less resource. In the same way, just keep the mind working. The higher the CPU frequency, the less resource. That means that the amounts of energy consumed will still be reduced because you are using less resources. Even you are running at high frequency. Vice versa, if you are running at lower frequency, we give you more resources, more CPU cycles. So the IPC will be maintained, but your energy consumption will be optimized in that way. So all this happens in the magical, all this magical thing happens behind the scenes in VPA. And we have that experimental prototypes in our GitHub. The very last C is carbon. So at the end of the day, the sustainability come to carbon, the amounts of carbon footprint that your workload consumes. In order to do that, we have a number of initiatives happening. One is that we want to visualize how much carbon you use, your workload use. That's very consistent with the same way that the people are using the utilities, your electronics. How much carbon your electronics consumes and how much carbon to make it have to make the electronics to produce. The same way we are using a carbon accounting. The other way we're using carbon is when you visualize the carbon as different geographic locations that certain differentials, deltas, depends on time of your day. Your carbon footprint from electricity grid is not the same. Surprisingly, you are using the same electricity grid, but the carbon intensity is different. And similarly, if you are running at different locations, they are also as different deltas. So let's come to the project Kepler. So what Kepler is about? Kepler stands for Kubernetes-efficient power level exporter. It's using the EPPF as the underlying technology to collect information from your hardware, from your workloads, and then using machine learning models in the background to calculate the energy for you. This is a very diverse and highly collaborative project started by Red Hat, IBM, Intel, and a number of reworks and many of the other community contributors. We recently donated the Kepler project to CNCF for sandboxing. And hopefully, we can have more contributions from the people around the world. So if you scan the QR code here, there's a website for Kepler, and there's a Slack channel. You're all more than welcome to join the project. So currently, Kepler supports a number of granularities of energy consumption. So we can tell you how much energy used by your processes, your containers, your parts. And hopefully, potentially, we can also aggregate to the certain higher-level APIs. We currently support CPU-level, GPU-level, and DRAM-level energy consumption. We are hopefully in the next release or two, we are going to support your networking storage and other accelerators, special accelerators. And we're also going to use the external power sources from your network switch, your certain other hardware using the data center. We currently support bare metal and virtual machines, meaning that we are using the same infrastructure, same metrics, and you can get energy consumption from your environment, regardless of where your workload runs. We are potentially going to support the encrypted, the trusted execution environments that's running your encrypted enclaves called TEE. This is one of the dashboards we built. I'm not an artist, so I just briefly explain what this is about. So on the left side of the panel, on the top left side is the carbon. So as I said, there's different fluctuations as during this certain time of the day. We can break down the carbon consumption into different levels consumed by your hardware. This is a little bit smaller if you are looking at the green side. This is the CPU package. And the yellow one is the DRAM. And the bottom one is because we are not using GPU at the moment, so you do not see any of the activities over here. This side is the power. So power is collected by Kepler. So we can collect as the node level, as your server level. We can also collect as your namespace level. And we are also going to the path. But the namespace is good aggregation. You can have a lot of visuals. If you create different namespace, create a different tenants based on your namespace, you can visualize over here. On the bottom, we translate that into the amount of carbon footprint related to your workloads. So this is a very small setup, by the way. So we can have better visuals. So on certain namespace, you can have higher energy consumption. And you can also have the higher carbon footprint. So this is one example of how you can visualize workloads energy as well as your carbon. So you can also create other workloads and other dashboards based on your new requirements. And that is also supporting the environment as well. Thank you.


Welcome to the first Exotic Wine Travel shorts. We're going to explore the grape Assertico. I just got three samples. Two of them are from the island of Santorini and Greece and one is from the Greek mainland. It's a high acid, exciting white grape variety. Do you like Assertico? Let me know in the comments below and I'll see you soon.


