So the main question of universality is about how to get rid of the Gaussian assumption on the matrix element. So you might like to have a theorem, which says that you start with a Wigner matrix, no matter what the distribution of the matrix elements are. This local statistics of the eigenvalues, so you take the eigenvalue, to the endpoint function, the joint distribution of the eigenvalues, eigenvalues, you take its k point marginal, you take its rescaling as we did before, and no matter what the origin and distribution was, you always should get the same Dyson sign kernel. So that's the famous Wigner-Dyson meta universality, and it has been recently solved over several years in a strategy, which we call the three-step strategy, which I will explain in bit more details. And this was done in for both symmetry classes, the real symmetry and the compression case. There have been related results, the first result, which went beyond the Gaussian calculation was Johansson quite early. So it was Johansson who did the Hermitian case, so Hermitian case refers to the complex Hermitian case, but with a large Gaussian component. So in the original calculation of Dyson meta Godin dealt with the Gaussian case, now Johansson could generalize it to the situation when the matrix elements are not Gaussian, but they still have a big Gaussian component, a large sizable Gaussian component, so you can think of it as a convolution of an arbitrary distribution with an independent Gaussian distribution. But this method works only for the complex Hermitian case, because it uses the Harris-Chandra formula. And then there was an independent result by Tau and Wu, which was done by the moment matching, the form moment theorem, which in the original version was also on the Hermitian case, because it went back to Johansson's result. And there have been similar developments for many, many other models. Here I will talk about bulk universality, but there have been results on the edge, then there are results also about the beetle of gases, which are a different type of ensemble. It's even not necessarily a matrix ensemble. And then there are also other types of matrix ensembles, for example, sample covariance matrices, this is exactly what Vishard invented, so it predates the Wigner matrices, there are sparse matrices, sparse graphs, Heldesheini graphs, regular graphs, and so on. So there is a huge zoo of possible models and also possible questions. I will just focus on the simplest situation. Ok, so let me just explain you this three-step strategy on one side, and then we will go more into details later, just to have an idea. So the first step in this three-step strategy is this local density law, the local semicircle law, which I already emphasized, mentioned before. So the local semicircle law, basically local density law, we talk about density law, when it's not the semicircle, we will see examples of that. So the local semicircle law, local density law cels, the following, you take an interval a, b, you always take it somewhere in the spectrum, and then you would like to know how many eigenvalues, what is the density of the eigenvalues in this interval, you take 1 over n times the number of eigenvalues in this interval. It's a very mature question. Now you divide by 1 over n, because in this interval, in this interval of typical size order 1, you expect order n eigenvalues, and then the Wigner semicircle in the simplest case says that as n goes to infinity, this goes to the integral of the semicircle density, so this semicircle functions as the square root of 4x squared function. So this just tells you that the number of eigenvalues in this interval divided by n converges to the integral of the semicircle in the corresponding slice. It's the area of this regime about the interval a, b. So that's a very natural statement. Here notice that this is of the type of a low-flage number result, because the left-hand side is still random. The number of eigenvalues in an interval is a random metric, and the number may fluctuate and thus fluctuate a little bit, but it doesn't fluctuate too much as this theorem says, because once you divide by 1 over n, then you get a deterministic number. So that's a low-flage number type result. Now, this holds the original Wigner semicircle holds when a and b is fixed. So fixed means that n independent, and of course that's sufficient to conclude that the local empirical density weakly converges to the semicircle low. Now, the local low here and the local density down to a scale 1 over n ask the same question, so local low, ask the same question if a and b is not fixed, but if a and b shrinks with n, so a and b may go to, let's write it in this way, b minus a smaller than 1 over n, much smaller than n. So for example, this is some power n to the minus, n to the minus a. There is a better letter. n to the minus c. OK? So now you ask the question when you shrink this interval, and of course when you shrink the interval then you may see more fluctuations. The right question to ask of course is not exactly this normalization because the number of eigenvalues will shrink as the size of the interval shrinks. So let me write it in the following way. Let me also divide it by the b minus a as long as the a and b is fixed whether I divide or not it doesn't make much difference but when b minus a starts going to 0 as n goes to infinity then it's better to take this normalization because in that way this integral here still goes to an order 1 quantity and it just goes to the semicircle at that point and then this is also the right normalization on that side to make sure that this quantity is order 1. OK? So we look at the question whether this limit holds if b minus a is shrinking that's smaller than order 1 and the question is that what is the smaller scale on which you can still hope for such a result. You still hope for a result that the number of eigenvalues in this interval appropriately normalized you got something which is deterministic. Now you can easily convince yourself that you cannot go too far below because you cannot take a too small interval because if this interval is so small if the size of the interval is 1 over n or smaller then you are on the scale of the individual eigenvalues and then if you take a very, very tiny interval here b like that an interval which is whose size is comparable with the eigenvalues and sometimes you may have an eigenvalue falling into an interval sometimes you don't and this quantity sometimes is 0 sometimes it's 1 so there is no deterministic limit so you cannot go you cannot go with c above 1 you cannot have c equal to 1 or above but the hope is that this is true stay holds for any c smaller than 1 so it means that you can go down to scales which is a little bit bigger than 1 over n and that's what we call the local law ok, so that's the explanation for this sentence now precisely how much what it means a little bit about 1 over n this depends a little bit how careful you want to do the want to do the work for all these talks I will just talk about an n power so I will take an n power which is strictly smaller than 1 so n to the minus 1 plus epsilon there have been results which tried to improve that so there are results which talk about scales of log n to some power over n and so on so there are improvements in that direction but I'm not going to talk about that ok, so that's the local law now let me mention immediately the local law comes in a stronger way to be useful here I just presented the local law in such a way that what is the eigenvalue distribution but actually it comes together with the resolvent in a minute but when we talk about local law in general we will also mean an entrivized form of the resolvent just put it here, the resolvent I will always use the notation g of z for the 1 over h minus z where z is a spectral parameter it's in the upper half plane so the upper half plane is always denoted by h and the z will be denoted by e plus i eta and eta is positive we always under in the upper half plane we take that branch of the resolvent ok, so this is the resolvent and the first question what you may want to know about the resolvent is this normalized stress I'll just try to put it up here so it's normalized stress is the 1 over n times lambda i minus z summing up for all eigenvalues and especially we would like to understand this for small for small eta the resolvent is a quantity which gives you lots of information about the matrix it's a very commonly used object not just for random matrices also for operator theory and the critical question when you talk about the resolvent is always the imaginary part of the spectral parameter the smaller the eta you can understand the resolvent the better resolution you get about the spectrum of the corresponding operator so in particular if you are looking for a local law you want to understand the resolvent you want to understand the distribution of the eigenvalues when you better get the resolvent under control on a small scale namely the scale on which you can understand the scale eta on which you can understand the resolvent will be basically the same scale as the scale on which you will be able to conclude the local law we will explain more details in precise formulas but here I just wanted to make a remark that typically when we talk about local laws in this setup you may think that you are interested only in the resolvent in the trace of the resolvent but actually we are also interested in matrix elements of the resolvent we also need need to understand the matrix element gij of z and when we talk about local laws then we also mean it in the stronger sense of course if you understand the matrix elements then the trace is understood already but the other way around it's not at all clear the trace has an extra averaging over the diagonal elements and in principle trace contains much less information ok, so this is number one this is the local density now the local density is still on the scale above 1 over n and it's still a low flash number type result so it doesn't tell you anything about local fluctuations eventually if you want to understand this sine kernel then you have to go to executive scale 1 over n nevertheless within this approach, within this three step strategy we found that even though the local density itself doesn't answer to the question the local doesn't answer to the question immediately it serves a very useful and very important, a priority bound for the other two steps and the other two steps is that the disembranding emotion which is when you do the following you take your Wigner matrix and you say that you don't study the Wigner matrix immediately but rather you add that tiny Gaussian component to that and then you study this new matrix the original Wigner matrix plus a Gaussian component a little bit similar in the spirit of Johansson and then you start you try to understand that now of course that's not the original model that's not the original matrix because you added a Gaussian component and the original philosophy was that you didn't want to do any Gaussian but nevertheless you do that it's a small Gaussian component and then you use the disembranding emotion which is a stochastic a stochastic differential equation to show that with the new matrix with the original matrix plus a tiny Gaussian component does have the right local statistics that's step number two and that's a separate theory and then there's a step number three when you have to go back because you wanted to prove something about the Wigner matrix with arbitrary distribution and you could prove something about the Wigner matrix plus a tiny Gaussian plus a tiny Gaussian component okay good but that's not the original thing so now you have to go back