Hi.  If so, you might be an introvert. Hi. So the whole entire internet this week has just decided to inform the youth on going back to college content. Uh, did that make any sense? No. I have seen like 15 videos this week recommended to me of like what I wish I knew before I was a freshman and I've already made one of those videos. So I'm making it again, but a little more niche. I'm so sorry. I actually look like such a mess in this video. My hair is wet. I took a shower. I was having a self-care day today.  So I was watching like so many video essays on YouTube about like plastic surgery and like feminism and I painted my nails. That's self-care. So and don't get me wrong. Like in this video, I want to make it very clear. I love people. I loved I'm doing Alexis Rose hands. I love people. I love talking to people. I really do. But it's gets very tiring for me. And I think that the transition from high school to college, it was a really big one for me in terms of like my work style, how I got papers done and homework because your living situation is constant. Like you can't go home from college. Like not really. I live 10 hours, eight hours away from home, the first two years of college. And it's a really big transition because like there are just some times where you're just like, I need to be away from people. Like I can't like everyone's always here all the time. And like if you have drama with your roommates, like that's a whole other thing. All right. So my first tip, hide from your roommate slash roommates. Yes. So this is what I mean by this. They come into a room, you do a spy role underneath the bed. Let's hope they have a bed skirt, because that is really easy way to just like, you know, they can't even see you if they, if you hear them coming, just hide behind the door. Because most of the time they'll just kind of grab something and then leave, right? No, I'm just kidding. Don't hide from your roommates. I'm going to admit something that's a little bit embarrassing. Okay. I'm a stalker. No, not this tip is going, this tip is going to help you. And I think that it's just human nature. It's not it's not weird. So I'm not a stalker. No, I'm not. But what one thing that is like really important when you have a roommate is to, and you're an introvert, like is knowing when you can have a long time. Notice your roommate's schedule. Don't like figure out like don't like, like obviously don't like ask, well, you could actually, you could really ask for a copy of their schedule. You could, if you swap schedules, you could do that, especially when you're like living in a room with another person. Like I think that alone time is like so very necessary. Yeah. And it's definitely important to communicate like what you are doing with your roommate to because, you know, maybe they want some alone time because everyone needs a minute sometimes, you know, everyone needs a minute, everyone needs a sec. Okay.  And it's like, it can be really overwhelming when there's like eight people living with you. And everyone wants to like tell it, especially the girls, like, my gosh, because we love to share the tea, we love to give the tea, we love to just like update people on our lives, like, and it's so fun. Like I'm not, I'm not going to get you wrong. It's so fun. But then there are sometimes where like you really have to do something.  And as an 18 year old, sometimes it's hard to be like, Hey, stop talking. I have to go to work because like, you don't want to be that person. And if you're, if you know in your heart that you're not going to communicate those things, so hide from your roommates. Okay, so we're just going to move on to number two. Oh, number two is do not be afraid of doing things alone. Okay, so I feel like college is just like high school trauma like relived like the first year, if we're all going to be honest with each other, like no one wants to sit in the dining hall alone. And there's definitely a stigma about that. Like in only in your head, because never not one time have I looked at a person eating alone and been like, damn, they look depressed. They look sad. They don't have friends. Never have I once done that not one time. Most people aren't going to notice you, especially if you're going and doing things alone, because sometimes you just have class or you're busy and you want to go sit somewhere and get some work done. And I definitely had like a complex about that. My freshman year of college, like it took a lot for me to go and do things on my own, especially living like in a new city by myself. Like I was like, I can't go to like a coffee shop by myself. You can use flash. But my sophomore year, when I had like so much work to do, I would basically just bring my laptop or whatever is the library or to a Starbucks and just like order like a coffee or something and sit upstairs and I would get so much work done. Honestly, sometimes the pressure of like a crowd of people like judging me, they're not judging me, but like I play this game in my head room, like they're judging you, so you have to write faster. Anyway, so I found this article on University College of London. They wrote an article, someone named Miriam Clark, a student at University of College London. She calls us a little introverts in this. All right, yeah, we can be little introverts together guys, we can. So I'm just going to read through some of the advice that she gives and give my own opinion. So the first number one is know your social strengths and embrace them. Okay, introverts tend to be great leaders quietly mentoring those around us with patience and encouragement. With our attention to detail and time we spend thinking things through, we also tend to be quite eloquent writers. Embrace these qualities and have your voice heard by writing articles or even running sabbatical positions. You can do it my little introvert. Okay, thanks Miriam. So actually I think that this is pretty good advice while that was just like boasting about introverts a little bit, which no one needs to tell me how awesome we are because I know. But that's definitely true. I joined like her campus which is like most college campuses. And it was a really fun like outlet for me to like share my voice and my opinions because I would like write these online articles. It was fun. I got to like meet some people through there and write my weird articles about the trauma of being a college freshman. So yeah, I think that that's actually a really good piece of advice. Okay, number two, let go of the idea that you're missing out. Say no to a social event because you'd rather spend time in your room with your cat dog Goldfish to recharge is not a bad thing to do. So what if you're missing out on the latest hot gossip? Your wellbeing comes first and then she wrote hashtag now regrets. Oh my god, thanks Miriam. So true. That's definitely for real. I actually relished and one of my roommates would go out without me after a while because like at first I was like I'm so tired. Like I can't go and then like I would feel bad and I would be like I'm so sad like I'm missing the party. Obviously like don't do that all the time. Like you should talk to people but if there is a chance for you to like be alone in your dorm room with nobody there with you like that's definitely like a necessary evil sometimes.  I love being alone. Okay, number seven, skip the small talk. Yeah, that's so true.  There I said it. I hate it. Like why would I talk about the weather when we could talk about your past trauma or my past trauma? I don't know. Like that's what I want to do. I don't really want to hear about the weather or like anything like that. Do I want to hear like the moment that shaped you as a child and to the person you are today? Like yes, I want to hear that. I want to hear that so badly for everybody and that's maybe an issue but I hate small talk. Like that's like makes like parties kind of hard and draining because it's like all small talk. Unless it like like at the beginning unless you like know the people but like yeah that can be definitely hard as a freshman to be like hey like what's what class? What's your major? Because like every time I have to answer that question I have to ask that question because like sometimes you can't think of anything. So like you do end up asking the person like what's your major?  I think like it's a collective death of the pieces of us. Number eight, stay true to yourself. So true Miriam. It's going to become exhausting for you as an introvert. Ignore your need for so if you oh if you it's going to become exhausting if you as an introvert ignore your need for solitude. Even if this means having a mini fridge in your room so that you can have quiet undisturbed night in. Do it. Offer yourself the care you need to be 100% you and you will have a much more enjoyable university experience. That is definitely so true. Like I think that it's so easy to get wrapped up especially as college freshmen nowadays like in social media and you know so many people have something to say online or showing what they're doing and you know not not everybody is fulfilled or happy in the same ways so you know I say this every time but you know comparisons bad so don't do that but don't do that. Do not compare yourself to others which I definitely did so I don't really know how I'll be there because I have been inside the mind of an 18, 19 year old and all you do is compare yourself online to other people. But yeah I think that all that advice was very interesting and yeah I think that it's important to talk about these things because I didn't even think about the fact that I was an introvert before I went to college really and I kind of wish that I had because it would have saved me like feeling bad about it I guess because sometimes on those nights alone where I was like having like a great time by myself because like I needed it I would be like well damn I'm like not I'm not like out being a party lady and I should have just you know been okay with those nights and enjoyed them and not not have stressed because you will meet people and you know you got to put yourself out there and that will include going to things that like you don't like but thank you so much for watching and you made it all the way to the end of this um I post new videos every Thursday and um I go follow me on Instagram if you haven't already um it will be linked down below it's like katieblake7 or something like that um and comment down below if you're an introvert who went to college like comment down your hot down your hot tips and please tell me that I am not the only one who like tries to hide from their roommate sometimes like because sometimes like you don't want to like say it you know you just want to like you just want to skedaddle