Good afternoon, Steve. Thanks very much for coming here this afternoon. First of all, let me congratulate you on becoming and being appointed the new chairman of Study Group 15. Thank you, Toby. I hesitate to call you a veteran of Study Group 15, but you're certainly very familiar with the workings of this very important group to ITUT. I was wondering if you could give us an idea of some of the highlights from the previous study period. And perhaps give us a, perhaps paint a picture of the work of Study Group 15. OK, Study Group 15, as you know, is the largest and most active of the ITU study groups. We tend to regularly draw about 370 delegates to our meetings. And Study Group 15 is responsible for really all of the major aspects of network infrastructure, starting from the core networks, the long-haul transport networks, DWDM line systems, if you will, down into metro networks, access networks, and now, in the last study period, extending that further into home networks. And really, as we know, the growth in data traffic is massive around the world. And at all levels of the network, that affects the work that we do. So some of the real accomplishments in the last period are pushing all aspects of those transmission rates in all parts of the network higher and higher. So in the core networks, we've moved to 100 gigabit per second speeds per wavelength on multiple wavelength fibers. And that works in the long-haul and in the metro networks. Down into home networking, we have a couple of technologies that we're responsible for. As you know, the DSL family of standards, bringing access into the home over copper cable infrastructure. Every time we think that that has reached the ceiling, people figure out a way to push it even higher. So the VDSL 2 with vectoring pushes up to about 250 megabit per second speeds today. And there's new initiatives pushed that even higher. We also have the fiber to the home technologies, the PON passive optical network technologies, which are a shared access technology. And those have pushed up in the last period into 10 and 40 gigabit per second shared access over a passive optical link split out into multiple premises. Let me pick up on one of the points that you made there about the transport layer. And the economist reckons that 95% of all international traffic runs over fiber optics. How much of a role do ITUT standards play in that and in facilitating that 95%? It's an enormous amount of international traffic. Well, first of all, I'm surprised it's only 95% because I think we're all aware of the undersea and the terrestrial fiber optic cable infrastructure that supports that connectivity. And I think that these days at the speeds things are running virtually all of that is fiber. And study group 15 deals with several aspects of that infrastructure. First of all, the cables themselves, the fiber optic cable installation maintenance of that cable infrastructure, a lot of that comes from study group 15. So what's actually running over that fiber infrastructure is an ITU standard or not, a lot of the fiber specifications do come from study group 15. So even Ethernet, you read the Ethernet standards, you'll find references to ITUT fiber types as what is being used by that Ethernet infrastructure. But furthermore, in the transport network itself, even if it's carrying something like Ethernet, to go any distance, generally, it's optical transport network standards, which are also under the responsibility of study group 15. So the way you manage and maintain and the way you combine traffic that goes over those fiber optic cables, a great deal of that is study group 15. Moving into the home as well, of course, a lot of the key access technologies are study group 15. You mentioned already that we're pushing the boundaries of what's possible with all of that copper that's still out there. And vectoring technologies in VDSL2 is about where we are with that now. But is it possible to squeeze more out of that copper infrastructure? Yes. In fact, there is a work item under development now, G-Fast, trying to push those speeds up to 1 gigabit per second. So yes, that work continues. And as at other levels of the network, we're pushing the speeds faster and faster. One more question that I think relates a little bit to the access technologies that we've talked about. But there's some work in study group 15 that focuses on the vertical section. This is the smart grid. And I'm wondering if that work is something that's going to progress into the next study period. And whether or not there are any other vertical areas that study group 15 standards will help to facilitate. The aspect of smart grid that we're involved in is communications for smart grid. Obviously, there are many different organizations that are involved with the whole ecosystem of how that works. And the reason that we are a natural place for the communications for smart grid is that we have the standardization both in the access network and in the home. In the home network, the transmission speeds within the home were working up into the 1 gigabit per second range. But in the area of smart grid, we have two technologies that we've been progressing so far in order to provide that connectivity and the control interface into the home. We have power line transmission modulation. And then there's also a short reach, narrow band digital radio transmission that accomplishes that that are technologies that we're providing to enable that smart grid kind of infrastructure. OK, but perhaps finally, though, are there any other areas that you'd like to highlight that you don't think that you've covered? Well, moving forward, when you push things faster, what you tend to do is to do it again. So we've moved in the last period, as I said, up to the 100 gigabit speeds per wavelength. We've done that by deployment of a lot of very exciting, advanced new modulation formats. And in the next period, together with some of the work in IEEE, we expect to be pushing that to 400 gigabits per second and beyond, essentially by evolving and refining some of the general approaches that we've done in the past. OK, well, thanks very much for your time, Steve. And good luck in your new role. Thank you.


Hi everyone, in this little video we're going to take a look at how you look at your grades and what some of the information you want to take a look at so you can better assess how you're doing in the course.  When you click on My Grades, you're going to have a listing of all of your assignments and basically what status that they're at, where are they in the process. So if you've submitted it, there's a good chance that it'll either say submitted and if it's been graded, it'll say graded. If it's upcoming, that is it hasn't been submitted yet, you'll have it listed here. Over on the right you'll have what the grade is and as you can see some of these have grades, some of them don't, some of them have the exclamation point which just tells you it needs grading. That is it's been submitted but the instructor hasn't graded it yet. And then some of them have this little word balloon here and the word balloon tells you that there is in fact feedback to be had. So if you click on that feedback, you will get whatever the commentary is that I've provided. So it might say something like this is really good but here are some concerns, here's how you might want to reevaluate it, etc. So you can look at any of your assignments and double check that they are, you know, why you got the score that you did. Now there's other assignments where you can not only just get the feedback but you can also take a look at the rubric. Particularly around discussions I use a lot of rubrics and so if I want to look at this discussion I can see, oh I can view the rubric so I can not only get the feedback but if I click here and view rubric, it shows me right here how I have done. So it's telling me, oh look, you know, for formatting I got 16.5, for organization I got this and so forth. So the rubric is really a good way for how I identify where the strengths and challenges are in your assignment and you'll want to take a look at those to better understand what it is you're doing. The other thing you really want to do particularly around major assignments is to take a look specifically at assignments where I have not only provided you feedback but if it's a paper I may have commented on your paper. So if we look at this assignment right here, I know I got an 81 but if I want to find out more about what were some of the strengths and weaknesses of it I can always click on the assignment itself and it's going to bring me to the assignment page and here is where it really gets nice in that I can actually see where the instructor has identified issues and challenges in the paper. Now if I want to see this a little more clearly I can shrink this left side and notice it expands a bit more. I can also just kind of make this a full page and that helps a little bit more. I can shrink the page a little bit and that shows me oh look this is where I had trouble this is where issues rose and then if I want to take a look at this paper if I want to download it I can click right up here where this little icon is and I'm sorry this little icon right here and it can allow me to download it as a file as a word file or as an annotated PDF. If you do download it as a word file please make sure that if you're going to be resubmitting it that you remove any of the markup that comes with it. So that's just a quick way to take a look at your grades and understand where your grades are coming from. Hopefully this will be useful and provide you with plenty of material to take a look at as grades start to come in. Thank you very much.