but it turns out that this you can do by perturbation but by some perturbation theory it's not completely trivial but it's in spirit it's a perturbation theoretical argument when you can remove this tiny Gaussian component it's a little bit funny because it sounds like that you added this tiny Gaussian component to get the universality but then you remove it nevertheless this is the way to go okay so now by now the step number two and three is considered almost quite standard because there are very, very general theorems which are available to express the power of these two methods of these two steps so basically there are theorems which you can take from the shelf which says that if certain conditions hold then there is a machinery and the machinery immediately gives you both the step number two and step number three and the input of this machinery is always some version of the local law some version is that some control on the trace of the resolvent here or even some control on the matrix elements actually usually you need both for the step number two you need only the trace of the resolvent but for the step number three you also need some information about the individual matrix element so the step number two and step number three these are very abstract very general results by nowadays which do not depend on the model at all they depend only on the input and the input is always some kind of local local law and only the local law which is model dependent so now the theory looks like that that if you give me a random matrix model under certain conditions under certain setup then you have to work for the step one for the local law and that depends on the model and you need some different methods to do that but after that if you got the local law then you just apply this machinery and you get universality so I will mostly focus on the local law here so here are models of increasing complexity which I am going to discuss in these lectures and here I am trying to come up with more and more general models according to this Vigners philosophy Vigners philosophy was that he used this Vigners matrix only to model something which was much more complicated we will talk about physics next time he certainly he knew that Vigners matrices are just caricatures and in order to believe more and more in this Vigners thesis it is better to extend the scope of all the possible models of all the models for which you actually can prove universality it still will not cover the original model of Vigners but at least it justifies if you study the problem if you prove universality for a bigger bigger class of models then it justifies more and more that Vigners vision was correct so this is the spirit in which we are we are constructing we are looking for more general models so the first model is the Vigners matrix which I already introduced is characterized by i, i, d entries and in particular the variance matrix which I will introduce here this I will call s, i, j this is chance there expectation of h, i, j square in the Vigners case the variance matrix is very simple each matrix element is the same is just constant it is 1 over n and now in that case what we are interested in is we are interested in the density ok, this I already explained but we are also interested in the matrix elements of the of the resolvent or even in general the structure of the resolvent so in the Vigners case you have the density which is semicircle the g, the resolvent is a sensory diagonal not exactly diagonal but a sensory diagonal which is very small sensory zero and then the diagonal elements are non-zero and moreover the diagonal elements are comparable, almost the same so that is the structure of the Vigners matrix and its resolvent now the next category is the generalized Vigners matrix which we introduced a few years ago here we keep the independent entries we drop the condition that the variances are the same which is this condition that the sum of the variances in each row is constant is the same for every row which for convenience you can choose to be 1 so these are the deviates from the completely mean field model when every matrix elements are identical but still kept this property that the row sum the variances is the same for every row and in that case the picture is basically the same you don't get any new feature the density is still a semi-circle the resolvent is diagonal and then the diagonal matrix elements are the same the next class is the Vigners pipe matrix which comes with still independent entries but now we drop any condition on the variances so the variance matrix is an arbitrary n by n matrix and then we study this model and in that case you see that the density is not semi-circle anymore it heavily depends on the matrix S is determined by this matrix S Salon, yeah? Do you need a lower bound than anything? Yeah, I mean of course I mean in order to have precise results sometimes you need lower bounds that's right, yes but you know this is I'm just giving you a no value for the moment there are no precise CRMCR okay so now for the Vigner the density is determined by this by this matrix S the second moments it's not a semi-circle but the G is still diagonal but the diagonal matrix elements are not the same in general so the matrix starts having a non-trivial structure and then the last model is what we call the correlated linear matrices we drop the condition of independent entries we replace it by non-trivial structure in that case of course the Sijj is not sufficient to describe the second moment in that case you have to replace the Sijj by all possible covariances so it becomes a fourth tensor which describes all possible covariances of the all possible correlations between matrix elements and in that situation even the G is not diagonal anymore simply you have a correlation between matrix elements and this correlation induces a non-trivial decay of the of the resolvent okay so these are sort of the four models of increasing generative which we are going to discuss and then there are many many other extensions of the original Vigner model which I'm not going to discuss so let me just flash it up for a moment the idea is again that you try to verify Vigner's vision in a way that you try to extend its scope and you try to come up with more and more general models in various directions so there are many many different directions then one can do it okay so now let me do let me give you an overview of the results there will be three types of results the first result is about not about random matrix itself it's about the density of states it's about what replaces the semicircle in the general situation and especially it's singularity structure so in other words how do this picture, how do the semicircle how do the replacement of the semicircle looks like if it's not the semicircle anymore because the number one the number two is what I already explained the local low, we would like to understand the g the resolvent and especially we are looking for deterministic quantity which describes the random resolvent with a high precision so in the case of the semicircle this is just the stirtjastrans from the semicircle which I will define in a minute but in a more general situation when the resolvent has a non-trivial structure its diagonal elements are different or even there are non-trivial off diagonal elements then we will altogether need a matrix a deterministic quantity which describes this fluctuating random object the resolvent so these are the local lows and the third type of results are universal which I already explained before so let me give you a few pictures those who are used to semicircle now you will see some non-semicircle lows so this is still the semicircle that's what you get if you have a generalized Wigner matrix situation there are some of the variances in the rows of the variance matrix is constant the next picture is when it's not what we call the Wigner type matrix some of the variances in the row is not constant anymore so here is a variance profile but you see the matrix with color codes in the matrix S the variance matrix the color codes indicate the different sizes of the variances we just came up with some ad hoc picture variances and then here is the density of states so definitely not the semicircle it's still symmetric the density of states is symmetric but it's an arbitrary picture and now here are some further pictures these are various pictures of density of states for Wigner type matrix here you see the variance matrix of the Wigner type matrix so I mean this is here we typically chose a block matrix when this variance the S matrix has 2x2 block structure and in these places there are different numbers and if you change these numbers then the variance profile then the density profile changes so here we model the situation when the density splits from having one interval one interval support into three interval supports by changing one of the parameters along this model so especially at the moment when the interval is split then you see a special casp singularity emerging so in particular you would like to understand what kind of curves what kind of pictures may emerge and the picture and the answer will be very roughly speaking that the curves what you see here these are analytic, real analytic curves apart from the singularities apart from the points when it comes to zero so in particular this point here is also a real analytic point it doesn't look so but it's a very very small but real analytic part but then when it comes down to the real axis then it develops real true singularities and the type of singularities square root singularity at the edge at this edge also in this internal edge it doesn't look so but these are all square root singularities like a semi-circular but then when you are exact at the casp then it's the one in the cubic root singularity now there is another feature this one feature which we investigate there is another feature you may ask yourself what determines what triggers the splitting of the interval splitting of the support into various intervals and after some investigations we found that actually what triggers this splitting is the discontinuity in the S-matrix so here I chose an S-matrix which I know there are some numbers 0.10, 0.2, 0.2 and 1 for example and because within this S-matrix there are discontinuities when you go from this point to that point the value of the variance jumps this is the reason which allows you to have different support if you smooth it out in such a way that you take this profile of the S-matrix in such a way that there is a little transition layer between 0.2 and 1 so you go continuously even a tiny layer like this red layer because then suddenly you restore the single interval support so then the picture is like that ok, so these are certain qualitative questions which we are looking for the third type of question that which I already mentioned but there are explicit formulas to express that so the singularities happen only when the density goes down to 0 and there are two fundamental type of singularities the square root h singularity and the cusp singularity but also we can describe very precisely forming so what happens when you are right before the cusp and what happens a little bit after the cusp so in one case you have a smoothed out cusp you don't see here but there is no singularity at that point it smoothed it out and then here you see a tiny when two square root singularities meet at a very tiny gap and we have explicit formulas for that and all these formulas look horrible but these are also universal formulas because they are basically universal singularity patterns which arise and also only these two types of singularities square root and cubic root singularities