o this in the cpa funds and maybe those funds are already appropriate in other ways but i have a hard time thinking about spending of capital items being at the same level which i know is the goal of some of our towns um while we're making significant operational cuts you know um i just have a hard time with it right if there's not cars under the solar canopies because we don't have enough staff to use the parking lot that we would be repaving maybe we should just all park it over at the middle school and we don't need the district office parking lot anymore even though it's got potholes and all that right and just to you know i'm a big supporter of the project but i i do want to say that juxtaposition of the cost of projects while we're you know potentially looking at cutting significant numbers of staff feels very uncomfortable to me uh if it's not about health and safety right directly about health and safety one could certainly make the argument that solar canopies in terms of greenhouse gases but um that's not the environment that we're in right now in terms of fiscal um and the same with the athletic fields you know i can't can't promise that if we're cutting 1.7 million dollars that athletics won't be affected athletics were affected the last time we had a big cut we do a number of games and so you know i get that these are popular projects i get that i'm supportive of them you know i definitely heard from families spent a lot of time a couple years ago if those are like you're on the committee you spent a lot of time on this a couple years ago about the condition of the fields um it's really important that that improves at the same time uh you know the juxtaposition is just hard for me so i just want to share that publicly i want counselors and select board members and finance commuters advocating for capital projects for the regional schools in general regional schools get short shrift not ours but in general on capital projects because you know when you're part of municipal government it folds really nicely into capital projects when you're part of a region and your its own entity you're not part of municipal this is what i was referencing earlier sometimes it it works actually disadvantageous uh and i think that we see that difference in the emmer's public schools and the regional schools and how that works out for capital uh especially as relates to technology and other matters at the same time you know i don't want to dismiss the importance of both of these project i want to be on the record i'm highly supportive of them but i think when push comes to shove and and we're seeing if we do go forward and see significant capital funds go towards them as we're reducing staffing um to levels that none of us here are comfortable with i think it's just going to be a hard moment for all of us you know i'll say it would be a hard moment for me to do that not because i don't support the solar you know part of uh parking lot and not because i don't support the fields i absolutely do they're both projects that need to happen um but i also think we need to make sure we have the staff needed to support our students in school and so you know i think that's going to be a very difficult calculus as we move forward so i just wanted to share a wildly long qualification uh an introduction to dr slaughter who i think is going to be much more factual and concise than um than my comments but i was uh i did have a reaction at the fourth internal reaction you know hearing that dialogue um uh as and we always have this capital and operational if you never spend on capital if you always put all your money in operational you know you end up in bad situations i'm on board with spending on capital don't get me wrong but when our when our potential budget cut number you know is as high as 1.7 million dollars um that's a hard that's a hard pill for me to swallow um so i'll leave that with dr slaughter he can explain some of the cpa pieces and other pieces moving forward because i think both projects are certainly overdue and very very important but i can't pretend that we're not in the context we're in right and so i i think you know relative to the solar i think that the original you know if we look back the the capital project that we were going to i believe partner with town of amherst a little bit on was a was more a solar study camp these was one component that was going to be looked at relative to that i think that uh no one has less interest in doing you know that we don't have less interest than we did have we have the same level of interest and need that we had before uh the economics have changed around us and so that's that's uh you know the unfortunate place we find ourselves uh and so i think our our our impacts on on things environmental will will need to probably be much more focused on uh smaller and more uh internal uh types of adjustments that we're trying to make um and i referenced one at the four towns meeting you know one of the things that have has happened through uh through uh some funding from the electric company is to to relamp a number of bulbs within you know the buildings and that's saving us uh you know energy and and and ultimately money but but also saving the amount of greenhouse gases we produce because we're just using less electricity than we were so i think we're going to continue to explore those things that that are available and become available to us and and continue to refine and and uh perfect as we can the things we can do uh as quickly as we can um shifting to this sort of cpa circumstance so you know in in the uh sort of planning last spring before covid hit uh we had a small chunk of of state aid that had come in for uh use uh in in the region that we were thinking oh we'll take a portion of that and do some preliminary work to get started on some design work for the for the fields to uh sort of um clarify and articulate uh you know pros and cons of of reorienting the fields and and talk about the pros and cons to some extent of of the surfaces that we could explore there and and sort of kickstart the program a little bit and then the idea was to take some cpa money and and start to move the design forward and of course with with the change in circumstances uh that we had last spring you know we've leveraged that money for other purposes um and so we didn't do that preliminary work um and so at this point you know the cpa money that we have received from the town is certainly uh likely to be utilized for that purpose and for you know continuing to the design phase uh beginning design phases for those fields and and track um but i think you know and i've said this before on some of these there are things you can do with those sorts of designs that that are always useful and available and good information but then there are other aspects of it that have a certain shelf life um so we want to be cognizant of that as we move forward relative to to uh to trying to move ahead with with that larger capital project is is can we and should we leverage cpa money can we use that uh and strike the right balance with the other communities to help in support uh through their cpa funds to help support that project but uh again i think at at this moment time given the the circumstances with the you know in the uh the upcoming budget um you know i think the town as well as as the the the town of amherst i should say as well as the other communities you know would would be uh a better reflection of our values to to spin more on our uh operating to keep and preserve our school district uh more as we see it and now we think we should you know and what we should offer and and uh be a little patient relative to our capital needs on on this front um uh the the fields you know have some health and safety concerns i won't diminish that at all and and we do want to address those as quickly and as best we can but relative to some some of the other more uh broadly impactful and and significant things we talked about earlier this evening about our operating budget i think it's it's tough to move forward on on capital projects of that size uh that that don't have the highest level of urgency and and these have a high level of urgency but not the highest level of urgency is how i would state it thank you any uh questions or comments from the committee miss dancer um so i i don't know if this question is relevant but i know that in the pelham town meeting last spring we did vote to provide some cpa money for for the field study so what would you then say to the town about that money if if we decide i mean are you saying we wouldn't use it for that so would we give it back to the town or so what i would suggest is it doesn't expire and so it really would you would be at the town's discretion as to what they would choose so if they wanted to repurpose it for another for another project that they feel has more likely to moving forward or that has more urgency you know that's certainly their prerogative and i would by any means i i think in the short term if if you know with that money that has been appropriated for our use um we're going to hold on to that and and you know if if they decide that it's still okay to fund that kind of a