All right, well welcome back from lunch everybody. Who's relieved that the hardcore bioinformatics is over. All right, so we're going to talk about sample collection biases integrating sample handling as well into all of this this afternoon. In a lot of ways it's still intense there's still a lot to think about, and not a lot of hard answers a lot of the time but I'll share with you what I've learned from a decade of making a lot of mistakes in this area. All right, bands the slides up here. Okay, so these are the learning goals, again, from today's lecture. But we're going to talk first about sample collection but it's really two main parts and then there's some subsections within sample handling we'll talk about. But really when you're thinking about sample collection and sample handling and microbiome, you really do need to be thinking about what it is that you're going to try to detect. What is your question and what kinds of biomolecules are you interested in, you know it's kind of like looking at the microbiome with all these different sets of glasses right like what you are going to see depends on whether you're set up well to see it. And so you need to think about that ahead of time. I think where a lot of people have gotten into trouble. In the field is taking from the literature which focuses heavily on DNA stability and processing and collecting their samples based on best practices for that and then nowadays are like, Oh, I want to do other stuff I want to measure carbohydrates and metabolites and proteins. Well, where you set up from the beginning of your study to measure those things or not. So, really my lab's work is heavily focused and I would say in general the work we're doing in the International Microbiome Center is heavily focused on multiomics. So we are all about preparing our samples preserving our samples to be able to do everything, look at every type of biomolecule at some point. And also we're adding culture capacity into a lot of our studies so that you know we get all this material it's super valuable and we can use it for all kinds of analyses in the future. And that takes a lot of work and a lot of planning and a lot of time. So if that's not your company, you know a lot of the data and tips I'll give you are also just set up for DNA because that's where we've really done most of our standardization. And a lot of people are only interested in making sure their DNA is okay and that's good enough and that's fine, but I'm going to kind of paint the picture from both sides today because we are definitely interested in multiomics. I'm just going to give you the overview then of how you set this up and you're thinking so if you're just interested in who's there. Then you're going to go for your amplicon sequencing your shotgun sequencing with your taxa right, and you just need DNA for that. But if you're interested in what they can do, you're going to have to go with shotgun sequencing right. These are all things that you guys have become very familiar with this week. And then from John's lecture this morning if you want to know what they are doing. What are they doing right now. That's where you're going to get into your mother transcriptomics and potentially and I think growing capacity in the field proteomics and metabolomics. And you can see that when you get into this last bit here there's all kinds of biomolecules that you may be interested in that you may want to capture. But it doesn't, it becomes not so straightforward how you prepare and design and collect your samples and process them to enable you to look at all of these diverse types of biomolecules in the future. But what we've kind of come to us, the sort of simple solution to all this in the microbiome field is this freeze as soon as possible store at minus 80 minimize free spas. Cool, right. How hard could it be that it turns out that there actually are a lot of things that kind of come up with this. So, this slide shows you then, you know, the schematic of sample collection and in my role leading the IMC core facilities I talked about yesterday. Certainly we have seen all kinds of samples. I have processed most of these types of samples except for plants and soil that I put them on here so I know there's a number of environmental folks but you know you might be working with, you know, whole animals, insects, worms, tissue samples. Of course we've got our fecal samples, different types of liquids could be sampling water or urine, breast milk. We've got kinds of swabs that are going to capture the surface material in the mucosal fluid from different body sites saliva. Anybody know what this one here down at the bottom is. Anybody know what that is. Yeah, it turns out to be a great way to get cervical vaginal fluid. So that's a common sample type now as well. You have all these different types of samples that you might be collecting, and you have different devices, and then you have different ways that you need to, you know, extract your biomass, get your microbial biomolecules out. Before you get there though you have all these steps so sample handling really has four components transport storage processing, and then your bio banking or your archival. So these are places, you know, our places where you can introduce bias, and you can't go back. We'll talk more about that. And then you need to extract these biomolecules that you're interested in. So like I said, sometimes there are mitigating factors or things that come up you're like okay my plan is to freeze as soon as possible. In practice, I want to store it minus 80 minimize my free thoughts, you get into the weeds here though. Well what if my samples at room temperature for one hour. What if it takes eight hours for career transport, what if the samples are held at minus 20 and a participants freezer for one week before it gets to my minus 80. There are so many what us when it comes to this idea of freezing at minus 80 as soon as possible. When you get to the sample handling part when you want to minimize your freeze thoughts. Okay, so I'm going to follow my sample I'm going to make my aliquots so I don't have to do too many more freeze thoughts but what if that process takes four hours and everything's on ice is that stable enough. What if I need to do a 30 minutes spin for my process and I don't have a refrigerated centrifuge that will work. What if I didn't add protease inhibitors and I said later I want to do proteomics. All of these things sort of compound the lots of what ifs. And where we're at right now microbiome science is that it's just such a broad field there's so many top sample types there's so many variances and an accessibility and being able to adhere to these best processes what you really have to do is plan incorporate good controls and systems of evaluating monitoring for batch effects monitoring for problems. And try to stick to your plan but like with the with the monitoring, you know, adjust as needed. That's really what it boils down. There's no simple. This will always work. And that leads here especially