can occur plus their smooth version ok so now here are the main results about the locales and universality it's still a little bit informal so the first result is about the Wigner type matrices we look at the Hermitian matrix Wigner type with a general variance profile and here now I already put in the proper assumption which was mentioned over that we assume that the singularity and the variances are comparable with one over n so there is an upper and lower bound a matching lower bound and then the optimal local holds optimal means in this sense that the local holds for any c up to any c smaller than one and also the bulk universality holds and then the more recent result is about the correlated case so let me formulate it more precisely so by correlated matrices we mean the following we take a deterministic matrix which you can think of as the expected value of the h or earlier we didn't include any expected expectation we always assume that the Wigner matrix has centered matrix element but you don't have to do that you can also include an expectation deterministic matrix here plus you take a random matrix which has now zero expectation because I already subtracted or wrote up the expectation separately and I put in the one over square root of n scaling explicitly that we made this W the matrix element of W to be order 1 and now we assume a polynomial decay of correlation for these W's so W's you should think of it as a random field so you have the W ij in the matrix element and by a matrix and the matrix element you can think of it as a random field and now if you have a domain A within the matrix then you can ask you can look at the matrix element you can look at the random variable W a which depends the collection of the random matrix element which are indexed by this set A and then you can take any function of that you can do the same thing for any function of another set B and then you can ask yourself how much the random variables here and the random variables here are correlated what is that covariance and then the way to describe it is that you take any function of these random variables phi and psi with some nice properties some smooth functions for example how this correlation decays in terms of the distance between A and B and that's how you describe correlation decay and the answer and the condition what we need that the distance decays is a polynomial decay and currently we can do power 12 which is not yet optimal but this is already a polynomial decay yes this A and D they are not the same that's right very good thanks yes the global density look typical pictures of global density sure of course yes everything is in order the scaling is done in such a way this one over square root of N guarantees that everything is order one but the picture is very different and also in that case the local I didn't formulate it yet it will come next time but in that case as I said it's the whole resolvent is a non-trivial object it's not just a trace it is a matrix a non-trivial matrix and then there is another condition which is sort of the condition the analog of the lower bound of s i j which we need and then the optimal local and bulk university holds optimal local means which I haven't specified yet it means that we can describe how the g resolvent looks like it is a deterministic matrix in which the resolvent approximates so here is a picture which I basically drew here


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Ableton on Air has been seen in the following publications, Park Chester Times, WWW, this is the Bronx.com, New York Power Online Newspaper, Muslim Community Report, WWW.H.com and the Montpelier Bridge. Ableton on Air is part of the following organizations, the National Academy of Television Arts and Sciences, Boston, New England Chapter and the Society of Professional Journalists. On this episode, we will focus on nutrition and nutrition planning, but before we do that, we will thank our sponsors, Washington County Mental Health, Green Mountain Support Services, the partnership with the Division for the Blind and Visually Impaired and the Association for the Blind and Visually Impaired and many, many, many others. With us to discuss nutrition and the importance of eating well and other things around that, we would like to welcome Shaquai Musa Jame, Imam of the Bronx, as well as one of the people in charge of the Muslim Media Corporation. Welcome to Ableton on Air, Shaquai. Thank you, Larry so much. Thank you for having me one more time. Today we're here to talk about health and wellness, and Friday, April 4th is the beginning of the National Minority Health Month, so this is the perfect time to start the month on your soul. Okay, so let's talk about lifestyle, lifespan and everything around that and the reason for healthy eating, because the Bronx or parts of New York have serious problems with obesity. Why? So it's a two-part question. What is lifestyle, lifespan, and why does New York have, or big cities, let's say, have problems with obesity? Thank you for the question. Lifestyle, lifespan campaign was launched about four years ago because, Larry, you're from the Bronx. We have over 40 hospitals and community health centers. We spent billions of dollars, yet consistently, Bronx has been ranking at the bottom when it comes to health and wellness in the state. Actually, we have been 60 seconds out of 62 counties when it comes to health. Bronx is part of New York City, the wealthiest city in the nation, the city that has more billionaires and multimillionaires than any other city in the nation, yet Bronx sites are not benefiting from the prestigious living conditions of these wealthy and well-connected New Yorkers. So four years ago, we said, if we have the hospitals, we have the health centers, we spend a lot of money and we still rank at the bottom, then there's only one solution to it, which is to change our lifestyle. And that is why we said, let's launch a campaign where we promote an overall health and wellness lifestyle so that we don't want you to go to the gym seven days a week at the same time, or use too much alcohol or smoke too much cigarette or eat so much junk. We want individuals to completely transform their lifestyle from unhealthy into healthy lifestyle. And then that will give you perhaps the ability to lengthen your longevity because your lifestyle determines how long you live and the quality of life that you live. That in a nutshell was the reason why the campaign was launched four years ago. Lifestyle, lifespan. Well, the problem with New York especially, I don't know about now, but years ago, like certain fast food chains, which we won't mention on this program, but if you have a fast food chain on every corner having a greasy burger, french fries and whatnot, you're going to have a problem. But most of these food chains now are having healthy options because they have no choice. So either you can get a salad, you can get something with fruit, or at this fast food chain. But is eating at a fast food restaurant a healthy option really healthy eating? Yes or no? Absolutely not. Actually, I call it fast food, fast death. The reason why... Fast food, fast food, fast death? Yes. Okay, go ahead. I'm sorry. Yeah. Yes. The reason why we are dealing with the pandemic before the pandemic, the pandemic of obesity, the pandemic of high blood pressure, the pandemic of diabetes, the pandemic of stroke, you name it, is because we are consuming junk food. Can you define junk food? Junk food is any food that is not natural. Processed food, unhealthy food, fast food, all these chips, these are all food that are not meant for our health and wellness. It is processed, it is designed to maximize profit, convenience, cheap, quick and everything. But what we're promoting is a whole plant-based diet.  Because our bodies are custom designed for what nature produce, not what manufacture process. There's two cuisines, two main cuisines. I'm going to actually three cuisines.  The Mediterranean diet, because I have family in Israel. The Asian diet, in terms of lots of fish, vegetable dumplings, that kind of thing. There's hardly any fast food type of things in Japan unless you're going to go to a Japanese McDonald's. Or also the Korean diet, with a lot of soups and ramen and noodle soups and that kind of thing. Do you have any, are there any other countries that are following a good diet? Because obviously America really has a problem with feeding people right. If you are living or having food-sustainable issues and living in a poor area, you don't get good food, yes or no? No, well actually I would say it all depends. We live in the Bronx. Bronx happened to be home to the largest produce market in the Northeast. Yet we still call Bronx food desert. That means... Define food desert. Yeah exactly, healthy food desert. That means we are not doing things right. So what the campaign that we launch is doing is to making sure that we put priorities in order. Number one priority is health and wellness. Because let me tell you one thing. The best, most effective and the least expensive healthcare is healthy lifestyle. Bad diet, unhealthy lifestyle are not sustainable. It costs billions of dollars. It's taking our lives away. It deteriorates the quality of our lives. And at the end there is absolutely no benefit, zero benefit. Now number two, you mentioned these different diets including Mediterranean diet. Very healthy, very nice, but at the same time they're not equal to whole food plant based. What I'm advocating to the best of my ability is for people to be introduced to the whole food plant based. So explain that. That we believe can only be gotten from animals. Animals got their protein from the plant. So we're promoting plant based diet. Okay, now years ago, in my fact we can show a clip in a minute and you know which I'll roll to it and we'll put it in editing. There used to be a, well there was a movie called, and it was two parts, it was called Super Size Me. Where it followed a gentleman, this documentary, where the gentleman ate fast food for an entire week from a fast food restaurant. And he gained weight, he felt sluggish, that kind of thing. Before we show the clip, why is it that when people eat fast food, hamburgers, greasy food, they feel that sluggishness. But yet when you eat raw plant based things, like a salad or you know, you have energy. Larry, our body is just like a car. A car, you have a place where you put water. You have a place where you put oil. You have a place where you put other liquid. The problem we have here is our bodies are naturally designed to consume clean water and nature produced food. But unfortunately, because of the commercialization of everything, you want it fast, quick and cheap. Now the fast food industry has now taken over. Our bodies, so where we should put water and healthy food, we put processed food and alcohol and soda and sugary drink. So there's no differentiation. With that, we can never ever maintain premium or criminal health condition because we're putting wrong item in wrong places within our bodies. And another thing as far as the sugar content, certain countries like Israel, for example, their version, Israel has a very sugary version, more sugary than America because they use cane sugar. So the more pure sugar you use, the worse it becomes. They have a version. Israel and some other countries have a version of Hawaiian punch, very sugary. I mean, also the more sugary a drink is or the more caloric, the more calories something is, also kids or adults can have learning disabilities from that. ADD, ADHD, like a jumping bean, if you understand what I'm saying. So what's one or how can your agency or your campaign work with these fast food companies to make