project we'll wait and see if we can fit it into a capital plan that that you know meets with uh our ability to actually fund the project fully and to do it in a way that's not uh you know in in lieu of our academic program that we we need so i think you know it's it you know we're happy to hold on to it we'll be smart with it we want to be smart with it um but at the same time it is the prerogative of the community so if they decided they needed it back for repurposing for another need that they have that's certainly within their realm and unretainable um dr morris yeah i think it may also be worth when we're talking about this decision to bring it to the next four town meeting um for those of you who went to the ones last year there was some skepticism from some of the member towns not so much about the study but actually be able to afford the actual project from capital funds um and uh you know i do think it's worth separate from what i said earlier about the broader context just making sure that um i don't want to do a study that's going to sit on a shelf right you know that happens a lot in organizations and there was some really broad disagreement last year from some of our member towns about the viability of an actual project not of the study so i i do think before going forward people's the town's financial situation certainly has changed at least some of the towns have indicated that and and i'd be curious about making sure all the towns were on board before we move forward with the study because i don't want to set up a a dynamic that we know is going to be contentious in the back and i not go anywhere and and spend a lot of town's money for no reason i think the towns need to be done i want to be really clear like the fields are really they need to be much higher quality uh but what i don't want to do is spend spend some cost funds when we know in the tail end we're knocking out the support of all of our member towns miss fitzer you're muted i just wanted to say that i i i similarly feel like awkward about this idea of funding capital projects while we're cutting our operating budget and and we just saw in the past budget cycle where we kind of hold it a lot of the spending we were planning on capital in order to to fill the gap but i i think what the interesting thing about the cpa funds is that it's not as though we could use those funds directly to fund anything on the operational side and if anything we've seen with our um with this current environment the importance of outdoor spaces and and i think the outdoor spaces around our high school and middle school are i mean probably all of our schools but i'm thinking about you know but the the report i saw from you know i heard the reports um about our fields and it's if anything i think some of them are getting more use at least in the spring you know then by community members who weren't necessarily school members and so i could see a case for if our towns believe that invest specifically the town i i'm from amers you know because the the fields are in our town but those spaces have benefited our community during this time when getting outside is the one of the only safe ways to get together so i i guess what i'm wondering is and this may not be feasible at all um but if we could use some of that money to improve the public outdoor spaces in a way that could be responsive to everything that's changed since covid because it just feels like to i understand why we don't want to but it feels like for the entire community it's not just our schools that use those fields but the having outdoor space in it the people can get together safely is really important and i'd hate to miss an opportunity to improve them next morris and i think i think you hit on a couple things that are important and i know we have to move the conversation along i know it's late but but i think that's a point of that i've heard over the years of contention between the four member towns that one of our member towns because happens to be located in the town of amers um there's a lot more community use of the facilities by community members in the town the schools are located in and that for some of our member communities and i'm not justifying i'm not taking sides on this one but i i want to be really plain and honest about what i've heard that the the benefit is purely to the the students who attend amherst regional middle school or amherst regional high school and much less so for the larger community i'm not saying no one from leverd ever comes that never comes down to use the track but i would guarantee that it's used more by residents of the town of amherst than residents of the towns of chute spare leverd and pallum so i think that's where it gets a little bit difficult to have that conversation uh and everyone has a valid point right you know like there's i don't think anyone comes to that conversation not wanting to support kids or not wanting to support the schools but i do think the fact that the school happens to be located in one of the towns has an impact on some of the community support and that's why i think bring it to the four town meeting uh might be might be warranted and might be helpful in terms of figuring out a path forward to do the things that carries that miss spitzer's talking about because i fully endorse everything she just said any other Mr. Deming just briefly i mean given my concerns about the current operational so i asked this item to be added to the agenda not because i want to add to our capital request for next year i i i go now seriously and i value the the the fields in the and the solar panels if these feel like such throwaway comments right yeah yeah i value them but like i do it's just that you know the reason for bringing this agenda item up for me was because i feel like whatever the questions that we get from our member towns even if we don't feel like it's something we can do this year we just need to be in a position to provide the answers right so if we're if we're asking a town to fund us above a level that they're initially telling us that they want to and then they ask us about xyz even if it's not directly correlated we just we need to be able to provide every answer about xyz so if we hit town councilors saying you know we want info about the solar efforts and see well here's here's here it is like as you've just laid out and and mr slaughter makes the um the excellent point about shelf life is that yeah we could grade the goal is not to have a field study the goal is to have a field right and so if one does not follow the other again eyes wide open right don't don't don't don't fund a study unless you're prepared to to go to go forward with it so i i think that that's the kind of information that you know we can have organized available to answer those questions and i think part and that helps i think just build trust right is that like you're telling us you have a desire interest or concern about some aspect of operation of capital well here's here's the information okay now let's go forward and have the more difficult conversations about funding level what not so i appreciate the information you put together it's exactly what i was looking for and you know thank you for doing that are we ready to move on okay so moving on we have a future agenda planning um i think we have we we have a sort of on our draft comm commons um uh meeting on the 5th of january um but what i'd also like to propose is um is that we um i if if feasible i don't know um how the committee is open whether the committee is open to meeting between now and the and january 5th um in executive session we had several several topics that we didn't get to um in our last one that i think um it would be helpful for us to continue that um and um wondering if folks would be open to to doing that um early in the next week um versus waiting until the first week of january and see my heads great uh tuesday same same bat time same bat channel okay um and then uh for january miss mcdonnell i mean i don't know if people have a more flexible schedule but i'll just say for myself i am not opposed to daytime meetings uh in the next two weeks um that may not work based on people's work schedules but um particularly if it's an executive session where there's not actually other things in the agenda you know in terms of viewership we're needing to do it at night i feel like that's reduced so just wanted to put as an idea out there that i don't think daytime meetings if pure especially if it's just a executive session it would be undoable it may be undoable from people's lives but i think from a public asex perspective i don't see a conflict hi miss fitzer well speaking as somebody who's a parent of three small kids who aren't going to have um school um and not just our public schools but any um child it would be difficult unless and especially given the executive session nature nature of it because i don't think it would be legally allowed to have a you know three-year-old in the background that me is there an age limit on that because if so i can't um i don't know if other people have a similar constraint but that's that's mine um the only reason i'd fight moving at much earlier as much as i'd like to not be on one of these calls later like yeah no that's fine i just wanted to put it out there um it certainly wasn't a request from me just wanted to put it out as an option um maybe we could make it six six we could start at six instead of six thirty if folks are flexible or we're six thirty the ideal time i know that some folks have hard hard time moving getting to meetings earlier okay um and then i think on january fifth we have a variety of topics already lined up um i'm going to try and pull that up i have it here alison if you like yeah so we'll have a lot of minutes to approve as mentioned earlier um we may have an AFSCME MOA to take a look at um you know perhaps an update on jlmc in the in the MOA we'll see how that that conversation goes um i want to come back to the