with these smaller details, but I will share as we go on, you know, some of the, the things that we've learned along the way and some of the things that the literature is showing to be efficacious when it comes to these processes. Okay, so with sample collection and we have some technical considerations. Now the goals here are to maximize our microbial biomass in microbiome science that's not always easy. Not just the biomass of what you're collecting but that microbial component like that's what we want to measure especially if you want to look at the taxa and the genes or proteins that are encoding. You need that microbial biomass more than you need that host biomass but sometimes you can't separate them right. So you want to maximize that at the same time avoiding contamination perturbation or dilution. But when I was working in this field I kept finding that a lot of clinical folks they insisted on every time that they wanted to take swab samples and they're like I'm going to put an Amis transport media. Why are you putting it in Amis transport media it was kind of like a, you know, blinders were on from some of the historical processes of collecting swabs for anaerobic clinical microbiology. I'm not necessarily always trying to culture these bugs so I'm putting it into transport media, you know, a dry swab don't dilute it don't change anything just keep the swab dry freeze it and then we go on to microbiome. So you do sort of find sometimes these ideas out there that don't fit they're not compatible they worked really well with the way that science was done in one way but now that we're doing microbiome we have to change our sample collection so you don't have to swap Amis transport media, you know, unless you need to culture or you need some very specific purpose for it so otherwise you just don't want to add anything in or perturb in ways that aren't specific for microbiome. So I know this is going to be a little slanted to humans because that's where I work and I know many of you work in that area as well but of course we are considering when we design our studies for sample collection. So we have a lot of historical considerations that are beyond the scope of my talk but even from a technical standpoint we still have to think about. We want to get the best sample quality and the best bio minus we can but we have to also couple that with participant safety comfort and compliance. And so, for instance, one of the things that's been explored and is still discussed quite actively in the field is whether we should be making people scoop this poop all the time like who likes doing that and honestly certain populations that's really difficult so like I'm running a study with autistic kids right and they have a lot of sensory processing differences that make it extremely uncomfortable for some of them. To go through this process of having to change the way I hate poop and maybe having it right front and center for them after they're done. And of course the parents have to be heavily involved but you know it's a lot it's a lot for certain people in certain populations to do this. So just get them to wipe like normal and then just give that to us and actually this was commonly used in one of the early studies called the American get project. But then there was this like, oh, well that's not a good process because they left it at room temperature and it's not a biomass and all this. So these couple studies have come out a little bit more recently gone back re evaluated it and have shown it works quite well. So it's hard to kind of convince clinicians sometimes that they should just let people send into toilet paper instead of like a nice big pumpkin. You know sample so you know it's one of those things that we have to think about this would be a lot easier if we implement this more commonly. There's nothing else that's coming up with my work since we do a lot of swab samples, mostly vaginal swab samples but we've also processed and done studies with respiratory swabs and oral swabs. And a simple way to get more biomass is simply to use a double tip swab participant doesn't have to do anything extra. They just have the swab has two tips instead of women. So, you know there's all different kinds of swabs and it does matter and you kind of have to test for yourself we found that phone swabs work the best for us. But we use these double tip phone swabs they work great. And then I think we get twice as much biomass and we can split it off as soon as we saw it the first time. We split one off like some of our studies we do really want to treat with protease inhibitors so we can treat have a previous inhibitor treated swab, and then our other swab we keep pristine for our DNA and other work that we don't want to add anything to it. So it's a you know there are some simple hacks to keep the burden on the participant down don't make them collect a bunch of different swabs but you still get more material to work with. And of course, their sizing weight constraints yeah you want a lot of biomass but I can tell you when you got a whole bar dish of poop it's almost like a little too much sometimes and they take up a lot of space. So, McCoy people know you're talking about. So, you know you have to think about these things as well in your design. I'll see you have to ship often these things and weight can really affect these things cost. So, you know, there are examples going into you have to plan around that, especially if you're planning to culture, or you're working with low microbial biomass material. So, you know there, you can get microbial DNA free materials tubes and so on, they're very expensive. They have to be treated with a gas basically ethylene something dioxide or something. So if you have an extremely low biomass sample where contaminate DNA, even a low amount is a concern it might be worth it but you have to think about these things ahead of time. Cold chain or preservatives so again, ideally, you maintain cold chain that's sort of the standard in the field we'll talk a little bit more about this on the next couple slides. So, this comes up a lot if you do want to culture, mostly.  