things more healthier? Is there a way? Yes. You see, the problem is the process. Let what you take whatever whether it is a plant or whether it is a tea or whether it is liquid is the process that destroys the purity of the product. We're talking about white tea, green tea, yellow tea, red tea, whatever. They all come from the same plant, but the process, the fermentation is what changes the flavor, the color and the benefit. Sometimes, you know, make it stronger or lighter, same thing with food. Now you have all these, you know, organizations and companies that are now, you know, trying to circumvent and come up with these products. Now, the goal is noble, but the problem will always be with the process. How much, you know, fermentation and define the fine fermentation, what is fermentation work that will destroy the quality of that particular product. For that, we said stick to the original. For example, you have teabags. If you go to China, you say teabags, a lot of the Chinese people will not touch teabags because they said the teabags itself will contain chemicals. We will drink tea directly from the tea leaf. And that is what we're promoting. We're promoting whole food diet to the best of our ability. No process. But a teabag, isn't it just tea in a bag? So teabag has been processed? No, the bag itself should not be part of what people should boil and drink. So that bag goes to the manufacturing processing in order to be a teabag. So, you know, the Chinese tea expert said you should drink tea directly from the leaf to your mouth. Do not have any, no matter how light it is. If you have a bag, then you destroy, you know, its purity. It's the same thing. It's the same thing, given an example. Oats, you know, not, okay, not the box oatmeal. I'm not talking about, like, the packages.  You can still make cereal from that in its raw form. Example, another thing that people are eating now, hemp, the plant, okay? No, I'm not talking about CBDs or anything. I'm talking about, like, the hemp plant. So it, correct me, so it has to come directly from the plant itself without going to the process itself? Correct? You know, Larry, most plants, most plants are custom made for our bodies. Even when you talk about hemp, same thing you're talking about grapefruits. The same grapes that produce wine also produce grape juice. I come from Africa. You know, the palm tree, we create so much type of product out of palm tree, including wine, all these things. So it is always the process, the mechanism in which you can transform a product into something else. That is where the devil lies. And to avoid those pitfalls, we said stick to the original product to the best of your ability. Okay, so tell us more about the campaign with lifestyle, lifespan.  Go ahead. Absolutely. The campaign, unlike almost any other campaign in the Bronx, was designed to completely seal individuals from the pitfalls of certain aspects that can affect our lives. For example, we devised a seven pillar of the campaign, including finance. You see, we have financial literacy problem. A lot of people can even balance their checkbooks. We have profession. You have a lot of people who think that they must get this or get that in order to have a good job. And then family, you have a lot of people in the Bronx who do not have both father and mother at home. This is a single family household. And then you also have, let's say, spirituality. Almost everybody is part of a religion. But how much do they know the real, real value of the spiritual affiliation that they are part of? And then we're talking about relationships. And again, we're losing our children from junior high in terms of premedital sex, drug, you know, gang problems. So what we designed is a seven pillar, all, you know, a gamut of healthy lifestyle so that once a person finishes the seven months training, that person has the very basic components of a successful life. We're not saying eat this, don't eat that. We're saying we're going to overhaul everything about your life into a healthier lifestyle. And these are the seven pillars that will transform you from wherever you are into a better living condition. And that's what the whole campaign is about. Now, some people may want to eat raw food. Some people may not. Some people may want to exercise. Some people may just want to walk. We don't determine what you do and how you do it. As long as you do it within the seven pillars, we're fine with it. Now, there was a TV program called My 600 Pound Life where it's on Lifetime TV where they follow people that are 600 or more pounds. I don't know if you've seen clips of that and they go to a doctor and they're horrible. They're eating all this processed food and some of them have passed away from having a large frame. Being overweight is never a good thing. Now, you said that the Bronx has the largest produce market. When did that happen and why did it take so long for people to kind of wake up and want better eating? You know, I would say 90% of it is ignorance. You see, the problem we have here is that we are ignorant about large things that are beneficial to our health and wellness. The produce market that we have in the Bronx is a combination of what was there and the Fulton Fish Market that was moved from the financial district into the Bronx. Now it is the largest in the Northeast Bronx. Let's say my 600 pound program. We unfortunately commercialized every single thing about our life to our detriment. We glamorized heavy stuff, big and tall, all these things. So, and this is all to generate viewership to market this and that, but I promise you one thing, there is no overweight benefit.  Because whatever we should not be overweight. I'm sorry to piggyback off that. Years ago, we were talking about like 17, 1800s, like the Mona Lisa or if they paint somebody that is overweight. Years ago, if you were considered overweight, you were considered rich, right? If you understand what I mean. So has people's perceptions changed? Yes or no? No, yes. Because being overweight is not good. No, no, no. Larry, I am an African. In some African cultures, women, no, both women and men, but beauty is attached to the size. Women will go through length to fatten themselves because the further the better.  Like if you go to South Sudan and Sudan, they have annual, you know, competition who get the biggest and they do every single thing to fatten themselves. As a matter of fact, a couple of years ago, somebody died from it. So what now for being too overweight? Yeah, of course, because yeah, I mean, but now science and technology give us more access to understanding the benefit of maintaining a right way. The benefit of maintaining, you know, right size within your frame. Now we're not saying big person, small person, everybody has a frame and structure, but what is not right is overweight. Meaning that whatever your frame is, maintain it, but do not subscribe to any ideology that overweight is good for you. But also, yeah, talking about that. Okay, so people with special needs need to have good diet, but you have diseases or disorders, as you say disorders, such as bulimia, anorexia and some others.   I'm not, I'm not beautiful. You know, that kind of thing. So psychologically it hurts people if they hear those things. So what is one way you can tell the, the special needs community about one's body and eating right, but at the same time, educating, you know, the right. You know, extremism is extremism. Extremism in overweight has almost the same effect as extremism in on the way. As you said, there are some professions, they saw over emphasize the tinniness to a point where that person is now on the way, and that person that can create a whole lot of health issues. So we are not saying you got to be tinn like needle. Everybody has a body frame, body structure, body type. Maintain it the way it was designed. Do not be overweight. Do not be on the way. Medical doctors will tell you what need to be done to maintain your primal health. It's called your BMI. Whatever. Just do what your doctors advise you to do. Now, in terms of the, you know, you know, the disability community, or as you call it different ability, you know, again, we have to follow the orders from the experts. Times is so advanced in our lives that scientists, the medical doctors are able to tell us what is best, best on the condition that, you know, we have whatever that condition is, the doctors will be able to customize, you know, remedy that will be suitable best on, you know, once condition. Well, I would like to thank you for joining me on this edition of Ableton on Air. For more information on Lifestyle, Lifespan, or any other program that Mr. Dramé runs, where can they reach you and how can they go about, you know, what websites should they look into? Yes, absolutely. My number is the best so far, which is<PHONE_NUMBER>. And the email is LifestyleLifespan247 at gmail.com. Okay. LifestyleLifespan247 at gmail.com. The website is being built, which is LifestyleLifespan.org. Okay. So for more information on eating well and taking care of oneself, you can go to the email is LifestyleLifespan247 at gmail.com. And contact Mr. Dramé,<PHONE_NUMBER>. That's<PHONE_NUMBER>. Before we end, we would like to let's take a look at a clip from, or the trailer from Super Size Me, which was the movie about overeating. Let's take a look at this. Three, two, one. Well, again, we would like to say thank you to Shaqe Moussa Dramé from Muslim Media Corporation and LifestyleLifespan for joining us on this edition of Ableton On Air. Thank you very much for coming on the show. And again, I'm Lauren Seiler. See you next time on the next edition of Ableton On Air. I'm Lauren Seiler. See you next time. Major sponsors for Ableton On Air include Green Mountain Support Services, empowering people with disabilities to live home in the community. Washington County Mental Health, where hope and support come together. Media sponsors for Ableton On Air include Park Chester Times, Muslim Community Report, www, this is the Bronx.info, Associated Press Media Editors, New York Power Online Newspaper, U.S. Press Corps Domestic and International, Anchor FM, and Spotify. Partners for Ableton On Air include Yachad of New York and New England, where everyone belongs. The Orthodox Union, the Division for the Blind and Visually Impaired of Vermont, the Vermont Association for the Blind and Visually Impaired, Center Vermont Habitat for Humanity, and Montpelier Sustainable Coalition. Montpelier Medical Center of the Bronx, Rose of Kennedy Center of Bronx, New York. Albert Einstein College of Medicine of the Bronx. Ableton On Air has been seen in the following publications. Park Chester Times, www, this is the Bronx.com, New York Power Online Newspaper, Muslim Community Report, www.h.com, and the Montpelier Bridge. Ableton On Air is part of the following organizations. The National Academy of Television Arts and Sciences, Boston, New England Chapter, and the Society of Professional Journalists.


A Hormel Food says it has stopped accepting hogs from a pork supplier in Oklahoma after an animal rights group released video from a farm the supplier owns there. The footage was released by Mercy for Animals, which called the treatment abuse. The video shows pigs in crowded pans and being hit with gas cans. The company says it learned of the undercover video Tuesday and is investigating the farm owned by the mash-offs, one of the nation's largest pork producers based in Carlisle, Illinois. Austin Minnesota-based Hormel said third-party auditors would go to other mash-off sites to check animal care standards. If you've enjoyed this segment of Lakeland News, please consider making a tax-deductible contribution to Lakeland Public Television.