attendance piece that misstands are and others have raised and and try to respond to that um i think the spring 2021 plan i mean the the next school year planning probably better to do with the second meeting in january than the first meeting uh just so i can get a more comprehensive read on the topics people would like to to talk about uh the time on learning and the live and not time of learning shoot the live instruction piece from desi uh and i've got snow days so that's an awful lot of things um there's another meeting later in the month where we'll get deep into budget as well as the 2021-22 school year um we also i would like to bring up um something that came from a partner agency mabe around the access test uh and some implications of standardized testing on ell students um they have a i don't know if it's quite a petition but a statement that i'd like to bring to this committee for for feedback and and potential endorsement of it's a packed schedule it is i'll try to be briefer than tonight my apologies about this evening hopefully the update will be much quicker since there will be vacation the next two weeks right so that'll help great okay um and just a reminder for um folks at the that we to circle back if there's anything more out in sort of school year 2021-22 that folks wanted to include in that consideration to email back and forth uh moving on um warrant report is that sir do you have any warrants from the region to report um it doesn't appear that i do so i think i reported them all last week okay um and we don't have gifts this evening um so we are we caught up a lot of time we're only 25 minutes past um um uh would somebody like to make a motion i moved to adjourn the meeting of the regional school committee i will second that moved by stance or second by mcdonald and there's no discussions i will move to a vote um mr demling demling i mr herrington herrington i miss kenny kenny hi miss lord lord i um miss seager seager i miss spitzer spitzer i miss dancer cancer i mr sullivan sullivan i and mcdonald i we are adjourned thank you everybody


we need to get in the huddle come on let's go that gun bro you're back here here marcus mariota just threw and caught a touchdown pass touchdown titan the lady walker my goodness the young maestro is special we've got a hundred dollar gift card from program we're going to bless you today thank you marcus of course thank you so much yeah very nice to meet you you know i think we compete to see who can do the most in the community no sir that is the maestro marcus mariota we came here for two reasons choose the bubble gum and kick them ass and we all out of bubble gum


Next question is from more life Jojo. Does the intensity of the squeeze or clenching of a muscle matter big time? Definitely big time your intrinsic Application of intensity right so how hard you squeeze the muscle? increases the intensity just like adding weight to the bar wood makes a big difference isometrics are depending on your intensity of Contraction if I flex a muscle I can flex it You know have good control or I can really flex it hard and the harder I flex it the more the louder the signal the more potential damage all that stuff so this makes a huge difference one way you can apply this to your training well There's two ways one is to do isometric training in your workout or two is Focus on the squeeze at the top of a movement and really squeeze the muscle hard But I will warn you you're gonna have to go lighter to do this you fatigue real fast when you do this It's not as easy as it sounds. It definitely makes it a lot harder Well wouldn't it wouldn't a simpler way to kind of explain this be like if I were to Clench my fist very lightly right so I'm squeezing my arm right now My brain is firing neurons to my hand right now to in order to do that and let's just say for this example A hundred of them are being fired in order to do that and then just by me simply Clenching down as hard as I can I now recruit even more from my brain to there in order to do more muscle fibers Right, so the and I and I'm also training that connection a stronger more More neuro neurological connection to to my fist and if that is training like I loved your analogy that used to give on The podcastle time which is comparing it to like an amp, right?  You're getting a higher wattage a stronger amplifier the better this better and stronger it can get the better that you can recruit muscle and the More likely you are to lift more weight and that goes back to the last question with like gymnast part of the reason Why they're so good is they have great CNS control I mean they have all that body weight training has connected them to their body so well So they have incredible amplifiers Maybe they didn't have big muscles yet because they haven't lifted hundreds and huge squat or dead lifting days But they've trained their body so well. They're so well connected They have an incredible amplifier all they need is to plug in some speakers and they're gonna get tremendous benefits from it Yeah, and for some reason, you know that concept is hard sometimes to for people to grasp right away of like you can Squeeze harder Just think of it as like if I'm going to grab an object that's lighter versus heavier and what that all entails like if I'm gonna pick it up And just use the same amount of contraction to pick it up as I would you know the heavier object You're gonna have to squeeze hard You have to try hard you have to recruit, you know all the way up your arm into your hips and stabilize with your legs like a Heavy object that you know requires More force production. So you have to be able to generate, you know more muscle fibers. You have to recruit more You know to be able to pull that off.


This week on Maker Update, a chonker synth, glowing glass, spinning cycles, assistive tech, cyberpunk, and how to build a testing jig. Hello and welcome to the Eight of Fruit edition of Maker Update for March 2020. I'm Tyler Wangarner, I hope you're doing great. We've got a fresh storm in, but otherwise I think that this winter is starting to run out of gas. And with some of the projects we're seeing in this month's show, I feel like people are starting to think the same way. So with Spring hopefully right around the corner, let's check out the project of the month. With the Circuit Playground Bluefruit out there, it seems like everyone at Eight of Fruit is developing wireless Bluetooth low energy projects. Liz Clark decided to combine BLE with her love of music to create this keyboard and mini amp combo. And I gotta say, it's as impressive as it is adorable. Half of this project is this goofy, chunky, single octave 3D printed piano powered by the Feather NRF 52840 board. And the other looks like a mini amplifier stack, housing the Circuit Playground Bluefruit. Liz took her inspiration from the Circuit Playground Bluefruit app, which has a piano tone generator module, and wanted to emulate the same function in hardware. When she presses a key on the piano, it triggers a momentary switch, which prompts the Feather to send a Bluetooth packet over to the Circuit Playground, which then plays a tone on the Stem of Speaker module. The NeoPixels on the Circuit Playground also add a swirl of color, which you can see through the speaker grill. For the build, Liz also had a PCB made for it, and she includes the Eagle files in her project write up, but also includes plans so you can recreate it using Perfboard if you're not that fancy. The Perfboard plans also have this one tactile switch holding the two sides together, which might be a metaphor for how we're all getting through 2020 so far. The code is written in Circuit Python, and there's a wonderful code breakdown describing how each function works. There's a huge wealth of knowledge here like state machines, how to set up an adaptable animation system, and more. Liz also provides STL files for the keyboard keys, PCB tray, the enclosure for the amp, and everything else. It's a great project to share with your friends because the result is so much fun. Now for the news, last month we mentioned that Circuit Python launched its 100th board. Now it's getting released on what is probably some of its smallest hardware. Kevin Neubauer designed this barely over 1 inch square microcontroller named the Circuit Brains Deluxe. It houses a Cortex M4 chip and features I2C, SPI, 18 digital GPIO pins, and 10 analog pins. It's an impressive design. Maker Laureate Gareth Brannwin has a new article series up on Adafruit answering the question what is Cyberpunk? Whether you've been reading William Gibson for decades or your entry will be the upcoming game for CD Projekt Red, it's a tricky genre to pin down. Gareth goes over the work of numerous authors and filmmakers to establish the core elements of the Cyberpunk aesthetic, and over the next few weeks we'll be exploring the entire Cyberpunk timeline, its impact on real technology, and its influence on hacker and maker culture. I can't wait to read more. More projects. Modern technology gives us a plethora of options for DIY lighting, but Aaron St. Blaine shifts hard into the realm of fantasy with these four seasons fairy bottles. She took a bunch of vintage bottles, decorated them with iridescent cellophane and some leather wraps for hanging, and then put some neopixels into each one. They're all controlled by a central circuit playground with touch sensors to call up different lighting programs. The result is a beautiful magical storybook setting. She raises the bar even higher and made this tree with animated eyes that react whenever someone gets close enough to it. Come for the pretty lights, but stick around for the awesome fantasy fabrication techniques. John Park has been exploring a few fitness adjacent projects lately, like this DIY cycling computer built on the alpha release of the Adafruit Clue, or as he calls it, the Piloton. To me, the most impressive part about this build is that he's using an off-the-shelf wheel sensor that