So some of the anaerobes in our vaginal samples, just by having the clinicians pop it into this bag before they send it over to us. So there are some simple solutions that can really improve what you can get out of these samples. And of course cost though every little thing even these little anaerobic bags that adds to the cost a lot. Okay so like I said, if you don't make smart choices with sample collection there's no going back right you got your samples you got what you got. So here's some things this is a really nice review that kind of goes through really from the standpoint of DNA based microbiome science but some of the difficulties with different sample processing steps and how that can lead to sources of error and bias. So with sample collection, you know in addition to things I just said you know like, I mean it kind of, I guess echoes them inadequate sampling is really about the biomass. Not getting the sample stabilized not having the right biomolecule stabilized. So in addition, you really want to try to avoid having any contamination in your kits that you start with your sampling kits, and labeling is also not always trivial, because sometimes these things are like like your swab is in a package right it's like in a sterile package sometimes. So your participant wants to open up the sterile package and kind of know that they have a sterile spot but there's no label then on that swab. So they put the swab into a baggie maybe you've put a label on the baggie that comes to your lab, you gotta make sure that that label gets moved back to the swab before it goes in the freezer right. So you have to really think about all of these little steps to make sure that you don't lose continuity of what's what and what's going on. Okay, so we're going to go into storage. And more now, like with handling. So, like I said this has three main parts and here we want to stabilize our microbes and biomolecules and then eventually partition aliquots and archive the biomass. So this is highly sample type specific. So it's always though that what I've also learned, you know, we tend to think fickle samples are not low biomass but you get some really young babies and all of a sudden they're kind of low bio so these things do matter. It's not just the sample type it's the age of the participant. Sometimes it's the sex. I didn't know when I started working with urine, I did not know that the biomass and female urine is like five to 10 times higher than the biomass and male urine. So you have very different requirements for volumes that you collect depending on whether you're working with male or female urine. So, you know, things I've learned along the way and sometimes of course with, you know, participants that might be very vulnerable going through severe illness, there's also that could be dramatic differences in the samples you get from those participants. All right, we've already kind of hammered the idea that cold chain continuity is ideal. And this really goes all the way through these four steps though, and maintaining that all the way through these four steps is not always trivial. So we talked about maximizing biomass in your sample that you've collected but sometimes you just also have to recover that and get it into the processing steps. So this is not always simple when it comes to swabs getting the material off of a swab is not super straightforward. And we've kind of actually worked on a little hack of my lab I wanted to get a picture in here and I didn't get it in time but there's these things called spin baskets and there's a variety of different ones and we finally found one from one manufacturer that works really well and we actually clip our swab and we put it into the spin basket and we centrifuge the material off and we've really increased our biomass recovery the use of spin basket so little details and your prep process can make a really big difference. As you're processing your samples, sometimes it's important to actually quantify your biomass because certain downstream analyses need to be normalized to weight. So a prime example of this is metabolomics. It really only works quantitatively if you can normalize to weight. So if you want to avoid your free spas. One idea that you know you can think about that we've adopted is when we do that first free spa and we go into sample handling the first time. We actually create many many aliquots, and some of them are weighed especially for feces. We also weigh other things like eluded material from a swab for those we track volume, and we also take an aliquot off so that we can count cells. So we have a cell count and a volume we can kind of infer some biomass numbers from that with feces we weigh. So, most of my studies I create at that first five to 10 aliquots, some of them are different weights or different amounts and they're sort of ready to go into different downstream analyses. If you hadn't quantified anything at the first free spa, then you're waiting till your second free spa to get that weight sample you need from metabolomics. So choices that you can make in your, in your own SOPs. Okay, adding stabilizers I mentioned this, the kind of proteomics we're doing we need to have protease inhibitors in there. Again, we don't want to add it to all of our samples because we don't want to necessarily, you know, inactivate enzymes that we might like sometimes we want to measure protease activity and sometimes we want to measure proteins that haven't been acted on in the web. So we partition our sample and make sure we add it at that first three freeze thought right away. So we don't want to go through multiple freeze thoughts before adding something that's going to protect a biomolecule. So that gives opportunity for things to change. And I can tell you an anecdote at the end if you want we have seen that this actually happens these enzymes are very active and things will change even on ice. Okay, like I said, we make many aliquots that are ready for downstream analysis. This takes a lot of time though. And it's worth the time and effort. It does sometimes save time in the secondary steps like you can just pull your samples from metabolomics if they're all weighed out. We often also put an aliquot into a bead beating tube, not with the lysis buffer because I can cause problems but just in the bead beating tube it's already there in the tube ready to go. We can pull our tubes because we do a lot of robotic extraction so 96 at a time we can pull our tubes add the lysis buffer. And we're ready to go. So we do a lot of that kind of pre planning in the sample handling procedures. And like I said, you do have to be mindful of biosafety for most human and animal work, and also DNA clean procedures so, but this means for us, since we're working with a lot of different sample types and a lot of them are lower biomass. We have a separate room, and it's a closed door room we call it pre PCR, and we just make sure that we don't bring lots of bug cultures in there and we don't bring amplified DNA in there. And we also work gowns, we do a lot of extra decontamination this space with bleach, because bleach will get rid of DNA ethanol won't. And we have a UV spectral linker in there and we UV a lot of materials not everything because some things will break down, but whatever we can UV or treat with bleach we do so that helps keep the DNA burden down in the space. We have a bio true biosafety cabinet to protect the humans from whatever diseases might be in the samples, mainly, and then we have some other smaller hoods that are just PCR laminar flow hoods to try to keep cells and dust and DNA right which is dust is mostly your profile DNA and human DNA. We try to keep all that dust out of like where we're working on your samples. So, those are, you know, pretty, you know, thought out and planned setups for a high throughput kind of microbiome processing but you know a lot of labs can adopt things like that having the sample