Just give us one minute. Welcome to today's open research webinar hosted by Elife. This series aims to give early career research as an online platform to continue to share their research as an alternative to in-person gathering. Anna Akmanova. I'm a deputy editor at Elife. And my own research is focused on the cell biology of the mammalian cytoskeleton. It's an exciting moment for me.  And I think it has a very important function to connect people in this very difficult time when we're all confined to our homes and trying to make the best out of it. And we are particularly excited here today at Elife to be able to give some of our young researchers who cannot travel to meetings anymore and opportunity to present their research and to connect to many of you. Today, we will hear talks from Sajal Davlon, who is a research associate at Advanced Science Research Center at the University of New York in the United States. James Thomas was Dr. Othello at St. Hutchinson's cancer research center, also in the United States. And Roger Amamoto is a post-doctoral fellow at the Harvard Medical School, also in the US. OK, so before we start with the talks, just a few explanations of how we're going to proceed. So each talk, which will take 10 minutes, we will have five minutes for questions for the speaker. Then we will move to the next talk. To ask a question, you can type it into the chat on Zoom or directly in the Google document linked here. The link is also present in the chat. We are joined today by Miran Den, Anya, and Naomi from Elife, who are working in the background to support you. They will help me line up your questions. I will read your questions out loud and include your name where possible. The open notes document is also a place for you to continue shared public notes. We welcome you to do so and to list yourself as a contributor in the list above the speakers for today's webinar. Thank you. Finally, I'd just like to let you know that we are also recording the webinar and live streaming it on YouTube. During this live webinar, we ask you, please, be respectful, honest, inclusive, accommodating, appreciative, and open to learning from everyone else. Do not attack the mean, disrupt, harass, or threaten others or encourage such behavior. If you feel uncomfortable or welcome on any of these webinars, please contact Elife by email. We have an inbox which is watched by Miranda Nye at Elife. Elife organizes a reserve right to ask anyone to leave and to deny access on the subsequent webinars on Zoom. If you need any help at any point, please send Miranda Nye or Naomi a chat message directly using Zoom. Okay, so we will now start with our first speaker, who is Sajal, and she will tell us all about it. The title is ANT1 Functions and Astrocytes to Regulate Sleep Homeostasis. Sajal, welcome to the webinar. Thank you, Anna. So I begin to share my screen. Just let me know if it works. Are we good? Can everyone see my screen? Hi, Sajal. It's Naomi. Hi. Do you hear me? Do you see my screen? Yeah, so we can't see your screen right now. If you try exiting and share screen again. Does it work? Not yet. Because I see it here. I did the share screen. If you close presentation, are you showing a normal PowerPoint? Yes. You're not presenting right now. PowerPoint window. How about now? Let's work out how to do this in the background. Should we go to James? James, would you be ready to go first? And then we'll come back to you, Sajal. In the background, we'll work this out. Yeah, I can present this one. Okay, excellent. If we can do that, let's do that. Sajal. Yeah. Okay, then we will first start with James. James, welcome and please share your screen. We'll see my screen here. Yeah, I see your screen. Great. Sorry, I was pulling up the chat to make sure I could see it. Okay, great. Yeah, thank you so much everyone for organizing this and having I'll start my video. Okay, perfect. So everybody's good. Okay, let me know if you can't see anything. Great. So yeah, thank you everyone for having us and yeah, participating in this during this difficult time. I hope everyone is doing well. Okay, so my name is James. I'm a postdoc at the Fred Hutchinson Cancer Research Center. And I'm going to talk to you today about RNA splicing and new tools that we've been developing to study RNA splicing. So here I'm showing you a very simplified gene model. So there's an Exxon here that has a stock code on and then Exxon downstream, which has a translation stock code on. And these two coding sequences are separated by an intron. And during the process of RNA splicing, the introns are removed and you can kind of think of the introns as quote unquote junk. So this junk gets removed. Now the exons are ligated together. And now this coding sequence can be translated to make protein. So you can sort of think of the coding regions as being the quote unquote important parts of the message. And the importance of these coding regions are consistent with the conservation we see. So here I'm showing you these blue regions here are regions of high sequence conservation. And if you just take a quick glance, it looks like the intron really has no conservation whatsoever or upstream. But really I've been hiding these regions of incredibly high sequence conservation across this gene. And these are actually regions known as ultra conserved DNA elements, which are defined as regions greater than 200 base figures in length that are perfectly conserved between human mouse and rat. And also very highly conserved and even more distant organisms such as the puffer fish. Surprisingly, most ultra conserved elements are in non-coding regions of the genome. And they're often located within or adjacent to essential genes. So DMRT3 is a gene that's known to be important for viability in mice. So if you knock out this gene, there's phenotypes you see in a mouse model. So you might think, great, let's delete one of these ultra conserved elements, since they have all these remarkable features, certainly the animals would have viability defects. But when this was done, using a single ultra conserved element knockout, the mice are viable. And there was no postnatal phenotypes. So this was very surprising. It turns out actually more recent studies have shown that combinatorial deletion between two ultra conserved elements actually begin to unmask some phenotypes. And this suggests that these ultra conserved elements could be functionally redundant. So here I'm going to show you another example of some ultra conserved elements in other surprising regions of the genome. So here I'm showing you the gene structure for SRSF3, which is an RNA binding protein. And again, the coding sequences are very highly conserved. But again, I'm hiding something. And that's this other exon that has incredible sequence conservation across it, but also very high conservation within the flanking introns. One other thing you might notice about this exon, which might be a little surprising, is it contains a premature termination codon. And we call this class of exons poison exons. And we call them poison exons because when an exon gets spliced in and introduces a premature termination codon, this is a signal for that transcript to be degraded by a process called nonsense mediative decay. So essentially poison exons poison the host transcript. So you get the exon in and the transcript gets degraded. These poison exons tend to be enriched in many RNA splicing factors, such as SRSF3. And they are speculated to provide, speculated by also some direct evidence showing that these provide a mechanism of auto regulatory feedback. For example, SRSF3 encodes an RNA binding protein, which can regulate the inclusion of its own poison exon. So this sort of provides the cell a feedback loop to maintain the levels of SRSF3 and other RNA binding proteins. A lot of these poison exons, we can actually see them in even more distant organisms, such as certain molds. Again, really highlighting the high conservation of these elements. So again, intriguing and almost obvious hypothesis is that these poison exons must be important for organism viability. The challenge though with studying poison exons and really splicing in general is that there aren't until, you know, we hope recently, you know, great tools to study these, especially in a high throughput manner. So again, this is an isoform that would contain a poison exon, and this would be degraded by nonsense, maybe 2k, and the isoform on the right would make a functional protein. So we want to ask the question, what happens when we just delete this isoform? Unfortunately, we can't use RNA to do this. You can envision designing an SI RNA to target the poison exon, but that just copies what the poison exon does in the first place, which is knock down the message. And really we want the message to go up. Another strategy would be to use an antisense oligonucleotide to block RNA binding proteins that regulate the inclusion of that poison exon, but ASOs are low throughput, and it can often be challenging to predict which ones will be effective at blocking a certain exon's inclusion. So we decided to take advantage of CRISPR-Cas9 to delete these poison exons, and more specifically use pairs of guide RNAs in a Cas9-expressing cell to delete the exon. And this was inspired by this paper and others showing that delivery of pairs of guide RNAs can lead to deletion of large genomic segments. So here we can get up to kilobase size deletions. So the model that we started with was to delete the exon, so remove that region of the genome. So now you take away the cell's ability to even make that isoform, because the exon important for that isoform simply doesn't exist. So again, GDNA editing can enforce the production of a specific isoform. And now we can ask what happens when we just lose this isoform. So we're calling this technique pickfarm. This thing is for paired guide RNAs for alternative exon removal. And I'll show you how we wanted to adapt this to our studies. So the first thing was we wanted to show that we can use paired guides to deletion exon. We then wanted to use this for high throughput screens.  