works on BLE. It's able to communicate with Clue, and there's a fantastic breakdown of how Bluetooth Low Energy works in its various modes to handle communication. The current calculation set is pretty basic, but John plans to extend the code to offer speed and cadence information in the future. We're a little past Valentine's Day, but this guide for making a heart-shaped neopixel necklace could easily be adapted to a number of other designs. The LEDs are hidden behind a layer of black LED acrylic, and it was inspired by this recent project by Sheryl and Gonda. But the real magic here is this technique for laying out your neopixel design. The Ruiz brothers used tape to stabilize the neopixel strip when soldering so they could maintain their shape and spacing. It's a tricky design, but with a great payoff. Brian Seapert of the Adafruit community used this LPS3X pressure sensor and some tubing to create a DIY Sip and Puff sensor. Sip and Puff is a common assistive technology that allows the user to create inputs like mouse clicks by creating positive or negative pressure on the tube. There's some clever use of zip ties to secure the tube and offer some strain relief when it's in use. If you're interested in learning more, the AT Maker's online community is a great resource to learn more about making assistive technology or getting paired up with somebody who has a specific problem and is looking for a solution. Time for some tips and tools. HackSpace Magazine just cut their North American subscription price in half from $125 down to $60. It's a great magazine full of projects, tips and tools at any price, but for 12 issues, this is an outstanding value. Plus, they're still including a Circuit Playground Express with a year subscription. Catney Rimbord wrote up this guided tour to the alpha version of Adafruit's Clue. This is their new dev board with a similar form factor to the BBC Microbit. There's a TFT display, Bluetooth Low Energy, Nine Doff Motion Sensor, Sound and Light Sensors, Pressure, Humidity, Stem of Connectors and more. Plus, it's Micro-Python compatible. The Ruiz Brothers also have this design out for a case for Clue either to protect it or wear it as a badge. I can't wait to get my hands on this board. If you're a hardware designer that's considering making a leap into production, a major hurdle in the process is developing a reliable testing jig. Dana Wall wrote up this guide to take you through the basics of building an electronics testing fixture. Worth it just to learn all the different types of Pogo pins that are out there. And finally, if internet commenters have badgered you into finally buying a set of mid-to-toil calipers, they've just become a little bit more useful. The calipers, not the commenters. There's now a Git repo with some Circuit Python code to interface with mid-to-toil digital calipers for digital data logging of your measurements. It's pretty cool. This month's Adafruit product spotlight is hot off the pickin' place and it's a doozy. The Adafruit Feather NRF 52840S is an Arduino IDE and Circuit Python compatible Bluetooth Low Energy board, but there's a whole lot more on offer here. It also packs in an SD Micro 9 DoF motion sensor, a proximity, light, motion and gesture sensor, temperature, humidity, barometric pressure, and plenty more. This one is probably going to go fast, so don't sleep on it. And that is going to do it for this month's show. As always, huge thanks to everyone at Adafruit for supporting Maker Update and to you for watching it. If you dug this show, give us a thumbs up or leave us a comment. It's always great to hear what you're working on. Remember to use tools, tell stories and keep on making. Take care.


So my name is Cristian Michaletti. I'm part of the faculty here at CISA, a colleague of Ali. And it's a pleasure to be with you and to share some work that we did in the past few years trying to give comprehensive perspectives on a problem that is ubiquitous when you're dealing with biomolecules that are flexible, usually densely packed as they are. Typically think of DNA inside a cell, whatever cell, a bacteria, eukaryotic one, even of the simplest possible organisms, which are viruses. So inevitably you end up with lots of entanglement. What are the implications of these entanglement for the biological processes of these long biomolecules? And to what extent you can learn about the properties of the molecules in experiments. So you want to identify a statistical mechanical approach to characterizing the entanglement that spontaneously arises in biomolecules. And this is what we have been doing using coarse-grained models and STATMEC. And the other flip of the coin is instead of to look at entanglement, let's say knots or links, that you can produce in a synthetic way. So instead of relying on the entanglement that spontaneously arises, you wanted to design that. So end up with special type of knots that you are interested in, not the casual knots that you end up in your earphone cords when you walk with them in your pocket for a day and then they're full of knots, but these are statistical. They're usually not the knots that you want, not in the positions that you want. So I will try to give you a physicist and STATMEC perspective on the problem. Probably a good way to introduce the problem and to quantify a little bit what I've been saying in a hand-waving way in the introduction is to consider the type of packing that you have of genomic DNA across the spectrum of living cells. So in eukaryotes, you typically have meters of DNA that are packed in a nucleus that has a transverse size of about 10 microns. Then you go all the way down to bacteria, you have millimeters of DNA in a micron-sized cell. And if you got the lowest possible level, a level to which you can dispute whether you are really dealing with living organisms in viruses such as phages, you have microns of DNA in a small container, a protein capsid that is typically about 15 nanometer in diameter. That is for the smallest viruses that you can have, phages in particular, which are viruses that attack bacteria. So a very early view on how DNA is organized at such scales is provided by the beautiful cryo-EM picture that you see here on the left that where people, for the first time, they managed to take cryo-EM micrographs. That is, you take tomographic cuts of your frozen viral particles, you measure the electron density. And by taking many cuts along many different directions, of course, from different viral particles, then you can use the theory and computation to reassemble all the electronic structure to come up with an average electronic structure that tells you how DNA is organized on average inside each particle. And as you can see at the bottom of this picture here, you can see that the DNA is, you can neatly tell, you can tell that DNA is organized in neatly loops or a spool, if you wish, that is a coaxial. So it's a coaxial along the axis of these viral capsid. However, one thing to bear in mind is that here what you see is the average DNA organization across many different particles. So you are bound to overemphasize the order, order the packaging of DNA inside. And so really how DNA is organized inside each of these particles can be different, can be disordered. That is something that you cannot really probe yet. An indirect insight into how DNA is organized inside. However, you can obtain from a very different perspective. People like colleagues based in Barcelona, in particular Joaquim Roca, have been able to develop over the years some very ingenious methods where they managed to get the DNA packaged all inside the capsid of the virus. And then it so happens that if you choose correctly your virus, the two ends of the DNA of the DNA strands are complementary. So the DNA is double-stranded. So it's the double helix. But at the very end, it's a single-stranded for a short stretch, a single-stranded and the two ends are complementary. So when these ends have the chance to meet inside the capsid, they anneal, they circularize the molecule. So instead of having a linear molecule, now you have a circular one. So if you happen to have any type of knots formed in there, it would be permanently trapped by this ligation. And then if you remove the capsid and run the material through a gel, you see the formation of different bands. So this is a technique that is called gel electrophoresis is these typically used to separate molecules of different lengths. So it works by preparing a gel that is very dilute. So the gel you should imagine has been like a wire mesh. So an array of obstacles to your molecules. This array of obstacles presents some entropic traps for your molecules and people typically use it to separate DNA of different lengths because if you put an electric field, you know that DNA is charged, you put an electric field at the two sides of the gel, you can make your DNA filaments migrate. And according to whether your molecule is small or large, they will migrate with a different effective velocity because they will be able to negotiate the obstacles of the gel to a different extent. So typically you use this technique to separate molecules by size. But here, though the particles of viruses have DNA of the same length and still you see different bands in the gels, you can see different spots, they are not separated by lengths, but what distinguishes one from the other is their topology, is they're not tight. How do people know this? Because over the years, people very carefully have cut out excerpts from this gel, they recovered the molecules in each of these bands, they coated them with proteins to make them thicker and large enough to be discernible by AFM microscopy. And with AFM microscopy, people have been able to observe and ascertain that each of these spots corresponds to a different topology of DNA, different nodes. So there are very different layers of interesting messages that you can see here. One is that you, and I would say the most fundamental