processing and in a particular hood, or you know making sure things are wiped down with bleach before you start you know there's lots of things that you can adapt, even in your standard lab. Okay, any questions so far, feel free to interrupt me, and we'll have time at the end for some chatter to because this is not as long of a lecture. I wish I had a really great answer. I'm working on a review and we're working on a big study in the as part of impact with the McCoy lab and lots of you here have been putting lots of effort into that. We're trying to understand more about sample handling procedures and preservatives, and it's tricky. So far it's mostly been evaluated for DNA. There's lots of commercial options, they're very widely used. Omnigene is probably one of the most commonly used but RNA later in Zymo have been used quite a bit as well. Omnigene now has options for RNA metabolites and of course RNA later, good for DNA and RNA people have used it for a long time. But yeah there's less literature and microbiome science about the effect of preservatives for RNA metabolites than there is DNA. So the DNA literature. It's really hard to compare and do a meta analysis on my next slide I'll show you there's one meta analysis that just came out that I'm working on understanding but yeah it's just hard because so many studies do so many different things. But most say that there are some minor differences when you add preservatives. You know this is not as easily seen with diversity metrics. That's all you care about but if you really want to look at enrichment of taxa, there can be some differences and, like I said, I think it's still to be seen whether we really are seeing any consistent differences with so many different procedures sample types, different it's just still hard to kind of hire you, but you know a lot of labs kind of go with one of these preservative methods for big studies and then they want continuity with their other studies. So certainly if you want like continuity and cohesiveness like sticking with preserved samples is good, but then for like cross study comparisons. So if you're interested, you know, in comparing to existing data that's out there where they didn't use preservatives and you did, you might have some bias there. Like there can be some potential for that for sure. And then interestingly, you know I do think there's evidence in the literature this is just one study but we're the one we're working on is kind of showing the same thing and there's other studies and I think the meta analysis on the next slide also keyed in on this. So just DNA work, there's quite a bit of evidence that room temperature for a week is pretty much fun. So that actually can simplify reduce costs a lot for microbiome studies. But yeah I think people have been a little bit shy about it thinking that it's better to go with preservative and you know I'm not sure that the evidence suggests that preservatives better than room temperature. So you're not going to be doing DNA. But the thing is the preservative, it's not going to let you do like enzyme studies or like, yeah, enzyme activity assays or proteomics you can't do that right now with preservatives anyway so you're already kind of limited either way. Interestingly to what we're seeing in our study we're going to present a little bit of our study impact next week and it's not yet published so I didn't want to put you data in but we're seeing an effective homogenization. And there is some evidence of this in the literature as well. And I think it's a little I'm still working on understanding it myself, why. And, and I think it matters a little bit when you homogenize one study this one by Zostac said that the length of your homogenization matters. So yeah it's a little hard to iron out still but you know there are. It's a factor me maybe it's a more important factor than some of these other things preservative or not. It could be that you know when you put things in preservative you're kind of homogenizing at that time right you're kind of diluting it and mixing it up with the preservative. So I think it remains to be seen really what's important is it homogenization is it the preservative. And you know what is the best practice with that. So this is this meta analysis that just came out earlier this year. And you know it's one of the first I think that's really tried to aggregate all of this literature. So take a look at it but yeah, it's, there's a lot to go through. Okay, I'll talk a little bit about extraction so historically. This was thought to be one of the huge the biggest influencers of inter study variation. And I think it's a question that part of this was just you know are you really going to lice all of the bugs in your sample. Is your extraction kit contaminated with microbial DNA and, and that's still actually not necessarily trivial to avoid. And, yeah, do you have cross contamination so if you're doing high throughput extraction there's always potential especially for cross contamination in this process. What's important over the years is that be beating is critical. The thing is though with this that it's effectiveness is sort of intertwined with the lysis buffer and how you agitate. So sometimes it's a little difficult to tease out but certainly in some studies. They've had like a kit that's designed to work with be beating I think I have the reference here coming up but they have a kit that's designed to work with be beating and then a kit where they just added a be beating step sometimes it doesn't work as well that they just add on. And we've also had had the problem and if any of you have had the problem where you try to be beating in on a kit that's not designed for it you know with just a phone mess. Lysis buffers can handle that degree of agitation without creating a huge phone mess. So you have to think about not just the be beating but kind of that whole package for extraction and the be type in size matters, and I think, you know there's not a lot of we're working on a study that's going to evaluate that and there's not a great literature on it. But certainly if you're interested in fungi and bacteria, this is where it becomes really important, because you need bigger beads to lice fungi than bacteria. So that's why a lot of kits now put a combination of sizes together. And so like what's kind of become the go to is this kaijin power soil or power people pro. These are essentially identical. They're often a go to solution and the study that I said where they had the difference I think it was the ball study, the difference between this power soil pro and just some other kaijin kit that they added be beating in power soil pro was way better in that study so these are just a couple of studies that have shown the power soil pro or power people pros better and I mean that lots of people homebrew their own, you know, floor form and they love it and they've shown it in their hands. We've tried off label kits for specific, you know, or just like smaller companies I should say, for certain sample