So to get pickfarm working, we first looked at an ultra conserved exon in an RNA binding protein called MDNL. I want to note that this isn't a poisoned exon. This exon is actually an ultra conserved exon that contains the nuclear localization signal for MDNL. So just keep that in mind. So we designed a variety of strategies to deliver paired guides to delete this exon. We can use Sanger sequencing to show when we turn on Cas9, we get the expected GDNA deletion. This is an RNA RT-PCR gel where we can show that at time zero, you get about 50-50 ratio of the two isoforms. But at day seven, you get a great reduction in the exon-5-containing isoform. And then most compelling to me, we can do a Western blot for MDNL. And without Cas9, you can see that both MDNL isoforms are produced. But when we use paired guide RNAs to remove that exon, we get specific knockout of the exon-5-containing isoform. And then again, I mentioned that this contains the nuclear localization signal for MDNL. And when we do immunofluorescence, you can see in non-targeting treated cells in here in orange, MDNL, accumulates in the nucleus of the cells. But we can see a loss of MDNL and many of the cells in our paired guide RNA treated cells. Great. So now that we knew the technology work and especially that it worked in a polyclonal setting, we can now apply it to high throughput screens. So for this, we can package a variety of paired guide RNAs into lentivirus, add the cells, add the lentivirus to cells and then let the cells grow over many population doublings and you then would count the guide RNAs before and after. So the blue guide RNA must have killed the cells and maybe the red one would help the cells to grow even better. We targeted 500 poison exons, 500 coating exons, and we typically target the three prime supply site of the exons. I'm skipping many details here, but the technology reveals coating exons that are essential for cell viability and also poison exons that are essential for cell viability. Regardless of what cell line we do it in, we often see qualitatively similar results. We can confirm that for specific substrates, both deleting a coating exon and the poison exon decreases cell viability. And then just in my last two slides, I'll just mention we also got curious in these exons which seem to have an anti-proliferative effect. But for this, we really switched the model. We added paired guides to cells, put them in a mouse, sequenced them, the guides from the tumors and found enriched exons. And then when we pick one of those exons, we can show that when we delete that exon, it actually increases cell growth in vivo. So here's the summary. Pick Farm is a tool to study isoforms. And I just want to highlight our lab and say thank you to everyone who contributed and our collaborators. And then I'll just put this up while I take questions. Great. Let me turn off. Great, I can hear now. Sorry, sorry I rushed through at the end, but I see this question stopping up. Thank you. Very exciting story about poison exons. I'm sure some of you have never even heard about them. So the first question comes from Valentina Snitkova. Wonderful talk, James. Are stop codons more conserved than surrounding nucleotides and poison exons? Yeah, that's a really good question. I would say that for the classically defined ultra conserved elements, these are defined as regions where all along a stretch of 200 nucleotides with perfect sequence identity. So in this instance, at least between human mouse and rat, it would be perfectly conserved. So we want to see enriched conservation for the three nucleotides itself. I think for other highly conserved exons, that might be a possibility, but I haven't listed that specifically. Okay, thank you. Then we have a question from Ashwariya Sigur, sorry if I mispronounce your name. Is the ultra conserved region in the coding region specific to start and stop codons? Yeah, that's another great question. It's not. So you might remember I showed the ultra conservation around the poison exon of SRSF3. And you can see there that the conservation extends throughout the exon itself, but even into the flanking and tronic regions, suggesting that there's other important regions beyond just the stop codon. One of the sort of models for why that's the case is again a lot of these poison exons can have auto regulatory feedback mechanisms. So SRSF3 would bind to its own poison exon to regulate its inclusion. So you might envision that the binding motifs for SRSF3 might need to be conserved in order to maintain the regulation. Thank you. Then we have a question from Jacob, our Colte. Do you see off target effects of your PGRNA class nine exon skipping method? Yes, we look at this a lot. And we look at it from a variety of ways. So both at the DNA level and RNA level. So first I'll speak to the DNA level. We did a lot of work using Sanger sequencing of topoclone fragments, I think, for maybe 20 different exons. And we can see that we get the expected on target effects of the pair of dives. So we at least we know that we're hitting the right spot. Most importantly for us though is the RNA level. And we want to make sure that we're just disrupting the inclusion of that single exon and we're not just messing up splicing for the entire gene. So you might envision if Cas9 is doing crazy things to the gene structure, you might get the accumulation of all these strange cryptic splice isoforms that would have undesirable functions in the cells. So to address that, we did a lot of RT-PCRs where we took primers and moved them back across the entire message. And we can show that we just get skipping of the intended exon. And we also did a variety of RNA-seq studies to look for low abundance splice functions. And for all the exons we've looked at currently, it seems to be highly specific. Do we have time for one more question? Yes. Alison Anders would like to know, do the ultra conserved regions contain conserved RNA structures? Yeah, that might be the case. It's not something I've looked at. I mean, I think that there's algorithms available to predict RNA structures. So I think an analysis like that is relatively straightforward and something we can do. I personally haven't looked at it. In terms of structures that I do know about, there's work, and I'm liking on the group that did this, but there's work showing that ultra conserved elements are enriched in certain domains of chromatin or kind of DNA-DNA interactions. But I don't believe, I can't remember if that study looked at only ultra conserved enhancer elements or all ultra conserved regions, but there's also some research showing that these could be enriched in certain DNA elements. So, yeah. Okay. Thank you, James. Time is up. We'll move on to the next speaker, but thanks a lot for the very exciting talk. We'll move to Rogi, who will share with us his insights on transcriptional profiling of specific cell types. Welcome, Rogi. Thank you. Let me share my screen. Can people see me? Okay. Thank you, Eli, for giving me this opportunity during these really difficult times. My name's Rogi, and I'm a postdoc at Harvard Medical School at the Kani Sapkola. And today I'm very excited to share with you our new method called probe seek, which allows transcriptional profiling of specific cell types from heterogeneous tissue using fish. As many of you probably know, you've worked with organs or different tissues, and they're usually composed of many, many different cell types. Deficted here is just one example. It's the retina, which allows us to see. And you can see I'm just depicting the seven major classes here, the rods, the cones, bipolar cells, horizontal cells, amocrine, RGC, and mule glia. But recent single self-strategies have identified more than 100 different subtypes in the retina. And this is not unique just to the retina. It's also happening in all the other different organs, different tissues, and different organisms. So when you're thinking about doing an RNA-seq experiment to, for example, let's see, let's say you want to understand what's going on in one of your cell types of interest during disease, then you have to really think about what kind of experiment you're going to run. So, you know, there are different common transcriptional profiling approaches that are already available. And I'm going to just illustrate this by using a group basket as an analogy. So imagine this group basket is your favorite tissue or organ. They just dissected it. Just like tissue, this group basket has different kinds of fruits, and there are some more common fruits like apples here, and some rare ones like kiwis.  And this is similar to a whole tissue RNA-seq. In which, you know, you get the very common abundant cell types and the transcriptional profiles associated with it, but not really the rare cell types. More recently, we have this fruit salad method where you can take all the different fruits, basically all of them, and then cut them up, and then you can taste each different ones. But to get to the kiwi, you're going to have to go through all the different apple pieces. And this is something like single cell RNA-seq, which is great, and it's very transformative in biology. But, you know, for you to get the 1% of the cell types that you're interested in, you might have to go through 99% of unwanted cell types. So, in the middle is something where I'd like to think of it as single fruit bowl. Let's say you're interested in the very rare kiwis, then you can sort them out, you can pick them out, cut them up, and then you can enjoy just the kiwis. And that's something that's like cell-type specific sequencing. So, there are, you know, there are different transcriptional profiling strategies for better cell-type specific already. For example, if you're working with a genetically tractable organism like mice or drosophila, you can, maybe you might have a promoter or an enhancer for your cell type of interest, and you can drive the expression of a fluorescent protein like GFP, and then sort them out. Or if you have an antibody, you're lucky, you know, you'd be able to take them out using anti-bodies against cell-type specific antigen, and again, back sort them. But oftentimes, even if you're working with mice or human or drosophila, you're not lucky and you don't have these approaches. And it's also a huge problem if you're working with any of these different organisms, these understudied organisms, and, you know, antibodies or promoters are just not that available for these organisms. So with probe-seq, we had one goal in mind. It was to obtain deep transcriptional profile from any cell type, from any organism, potentially. And this is the workflow for probe-seq. And as a proof of concept, we decided to go with a fresh mouse retina where we know all the different cell types that are involved, and we decided to take out a very specific subtype of bipolar cells in this layer here, and it's about 2% of all the cells in the retina. So what you do is you can dissociate a fresh mouse retina and then you can dissociate and then fix in 4% BFA, and you get this fixed single-cell succession, and you can use gene-specific probe sets for your markers of interest. Here we use VSX2 and GRIK1, and VSX2 labels all the different bipolar cells and muller glia, and GRIK1, which labels a subset of those cells. And these probes are what we use for stable fish as well, and you can do this in tissue. And this was developed, at least in part, by our lab, the SEPL lab. And once you do this overnight incubation with these probes, you can put these green or red fluorescent oligos onto these concatomor tails, and you can use a different combination. And you can fact sort them and then put them into different tubes. Here we have the VSX2 positive, GRIK1 positive population, and VSX2 positive, GRIK1 negative population, and VSX2 negative population. Then you can de-crosslink and perform RNA sequencing. Now if you do this in tissue, this is stable fish, using the same probe, you can see that VSX2 is confined to the INL layer. These are all the bipolar cells in Heliglia, and GRIK1, which labels a subset of them. And if you use this on fixed single cells and go through facts, you can see that there are different peaks. This is debris peak, and then you can see this is a single cell peak, which is what you want, and other contaminants. And if you take just the single cells, and then you can see this negative