one is that you find out that almost all viral particles have unnoted DNA. So the number of viruses that have unnoted DNA inside is less than 5%. Not only that, but if you look at the type of nodes that you have, it's a very special type of distribution that is very unlike the one that you have in random polymers in equilibrium, or as I mentioned before, in your earphone cords, when you walk with them the whole day. So these topological fingerprints are a robust feature. It doesn't really tell you how the geometry was inside, but it is reflective of how DNA was organized inside. And it's very robust. Topology is not geometry, it's very robust. So these are very valuable information that you want to be able to reproduce with the theory and computation. And so the question is, these nodes, what do they tell you about how DNA is organized inside? Well, here I don't want to give you the fine details of a story, I just concentrate on the broad picture so you can understand at least what is possible, what results are known. So the punchline is that if you create a sufficiently detailed model of DNA, then you are able with simulations of the DNA packaging inside the model viral capsid, which we treat as a sphere, you are able to reproduce the node spectrum. And only if you get the correct ingredients, and these ingredients are, first of all, escluded volume interactions, of course, you need to treat your chain as a thick chain. Then you need to take into account that DNA is very stiff on the scale of the viral capsid. The persistence length of DNA is a 15 nanometer. The thickness of DNA is a 2.5 nanometer. The persistence length, I remind you, is the arc length over which you pay one KBT in order to bend your molecule. So imagine your molecule is very thin, 2.5 nanometers, and you have to pay KBT in order to bend it by one radian over a length scale of 15 nanometer, which is much, much longer than the diameter, it's 20 times longer than the diameter. So essentially the DNA behaves almost like a steel, a thin steel cable that is very thin. And despite this, you have to pay a lot of energy in order to bend it over a size comparable to its diameter, really a lot. And so these are two key ingredients, but these are not sufficient. If you want to reproduce the node spectrum that you see in biological samples, then you need to take into account the double-edical nature of DNA, which is such that if you tightly press two DNA strands, one against the other, they don't want to be at a random angle, but because they have the helical grooves and you have the charges of the DNA arranged the helically along the DNA contour, the two contactless strands want to be at a finite angle from each other. If you take into account these organization principles in packaging simulations, then you can match very, very well the distribution of nodes inside viral capsules. Not only you can account for the abundance of nodes, but you also account for the different relative population of nodes. So in this way, you have a validation of how DNA is organized inside. And then by looking at your molecule, you can have an idea of how the DNA was actually inside each of these viral particles. Now, a more fundamental question, however, is this one. Since we know, we know, not from the models, but we know from experiments that most of the viral particles have a noted DNA, the question is, how come that you can afford to have a noted DNA inside and still the DNA is able to be ejected through this very narrow channel, a channel that is narrow enough to allow only one portion of DNA to go through and not several filaments. So the situation is like, imagine that you have a complicated node here, and then I want to eject these through an aural pore. No, I don't know if you can see my camera. The question is, how come that I can get it out that the virus is able to eject it without jamming the pore in this ejection process. So if I try to do it with this rope, I'm not able to get these through my fist because here there is lots of friction that develops and the knot tight ends, I cannot get it out. How come that we know the DNA is noted and still is able to be ejected from the pore? The problem is also relevant for another context, entirely different, a technological one. Nowadays, there are chips that you can buy that will sequence DNA using solid-state nanopore translocation. So the idea is the following. And it goes back to a seminal idea put forward by Deemer, I think at the end of the 80s, but only let's say in the last 10 years, this has become a reality. Imagine that you have a very narrow pore in a slab. It could be a biological pore, but you can do it in a solid-state slab. You do a pore with a very thin diameter, a diameter comparable to the thickness of the molecule that you want to study. Let's say single-stranded DNA, just one filament of DNA. And then because DNA is charged, you put a potential difference across the two sides of your pore and the DNA will be sucked in and will go through. Everything happens in the presence of ions in solution. Now, as the DNA goes through, if your pore is narrow enough, you can measure the current, the ionic current that goes through. The ionic current will be modulated by the obstruction of the pore that you have. Whether the pore is completely filled or it's completely empty or in between, you will have different levels of the current. So by measuring the current, you may have the hope of being able to tell what nucleotide is passing through at the moment. And this is in fact what it can be done. As you can see here, you have as a function of the position of a DNA strand going through the pore, you have the different levels of currents that you can see. And you can see that there are about four different levels of currents and they correspond to the four different nucleotides.  And so you have more than four levels because you measure properties of two or three bases at the same time. But the idea is the same. Like just taking a simple measurement of current without reagents, without amplifications like in PCR. So it's a very cheap technique. You are able to profile the chemical identity of the sequence just by knowing what is the current. Now the issue that crops up also in this case is what happens when the filament that you are fishing is noted somewhere, because it's bound to be if you have long filaments. And then you try to pull it through the pore and it's going to jump the pore presumably. As in the example of this force of translocation that I'm doing here. So we got intrigued by the problem and also its connection with the viral case. And so we wanted to study it with models with the idea of telling experimentalists the following. We wanted to identify the length of DNA stretches that was safe enough to be driven through the pore without getting jumped because of being spontaneously noted. So if it is short enough, it will not be noted with high probability. So what is the safest length that you can measure? And let me give you, since you are mostly physicists, let me give you take this opportunity to give some background, a simple background on the problem of polymer translocation, which is a fascinating problem. Actually, we had a hybrid workshop on that for an entire week last week. It's an extremely active area. So let me give you the basics of the physics because I think it is something that is worth knowing about. The problem is the following. I'm sorry, can I just interrupt you one second and ask a question? So this, maybe you said that name is this nanopore sequencing of DNA. So the advantage with respect to that data already many existing techniques for sequencing is just the fact that you don't need PCR to verify or is there something else? It's very cheap. You don't need reagents. You can do it in parallel. On a little chip, you can have thousands of these little pits that you can draw things through and you can measure uninterrupted stretches of DNA of the length that you want, the length that you can manage, which has been constantly increasing instead of having to chop it up and then put all the pieces back together using a software, which can be a problem in regions that are highly redundant like central nears of chromosomes and so on. Okay, thank you. So coming to the problem of translocation, here what you see, let me show you the phenomenology first and then I'll tell you a little bit about the basics of the theory. This is a chain. It's an unnoted chain. This is a chain of 15,000 bits. So it's a model chain. There are 15,000 bits. So it's very long. It is fully flexible. It is colored from which shades, color shades from one end to the other so that you can see somehow have an idea of the sequence. And it is charged. It is driven through an anopore. The anopore is here. It is driven by an electric field that is applied only here, where my cursor is. There is not an electric field that goes all the way through the simulation cell, only here. Now I let the simulation evolve and then I will play it again. So to point out the physics. You see that now we have a global that is much denser that we started with. This is not because we have a bottom here. You might think, okay, there is a bottom you cannot go beyond. No, it's not the case. Let me start again. So this was on an equilibrated chain. Let me backtrack a little bit. Okay, so this is the equilibrated chain. This was in equilibrium. It has a certain size and the size of the chain scales like the length of the chain to a power that is called the new, which is about 0.6 in three dimensions. I believe my colleague, Angelo Rosa, yesterday, you heard about it when he talked about chromosomes. This is the typical, it's called the metric exponent. But as we are driving it out of equilibrium very fast in the other side, when it goes to the other side, it does not have time to relax. And therefore it is much more compact. Okay, so this is first of all, an out of equilibrium process, which is very interesting. Out of equilibrium stuff make is one of the most active areas of research. But then the thing that you see that is going on here is that you see that there is a longer and longer portion of the chain that gets rectified, gets linear. In fact, what happens is the following, the out of equilibrium disturbance that you are applying here at the port gets propagated through the chain, through the backbone.  