types and we have had hits and misses sometimes a lot of DNA contamination in the kit. Sometimes it's okay, but almost universally with a variety of sample types we don't see better yields or sort of better performance from anything else when we compare to power soil or power people pro. So, you know, there's other options, you don't have to use this but it's kind of become a pretty standard go to and a lot of my colleagues at the IMC feel the same way. And the reason it works well it has to do with these mixed speed sizes, lysis buffer, my postdoc lab always measured inhibition in their DNA and we found back then before it was acquired by kaijin it was this company mobile by by far it was the best at having low inhibition. And all of our library prep methods whether you're doing shotgun or amplicon require PCR so if you have PCR inhibitors it's going to reduce the efficiency of the process. And it can be hard to avoid having inhibitors in microbiome samples but this particular kit does a good job of reducing them. I don't have any references for this I haven't found a study I need to go back and look more but I've been in multiple labs and have tried to do plate based beat beating beat beating and it's just always been associated with high cross contamination so we avoid it we've tried it even again recently with whole genomes, and it was still too much cross contamination so unfortunately I think best practices is to beat beat and tubes, and least in our hands. So what if you can't be beat so for long read sequencing where you need these long DNA templates, you really can't be beat. And so I think it really remains to be determined what's great for microbiome this paper came out last year from the Arab, the Aaron lamb. They're still working on it they mainly were evaluating for microbiome samples, well length of the reads and the amount of DNA, not perfectly evaluating yet whether you're getting all the taxilized and so on so well ways to go with that maybe, but it's coming along. Okay. So, those are the main sources of bias really your sample handling and your extraction. When it comes to your sequencing library though there is some evidence of literature and again we're kind of seeing this in our own hands that library prep truly for shotgun can lead to some bias so depending on which kit you're using. There's a long standing notion that there's GC bias in your library prep. And this can vary from kit to kit based on how the library is made how the DNA sheared and how it's amplified from there. So, I don't know for sure yet if it's GC bias or if it's other aspects of library prep that are contributing to some of this microbiome variability. But this is starting to emerge and something to keep an eye out for. Okay, so this is just a different way to show what I just showed you all the steps right all the steps collection storage extraction. Then here all these lighter kind of orange circles then show all this bioinformatics that you've been thinking about for three days. So, in this long sequence of events that go into microbiome multiomics where do you think you need to think about positive and negative controls, which steps, starting at extraction extraction. Anybody else have any other ideas. Go ahead. I like that idea. Yeah. So, it turns out. Yeah, pretty much all of these steps. They should be thinking about controls, but you know kind of in line with what you're saying. Sometimes these controls are started at the beginning and carried forward and sometimes they're introduced at certain steps, and you really kind of have to do both to adequately control for all the steps. These should be positive and negative controls, and they should be done routinely, and they're not, they are not always. So for positive controls what we're talking about are known or expected biological content or DNA or wholesale community standards and we'll talk more about that in the next few slides. For negative controls we're talking about collection materials that are sham or lacking biological material. So for instance, you know, like some of the ways that this has been done is for swabs let's say. If I have vaginal swabs that are being collected in the hospital. I definitely want the exact same kind of swab and I want to carry it all the way through all my storage procedures and biological extraction. Some studies have gone so far so like give the sham swab to clinician have them unscrew the tip open it up to the air in the hospital environment where the regular samples are being collected and then close it. So you don't introduce it to a patient but you introduce it to all the other environmental features. Again, it's more important that you go to the extremes with all of that if you're doing very low microbial biomass where that background signals really going to be important, but for sure you should think about having some sort of sham or negative control that goes all the way back to sample collection, if you can. A lot of times you can carry those forward all the way through you can run it through extraction and library prep and so on. Other times like I said though you might introduce your community DNA or wholesale sample so wholesale that extraction, introduce your community DNA library. That's because then you can separately control for those two different things if you introduce the cells at extraction, and then there's some bias in the extraction. What if there's also bias at library prep, you're not going to be able to tell from the extraction control, what was the bias specifically from library prep. So that's why you introduce a DNA control at library prep as well to differentiate between two. And the purpose the purpose of these is to identify those unexpected problems things that happen that will reduce your sensitivity introduce contamination or bias the results and to detect batch effects, which could be a big clue that something's going through. Okay, so a little bit more about defined community standards and we'll wrap up and do some questions. So like I said these have to be defined you have to know exactly what's in it. You have to definitely advocate against trying to hunger them. I know sometimes in certain fields you have to because what the things that you're interested in are not available in a sort of manufactured and standardized standard. But it's very difficult to actually know and be able to control the ratios of the bacteria or the DNA or whatever and keep that consistent. Yeah, we prefer using sort of the same ones that are produced in mass by companies or some other standards entity that I'll talk about. You can get them like I said as whole cells so mixed communities of bacteria they're a frozen pellet that's how they come to you so they've already been frozen but they haven't been laced, or you can get the DNA. So the genome copies of the bacteria are mixed in equal ratio, essentially, or known ratio. So controlling the things that are important to control for with this is, you know, are you lysing both ground negatives and ground positives well in your