population, and there's this bump that is for VSX2. And if you get just that, now you can see there's a GRIK1 negative population and a GRIK1 positive population. That is very prominent here. And if you look at them under the scope, well, the double negatives, you have the host staining, but you don't have the punctile of VSX2 or a GRIK1. Now the VSX2 positive, GRIK1 negative cells, they have the punctile just for VSX2, but not GRIK1, and you have the double positives. And based on the markers we use, GRIK1 and VSX2, we expected bipolar cell subtype 2, 3A, 3B, and 4 to be in the GRIK1 positive population, and other bipolar cells and mule glia in the GRIK1 negative, and all other cell types in the VSX2 negative population. If you do RNA sequencing and you look at different markers, which is in these different rows, you can see that in here on the right side are the GRIK1 positive population. You can see that BC2, 3A, 3B, and 4 markers are enriched specifically, but in the GRIK1 negative, all the other markers are enriched. So it seems like probe seek is profiling the correct cell types. And the question we wanted to know was, okay, well, if people want to use this, how good is probe seek compared to normal bulk RNA sequencing? So this is kind of the gold standard. You have a promoter. We use GRIK1 promoter, driving GFP. So here we electroporated it into the retina. And if you do that, about 72% of GFP positive cells are GRIK1. And then from that retina, it's from the unlabeled, unelectroporated area, we did probe seek for GRIK1 positive cells and perform RNA sequencing. And in the electroporated area, we fact purified live GRIK1 GFP positive cells using a very standard trizol approach and performed RNA sequencing. And the results were very good. We thought it was very good. If you compare the transcriptome of the live GRIK1 positive cells and the transcriptome of probe seek GRIK1 positive cells, we saw on an average a correlation of 0.78 indicating that high quality, you know, transcriptome can be obtained by probe seek. And if you're interested in just, not just using two probes, but if you're interested in four or five different probes because you're looking at very specific subtypes, then we thought we can do a serially multiplex probe seek where we had GAD1, GSX2, GRM6. We had an overnight incubation of all three of them, but we put fluorescent probes only on two of them, GAD1 and GSX2. And you can see this population of GSX2 positive cells and GAD1 positive cells. And then you can very quickly strip these fluorescent oligonucleotides, but not the probes themselves. And that only takes like five, ten minutes. And you can see that the labeling is gone. And then you can rehybridize the fluorescent oligonucleotides onto GRM6 here. And you can see a new population appear. So I don't have time to go into this, but we also did this with frozen human retina nuclei as well as Drosophila mig gut and fresh chick retina. And they all worked great. So that shows that maybe this can be possible with any organism. And in the future, I think that probe seek can be combined with a tactic to look at open chromatin in the specific cell type or do single cell RNA sequencing after probe seek just to get a more finer, finer view of your cell type of interest. And with that, I'd like to thank everybody in the SEPCO lab for being very supportive, especially Connie for being a great mentor and the Taven and Demecki lab biopolymer facility for the sequencing, immunology facts for and the imaging and the funding. Thank you. Thank you very much, Roger. Thank you for sharing with us the smoothies, the TVs, the fruit salad, and particularly the probe set. So I would like to start with the first question, which comes away from Chantane. Is this method also working for plant cells? So that's interesting. If there is a way to dissociate plant cells, well, I have no experience with plant cells, but if you can dissociate them and if you can get these things into the cell wall by permeabilization, I'm not quite sure which permeabilization works for plant cells, but plant cells have RNA and if the Nc2 hybridization works for plant cells in tissue, then it should work for plant cells. Okay. The second question is, what are some challenges that you would encounter if you combine probe set with single cell RNA sequencing? Yeah, I get this question a lot. So I think, so right now, you have to fix the cells and if you want to do a high throughput droplet-based method, then all of the end of de-crosslinking at the end for probe seek occurs with protease. So if you wanted to combine single cell RNA seek with the droplet method, then somehow you have to de-crosslink beforehand and then put them into the droplet, or you might want to use like a methanol fixation and that's something that we can try. But I think, at least right now, it's probably already possible to do a plate-based single cell RNA seek method. I see. Another question, what are the, like, what is your pipeline for introducing fluorescent nucleotide into the cell? Is it enough to permeabilize the cells after fixing or do you have some other tricks? So the probe has to, probe can go away and only once it's fixed and hybridized. So there's a tritonex step and if it's not permeabilized, I don't know if it can go into the cell. I see. Then we have a question from Julia Piazza, who found this talk very cool. What's your pipeline for... So, actually I think it was the same question, sorry. Can you comment maybe a little bit more about the tricks that you use because I mean you must have been optimizing this a lot. It's interesting because so I had a previous paper where we were using antibodies and basically if you, once other lab members in our lab got the favor of fish to work in tissue, a lot of the same things that they were doing in tissue, if you applied them to single cells, they worked almost perfectly. The only optimization that we had to do was they were using tween in their, basically all the wash steps and then I found that if you use tween, they clump up a lot. So we just have to get rid of that. But it wasn't that much optimization that has to go into it. Okay. Then we have a question from David Sun. The abundance of these gene markers, is it important, does the marker has to be very abundant for the method to work? Yeah, so all of the markers that we used in the paper, they were very abundant genes. The two things that we found that were important were abundance of the genes and the length of the genes because these probes tile the RNA of interest. So the longer the transcript, you have many more probes that can bind to it. So we found that both of those parameters are important. The good thing is that now that everybody's doing single cell RNA sequencing on basically everything, we have 10 to 100 of different markers that we can use. Okay. Pretty cheap. I think if I understand correctly, we have to wrap up. Is it correct Naomi? So thank you very much, Roger. We now finally move to Sejal. Welcome Sejal and thanks Roger. And so the last talk will be about this mysterious long abbreviation and its function and astrocytes to regulate sleep home spaces. Can you see my screen? Hey Naomi. Is it good? Yes. Okay, perfect. I can see that. Okay. Hi everyone. My name is Sejal and I'm a postdoctoral researcher at the City University in New York with the work that I'm going to talk to you about today is a work I did as a PhD student in John Van Maal's laboratory at McGill University. So as most of you know that the most abandoned cell types in the mammalian nervous system are astrocytes. So here is a picture of this single astrocyte in the cortex of a rat. As you can appreciate here that these cells have very profuse remarkable morphologies. And because of the structures they are key for neural circuit assembly and functions. So for example astrocytes, they release different molecules during development that are important for synaptogenesis. Because of their morphology where they sit very close to synapses they have important functions in neurotransmitter uptake and processing. For example, in expressing neurotransmitter receptors or enzymes. And because of this molecular feature they can respond to neuronal activity as well as they release different neuromodulatory factors in response to this neuronal activity. And because of all these key functions that astrocytes serve in the neural systems they are very important in different thesis mechanisms. So overall the field is interested in how do astrocytes differentiate and acquire specific molecular profiles? How do they influence CNS function and behavior? And perhaps maybe we can use this knowledge and exploit astrocyte to better understand and develop therapies for human diseases. So I study astrocytes in drosophila because they have cellular and molecular features that are conserved. And drosophila it's a beautiful genetic model system that is easy for in vivo manipulation and you can readily observe phenotype. So if you were to take a cross section for example here it's the larva it's a brain of first and start drosophila larva. If you were to look at the cross section you see two histological segments. So here it's called the cortex which is shown here in green and it covers all the neuronal cell bodies. And you have this neuropile region that has axon student rights and synapses but they do not have any neuronal cell bodies. Now at the intersection of these two histological segment you have the astrocyte cell bodies that infiltrate that beautiful membranous branches and connect with synapses and serve and functions. And here is if you were to label astrocytes in vivo with GFP that's what it would look like. So again the question that I'm addressing is how do astrocytes in drosophila influence CNS function and behavior? And I'll give you a small example today that's my PhD work where I studied this gene called A1 and how it modulates sleep in astrocytes. So before I jump into the results I just want to give you a brief introduction on sleep. That sleep is modulated by two regulatory mechanisms. So if you look here you have sleep traces of drosophila where on the x-axis 0 to 12 it's a light phase and 12 to 24 it's a dark phase and you have the sleep profile of individual flies either in black or in red and it resembles some of our sleep pattern now that we don't have to go out to work. So this sleep is more or less governed by circadian rhythms something we call a baseline sleep. However you can imagine a scenario here that if a fly is sleep deprived overnight compared to this control fly that is not sleep deprived the sleep deprived fly exhibits higher amount of sleep the next day which is something we call a recovery or a rebound sleep and this is governed by the homeostatic mechanisms. So one of the key questions in the sleep field overall is try to understand the mechanism that governs circadian or the homeostatic rhythms and there is one example that we know of this mutant which is in a gene called A nut 1 these mutants they do not have any defects in their daily sleep baseline rhythms which is the circadian sleep however if you were to look at it in the bottom right corner you have the wild side flies when they are sleep deprived in this black bar they have a higher amount of rebound sleep the