And once that is fully rectified, it goes through extremely fast. The interesting thing, so here I'm giving you some sketches that you can use a scaling argument that will tell you that the time that you need to translocate the chain grows with chain length, with a power that is greater than one. So naively, you might expect that if I take a chain that is twice as long, I will require twice as much time to translocate it. No, it's not the case. You require much more than that. It is actually a process that slows progressively down.  So it's a diffusion along one chain but embedded in the three-dimensional space with a certain fractal dimension. So it's a complicated process. And you end up with this mind-boggling result that the translocation time is nonlinear as a function of chain length. This is what theory tells you. And in fact, this is what simulations also tell you you need to be careful in order to be asymptotic but indeed the exponent that you can see is very close to that and definitely is not a linear relationship. So I try to give you a flavor for the interesting physics that goes on in the translocation process but the point that I want to emphasize is that all these theoretical arguments are necessarily for unnoted decent angle chains because there is no way that in theory you can use paper and pencil to come up with expressions that generalize to notes. What happens if you have entanglements there? And this is where simulations come to help. And so I'm going to show you what happens when you translocate a noted chain. So this is a chain. It is noted. The note is in the red region. There are, we have developed algorithms that will identify the noted region for you and if you are not convinced that there is a note what you can do is you can just pull the chain. You can do a simulation and if you pull the chain indeed you find out that there is this type of note which is the so-called Savoy note or figure of eight note in that particular region there. So what happens when you translocate the chain? You can try to take your bet. This is your noted chain or our noted chain and this is its translocation process. It goes through. No problem. Okay. So when I saw this result I was very surprised. First of all, I looked at the numbers in my data files and so I thought something had gone wrong during the simulation. Maybe we had a crossing. We got rid of the note for some reason but no, this is not the case. And in order to convince you of that I'm just showing a close up of the same simulation but taken close to the port now. Now I zoom in on the port. So the note is initially very up above my screen. And as I started the translocation process. Okay. Now the translocation process has started. It goes on. Now you see there is this blob here. This is the note. The note was initially far up and then as we pull the chain because we have this tension that propagates the note of titans gets squashed against the poor entrance. It stays there, stays there, cannot go through. It would like to go through but the poor is too tight to let it through. It stays there but you see what happens the chain is able to slide along the noted contour and pass to the other side. So this was very surprising, very surprising in a way difficult to accept. But then of course, if you was that you have the result so you see I'm completely honest about this. Our initial goal was to tell the experimentalist which lengths were safe to process in order not to incur into notes. And then we found something very serendipitous that even if you have a note, that is not a problem. Why is that? Now that you have the benefit of looking at the structure you understand what is going on. Essentially in the noted region where I was zooming in the beads are pulled against each other but to some extent the chain is not very, very tight yet. So the forces that I was applying were sufficiently strong to tighten the note but this tightness was somehow counterbalanced by thermal fluctuations that opened up the note very frequently. And so the chain had enough slack room to slide along itself. Now that you understand this you may have the idea and this is what we were inspired the next to apply a much higher force. So a much higher force should not allow the noted region to breathe as much under thermal fluctuation. So the beads would be tightly pressed against each other and in fact the note now does not go allow the chain to translocate. Now you see the note is tightened, it stays there would really like to enter and go to the other side but it cannot but now the chain is too tight to breathe because we are pulling almost 10 times stronger than before and now the note is not able to pass because the chain is not able to slide anymore along it's not the contact. So at the end of this process essentially we can recalibrate our intuition from the macroscopic scale where we have lots of friction to the microscopic scale where the friction is less extreme and essentially everything is nicely summarized by this slide here which tells you that even if you have a noted chain if you pull with a low force it goes through no problem almost like an noted chain you pull a little bit with a slightly higher force it goes through faster as any chain would do but then if you start applying higher and higher forces eventually you slow it down until you jam it completely. So the more you want the less you get if you want to remember this result in a pictorial way. And these can be generalized I don't want to get into unnecessary details but this can be generalized holds for different type of notes there are no commutative properties if you have one note after another note the translocation properties depends on the order in which you encounter the notes there are different notes families all these is actually not only a curiosity but it's actually relevant in connection with the problem that we looked at because in the bacteriophages there are different families of notes that occur spontaneously and now we have the insight into wow why it is actually possible to be highly noted and yet being capable for the DNA to be delivered through an aropore that is not large enough to let the note goes through if you compute the pressure holding the DNA inside there and therefore the ejection force you estimated that this force would never be enough to jam the translocation process. So there are various reasons included the fact that notes are under confinement are delocalized and they are not tight but even if they were tight the ejection forces would not be strong enough to jam the chain. So we have resolved in this way a conundrum that had been in the community for a while. So let me check how much time I still have 10 minutes, right? Ali? Yeah, that's correct. Okay, including questions or not? Including questions. Including questions, yeah. Okay, so, okay, I guess just to for the sake of having a complete story maybe I will just talk about complete this part about translocation and not cover the one or synthetic notes. Very recently, there have been some experimental breakthroughs that have allowed the people to use the same setup that I mentioned with very narrow pause to do sequencing and use that instead to detect with unprecedented details the amount of notes that you have. Why is that interesting? Because the technique that I mentioned here the one based on gelatophoresis has some intrinsic limitations. You can use it for DNA stretches of up to 10, 15 kilobase pairs, thousands of bases, not more. The reason is that the longer is the DNA the larger are the electric forces acting upon it. And if you keep increasing the size of DNA eventually the DNA will snap. And so you have to stay below a total contour length if you want to use the technique at all. So people are really blind as to what are the incidents of notes in lambda phage viruses which have a DNA of 50 kilobases for instance. So this technique will not be confirmed. What other techniques are available? Well, zero until about five years ago but very recently about five years ago Kisdeker and coworkers introduced a very elegant technique that uses the same principle of nanopole translocation to detect notes. And so let me tell you how we used COSYR models to complement the insight that you can get from these experiments. First of all, the phenomenology. They use a very wide pore, 10 nanometer in diameter. I told you that DNA is a 2.5 nanometer in diameter. So these are pore much wider than your rope. When the filament goes through, the pore is partially occupied.  