extraction process. So I'm going to use these to look at are you recovering low biomass taxes so some of these samples purposefully have low biomass entities in them and then you can see did I extract and detect the bacteria in my control. Give me some expectation of really how low biomass I'm detecting in my true samples my experimental samples. You can either or determine what is the optimal sample handling procedures you need controls to work that out. Like I said with library prep, you can examine this taxa or GC bias with sequencing controls can help you detect and account for barcode hopping. Let's get into the bioinformatics to look at your taxonomic assignment is it sensitive and specific for all of the taxa and your controls to good measure of whether you're on the right track. One, two, five. Okay, so these are just a couple sides this is actually some data that hen has been putting together for me and other stuff in the IMC data science core. So these just we're starting to compile all of our standard DNA, sorry standard sequence runs from the last few years of experiments the last few years of sequencing data that we've generated and run and just look at the varies. This is where the field doesn't have a good answer is how good is good enough. Is it good enough to just see all the bugs you're expected to see to have the ratios look pretty much like they were expected to actually put a mathematical number on this in terms of, you know, how good did I do in representing a bacterial profile that was expected and known the math for that in the sort of standard application of that is still kind of lacking in the field. So to me develop there are some papers and algorithms out there, but it's not universally applied in the field at all and for sure. People are not accounted in that way at all at peer review. Most of the time in peer review that they only want to see did you use controls. They want to sort of use controls to maybe look at batch effects and a lot of times I've even seen authors come back and say, we know we didn't have batch effects because our controls we detected every bug we and just in our controls and every batch. abundance doesn't matter to them it's just we detected it and that flies to peer review. So, it's hard to fix it like you get these things to change but so far that's where we're at. We should be running controls we should be looking at the data but how good is good enough it's hard to say. So this is what we're getting in the IMC we can see the expected values for the Zymo community here, the standard Zymo community on the left. You can see the expected values and then you can see what we've been getting. Interestingly, you can even control the expected values by lots, and they have also ways to change and adapt your expected values based on copy number so you can tweak what the expected is, you know, using your Zymo control compare exactly to what you got. So we've got 16 as it's pretty consistent. We've also used some other controls that are a little bit more complex, but also then still commercially available. We've used. This is the Zymo gut control we've also used some of the ATCC ones. So you can see it's a little bit more complex and then a little more variable and like how well we replicate what we expect from the control. So you can see overall still even more kind of variability from experiment to experiment. And yeah, I don't know it's like certain experiments kind of our stand out so what we're doing right now is trying to dial into exactly what library prep methods and extraction, sorry library prep extraction doesn't vary too much but library prep definitely has varied over the last few years as well as sequencing platform we sequence on Nova and high seek. So we're looking at those variables to see if we can sort some of this out. Okay, so where can you find standards to use in your experiments so this is the international microbiome and multiomic standards alliance, of which I'm contributing member and their goal is to just continue to develop and build this understanding of best practices in the system. They have a great resources webpage where they have links to all of these commercially available publicly available standards. They also have like links to all the main microbiome standards organizations that have other kinds of things like data sets that you can use to control. So then I'll talk a little bit about what we're doing here in Canada with the impact. So we've been working on developing our own microbiome standard. The reason is that these, particularly the host whole cell ones. They're quite expensive. You don't get many uses out of each order. We can't actually import very easily a lot of the host whole cell ones that are manufactured in the US or other places they tend to have some bugs in them that are like fish pathogens and then we can't import them so we're home growing our own Canadian animal here. And we're also trying to make this more complex, whether or not this is a good idea remains to be seen but you know most of the others have no more than 20 taxa and we are spiking in 45 unique taxa, some of which are very novel and so it will also be like a good challenge for your microbiome informatics are you detecting these bacteria. On the other thing that we're doing this is a collaboration with an Ellen Burcos this is kind of her brain child but you spike in the strains into a matrix of human feces so you actually have like all the bacteria and the stuff in the human feces you have to get through that like component and control for that component, and then detect your spike in bugs. Right so it's kind of like making it much more of a realistic challenge to to our samples. So this is how we have been making these we got healthy human fecal samples we have been doing shotgun sequencing on them. We've also got the isolates of all the spike in strains and we've just finished the whole genome sequences on those. And these then were sort of pooled and Ellen Burcos lab and allocated and we've been distributing them in Canada for a year now to probably 25 different labs so far. So we are hoping to distribute the reference data sets everybody can map their reads back. Next, and then, you know, we'll see how they're performing and see what we might learn when we challenge. Our methods even harder. This is kind of what it looks like it's a lot of bugs in theory they're equal abundance we know they're not which so what would be sort of telling people based on our own sequencing. What's what we found in terms of our typical abundance ratios. So I'll just close with the impact websites. If you haven't seen I suppose you have because most of you were registered here but the resources tab up there is going to have the information of where you can find those standards if you want to sign up and have them shipped to you. So links to SOP some of them still need to get uploaded here in the next little bit but some of our sample handling procedures where you are definitely trying to make them public so you can benefit from them.