Your string of DNA can actually enter the pore in a back-folded way. In this way, it starts by, the current goes down by two notches initially. Then it goes back to one when it is occupied only by one filament and then back to the baseline level when it's done passing through. And then you have signatures like these, which I ask you to think about it for two seconds and try to explain. We start from the baseline level, go down by one notch. So there is one filament inside. And then suddenly we go down by two notches, go back to one and then to zero. Or same situation here. You start to go down by two notches immediately. So this might be a situation like this one here. And then suddenly after having gone back to one strand, you go immediately down to occupation of three strands. So go back by two notches and then what? What is happening after the blue? And the only way to make sense of these signatures is to assume that you have a knot there. And these signatures you find in linear DNA and you find in circular DNA where knots are permanently trapped. So you cannot really see the knot, but it's the only way to make sense of such signals. And this technique allow you to answer a number of very interesting question. Well, first of all, you do counting. You can see how many of these instances you have, which allows you to count how many knots you have, the abundance of knots that you can get also from generator freezes. But then you have other information like when I had the signal of a knot passing through, when was that occurring at the beginning of the process in the middle, at the end, uniformly in between because the knots are expected to be random. And also what is the duration of these signal that you have the knot passing through? Is it brief? Is it wide? Is it a tight knot? Is it a broader knot? So these questions are all questions that you can answer, at least in principle with this technique. Well, first of all, regarding the first one, measure the incidence of knots. So this is what they were able to measure. They measure the spontaneous incidence of knots in DNA of up to 1,666 kilobase pairs, sorry. And with generator freezes, you are stuck at 10 kilobase pairs. So you are about here. So that's an order of magnitude improvement. And I don't think that is really an upper limit so you can go beyond. And then there's also some interesting features like the statistics of when you see the signals due to the knot is skewed towards the late passage, late at the end of the process. And then most of the passage events are very short, very short to the point that if you estimate the knot size, that would be tens of nanometers. So that's very tight because the persistence length of DNA is 15 nanometer. That means your knot is much smaller than the persistence length. So it's very, very tight. We tried to, am I on time? Ali, I don't know if I heard. Yes, you're fine, you're fine. Okay, so I'll try to wrap up in a couple of minutes. So we use a model, so that is quite detailed. So a model that accounts for the double medical nature of DNA and also for the possibility for the strands to open up. Because we assume that if you have a topological entanglement doesn't may actually snap or cause a bubble in the DNA to open. So we didn't want to miss that. It didn't come up to be an issue at all. So this is a model of the double strand DNA. So it looks like a filament, but it's actually the whole double helix there with the two filaments. The knotted region is red. You can see what happens. It tightens a lot before entering. So it makes a few attempts to enter the pore, and eventually it goes through and when it goes to the other side, it opens up. So let me show it again, because it clarifies a few things that were not clear from experiments. This is a knot in equilibrium. By the time that it reaches the pore and then you measure it, the equilibrium properties of the knot are gone. So what you measure, unlike the expectations that experimentally said, is not indicative of what the knot was in equilibrium. It's much tighter. There is no weight. So by measuring it, you disturb it. So it's not a delicate procedure. And then while experimentalists use currents, we can measure the occupation of the volume, which is about the same thing. So we can measure when the events occur, what is their duration, and what we... Okay. And experiments saw a skew towards the late passage events. We see the same skew, and this is because the propagation of the tension front always tightens the knot before reaching the pore at the essential crossing that is the furthest from the pore entrance. So even if you start with knots that are randomly distributed on the chain, by tightening it, you're pushing it away from the pore effectively. So this is what causes the late passage events. So this is something that you can understand in simulations that was not clear with experiments. Not only that, but what about the size of the knots? Well, we find out that about in half of the cases, the knots were not tight at all. The knots were actually occupying the whole region in the sea side, so the before translocation side. And what was tight was not the knot, but only the tight gathering of what are called the essential crossings. So you see, this is a situation that is very different from when the knot is only on one of the two sides of the filament. Here the knot really sits across the two in a very dramatic way. So again, this is indicative of the fact that if you measure the signal, that is cannot really be by itself revealing or what is going on behind the curtains. So this is essentially a push to design, if possible, setups suitable pores of suitable size and geometry, suitable applied forces in order to be able to distinguish properties that otherwise are all lumped together. Okay, so I don't have time to show you the molecular knots. So let me just go to the acknowledgments and acknowledge the people that have been involved in these studies. And I will be happy to take questions. Thank you. Thanks very much, Christian. I believe there's a question already in the chat, Christian. Okay. So, Patricio Barletta, if you increase the temperature, you are able to pull harder. That was back then when you mentioned, hi, hello, thanks for the very interesting talk. That was back then when you mentioned that it was random fluctuations that allowed the knot to relax. Yeah, yes. I would say, in brief, I would say, yes. Maybe I would phrase the answer, a longer answer in a slightly different way, but I think you got the right idea. Essentially, everything is controlled by a dimensional ratio that has to do, that involves the force and the time scale, which is essentially knot side divided by KBT. And so it is this ratio here that determines whether the knot is able to breathe and therefore you are able to get it through, or if it is completely stuck. Right. And in the case where you put experimentals that we're trying to solve, please, in the case where you are in control of the force field, what if you change it back, you switch it when it hits the knot, you switch it back, you let it go up, and then you pull harder if you do that. Okay. This is an interesting question. I think there are groups that have looked at these intermittent mode of pulling, if I understand. So you say, apply a force, zero, apply a force zero. So instead of having a constant force, you do a square wave modulation, let's say. Right. Sinus order. So there are groups that have done that. We have also done it recently, but I think the first group that did it is, Shinchuk, I think, and they saw that that allows you to use, so if you use impulse, that allows you to get the knot through, even using forces that applied at a constant level would jump the knot. Because essentially, when you switch it off, you are precisely giving the opportunity for the knot to breathe a little bit. And so the next time that you pull, if you gave it sufficient time, it would have opened up and you gain one inch and then again and then again. Thank you. And I was wondering, you mentioned, are these knots present in just bacterial phase? No, no, no. Of course not. I think definitely they are present throughout all genomes. And the, so I think bacterial phages, maybe are not even the first system where they were detected in the first place. They are reported in bacterial DNA. So in bacteria, those have been studied really a lot. And now recently, people have been able to devise the technique to detect them in eukaryotes. So I think our knowledge of the abundance of knots in genomic DNA is just limited by the technological difficulties of accessing them. So the same group of Yakim Roka has done beautiful experiments. They, two years ago or three years ago, they in 2018, you look at a couple of nucleic acid research papers, they measured the occurrence of knots in mini chromosomes of the east. So which is eukaryotic DNA with all the nucleosomes, et cetera. Thank you. Yeah, maybe if you can answer the next question. Right, okay. So it is very surprising that not can be resolved even under law force. I would say because of the law force, I missed the sound. I cannot imagine how it comes in energy or entropy barrier, the long DNA. Not sure I understand, but anyway, so the idea is that the reason, I hope it was clear from the video, the knot was not resolved. So it's not that we got rid of the knot. The chain was able to slide along its knotted contour, which is different. And the reason why it was able to slide is because friction at a macroscopic level is not as strong as you see in this piece of rope that I'm not able to get to sliding anymore at a certain stage, you know? And so the friction depends on how tightly your molecule, in this case, our beads, which I remind you, the beads are not there as physical beads. The beads enter through a spherical, symmetric potential centered on the center of the beads. So if your beads have wiggle room because of thermal fluctuations, imagine a bead that has to slide along the contour. It will feel a bumpy ride because it gets in the way of a very scalloped potential, no? Festune the potential due to the interaction of the other beads. But if your fluctuations are larger than the barriers in this washboard potential, it will have some friction, but it will still go through. If you pull very tight, you increase the barrier so much that even if you apply a slope, if you're driving, you're not able to get it beyond the barriers. I hope it's clear enough. Oh yeah, thank you, it's clear. So the sequence slide through the knot, instead, the knot was disappeared before it passed through the... Right, the knot was there, it didn't get unnoted. It was translocating despite being noted. Okay, yes, thank you. Okay, thank you very much. And I guess Christian will be at the gather to this afternoon. We don't leave one caveat. So I've got a commission for an examination at 2 p.m. So as soon as we finish, which might take up to one hour, as soon as we finish, I will join you on gather. So I will not be there immediately. That's for an institutional duty.