A nut 1 mutant flies they exhibit even higher amount of recovery sleep compared to the controls so how does A nut 1 participate in these sleep functions so first of all what this A nut 1 gene is it's an enzyme called aryl alkylamine anacetyltransferase that acetylates an inactive monamine such as serotonin dopamine and octopamine so the key question is which cell types in the fly brain express this enzyme and in order to probe that I developed an antibody against this exposed region and we tested this antibody so what do you see here in the control animal that the antibody beautifully lights up different cells in the neurosystem of drosophila larvae however the animal that has a complete denlation of A nut 1 gene does not have any staining suggesting that this is A nut 1 specific antibody so I looked at the A nut 1 positive cells in the drosophila adult drosophila brain what you see here in the first panel in green you have the A nut 1 positive cells and magenta ages the antibody to reveal the brain morphology I further did the marker analysis using a panneuronal marker LAV and a panglial marker called repo which suggests that both neurons and glial cells express this enzyme based on the morphology of glial cells I can already pinpoint that these cells are astrocytes which I further confirm using a marker analysis where alarm which is a promoter which only expresses in astrocytes if I were to label astrocytes with nuclear RFP all astrocytes in the fly brain are positive for A nut 1 and this is sort of a snapshot summary of how we characterized other cell types where among all glial cells astrocytes are the key cell type that expresses A nut 1 and there are different subset of neurons that also expresses this enzyme so here is the model until now we have a very good idea about the circuit and how they generate behavioral output through different receptors how do astrocytes participate and here is the one example where I found the expression of A nut 1 in astrocytes so the question was do astrocytes alter the level of monoamines via A nut 1 and affect the behavior in this instance sleep and then the second question would be what monoamines are processed by A nut 1 so the first example, the first experiment I did was what happens if you were to knock down A nut 1 from astrocytes only and this was achieved with GAL4US system and RNAI so here is an example if I were to knock down A nut 1 only from astrocytes you can only see the neuron specific expression but all the astrocytes expression is gone the number of astrocytes remain unchanged in this manipulation here is the sleep data that was done in collaboration with Amita Segel's lab so in black and blue you have the control traces of flies as I showed earlier and in green it's the experimental animals so what we see is during daytime and nighttime the amount of sleep remains unchanged so the circadian sleep component remains unchanged when we knock down A nut 1 from astrocytes however in these flies if we were to do overnight sleep deprivation as shown here you can appreciate that the green bars here are the experimental animal and this is the recovery sleep 6 hours after the sleep deprivation which is significantly increased compared to control GAL4 and UAS flies and this is a very robust phenotype now this phenotype essentially is what was reported before for this A nut 1 low mutant phenotype so what I'm showing you here is here is the gene A nut 1 that do not participate in the circadian mechanism of sleep which is important for the homeostatic sleep and just to end here if I have time so what happens to the level of monoamines so when we look at the control verses the A nut 1 low mutant flies we do not see any difference in serotonin and dopamine levels under baseline sleep conditions however if that window is in the sleep deprivation there is a robust increase in both serotonin and the dopamine levels and if I were to repeat that experiment just by knocking down A nut 1 in astrocytes we do see a trend in these green bars where there is an increase in serotonin and dopamine so just to wrap up here what I showed you today we found this enzyme A nut 1 that expresses in astrocytes this enzyme is important to process monoamines this enzyme is important to drive homeostatic sleep in astrocytes I did not show you this data but it is important to drive baseline sleep and then this enzyme controls the levels of dopamine and serotonin under sleep deprivation conditions and with that I would like to thank my lab and our collaborators and thank you for your time and I'll be happy to take your questions thank you very much Sejal it was fascinating I never have been thinking about sleep deprivation and flies in my life but it's absolutely fascinating first question from Ariel how can the understanding of sleep and drosophila be applied more widely to humans or other vertebrates absolutely that's a fantastic question and you know just a bit of a historical perspective so you know in 2017 the Nobel Prize went through the discovery of clock genes in drosophila and turns out it's the same mechanism that operates and flies in circadian rhythms and sleep it's the same mechanisms that we humans have so for example the molecular clock is incredibly conserved just talking about the a01 which I investigated in drosophila astrocytes so a01 it's also present in the human brain especially in the SCN which makes melatonin the enzyme that precedes the first step in the melatonin synthesis so there's definitely conserved elements in terms of a circuit mechanism the monoamine circuit is also very well conserved so the kind of work we do in drosophila to understand sleep it's definitely very informative for humans sleep let me see we actually had a very related question are there any human diseases associated with mutations in a01 or any disorders has it been looked at genetically? absolutely that's another very nice question so we looked at the mutation data and there is actually a syndrome in human which is in the a01 related gene which is called the SNAT and these people have something we call delayed sleep onset phase so they are sort of like in a way nocturnal, their sleep phase runs from late at night and they have definitely problem waking up early in the morning so they start that day a little bit late so that is the one definite example we have where a01 is related to human sleep disorder we have a question from Catherine can you see the same effects in serotonin or dopamine mutant background do they have disrupted sleep overall? yes so there is like a long list of mutations in genes that are important for serotonin and dopamine pathway for example transporters or receptors and they do exhibit a lot of sleep defects what was really fascinating from that literature was most of them have baseline sleep deficits and you know when you if you think about it if you already have a baseline sleep defects and on top of that if you were to do sleep deprivation experiment the results are a little bit confounded so this is the only molecule that had that clean separation that you do not have a baseline sleep defect but you only have the homostatic sleep defect I think we have to wrap up is that correct Naomi? yes so unfortunately our time is up we had one more question left and we will have to stop now so first of all I would like to thank three speakers Sejal James and Roger for sharing their work with us today which was completely fascinating and very diverse and I would also like to thank all of you for listening and for asking questions our online research talk series will continue next week with three neuroscience talks on Tuesday at 9 a.m. British time in partnership with Neuromatch conference for online unconference for those of you who don't know what unconference is conference run bottom up and not top down for computational neuroscience starting on Monday you can check out their agenda online even if neuroscience is not your area of interest we hope that you will come along to support our fellow early career researchers and on Thursday we will be back with talks from Catherine, Madan and Elka so for updates about this series please follow us at elive community and you can find this schedule and the registration information online thank you very much thanks for joining and we hope to that you will join again thanks very much for sharing Anna thank you very much thank you very much thanks everyone for coming along join us on Tuesday yes absolutely have a nice day everyone thank you very much Naomi Miranda Anya for organizing this so well it was fantastic it was exciting and thank you very much all the speakers for sharing your science and starting this series and some of them these difficult times I agree thank you everyone take care bye bye


Welcome back, guys. Welcome back to the beautiful girl. This is Anokali and as always, we have mad vibes, mad energy. Miskewimbom ki gostiyom gosti ke. So that was Kago Mungai. And of course we are going to a very amazing segment of the show. We have our guest artists with us. A beautiful singer, beautiful performer. We have the amazing Suwagati with us. Hello. How are you? I'm fine. Are you doing well? Yes, I'm doing well. Because this is Suwagati, if you don't know Suwagati, I'm doing the intro for you. Next to me is Suwagati and then I'll let him introduce himself. You can call me MC Reimo, the manager. Suwagati. You're manager to Suwagati? Yes, of course. Nice. You're the manager. Yes, of course. You're the manager? Yes, of course. I'm the manager to Suwagati. Whatever. Whatever? Yes. Because you're coming to Anokali. How long have you been in music? I've been in music for 5 years in the industry. I've been in music for 6 years. You've been in music for 5 years? No, I've been in music for 2 years. Oh, you've been in music for 2 years? Yes. Oh, nice. So you've been in music for 2 years? Yes, of course, yes. So Suwagati, for people at home, what kind of music is Suwagati? Suwagati is a gospel singer. That's true. Yes. So you're inspiring people? Yes. I'm writing an inspirational song. It's a traditional song. Yes. Was the specific language you spoke? Yes. Because you were a couple of lures? Yes, lures, Swillian English. I was a knick. A knick? Yes. I was a knick. I was a knick. I was a knick. You were a knick. Yes, I was a knick. You were a knick. Yes. So you were singing in vernacular? Yes. Okay. We were at the first battle. Maybe we were singing in vernacular. We didn't understand anything. But we had time to make up for the industry. So you were a knick. Yes. We were in a vibe too. Yes. We were in a vibe. Yes. We were at a flow. Yes. That's amazing. We were mixed in vernacular, both Swahili, any language in our language. Yes. Okay, nice. So you were a knick for 5 years? Yes, 5 years. You spoke in Liansa? Yes. Did you speak in Liansa? Yes. I spoke in Wutua in the village. I spoke in Ziki, Saana, Burned in Nairobi. What was your name? Nairobi means I lived in Nairobi. I lived in Nairobi. What was your name? Miki kutani na manejawa kuku ama.. Uku? Uku? Ukuku? You manage work as a manage artist? Yes, yes story Ah, Imba story Was it a talent?我是 talent management na kwa kwa hisha protekte fucha.  So watu kumawu yo, bibi kumawu yo aksha kwa kwa industry, lafu ni mitua kona, aneishinawa to lafu banaakia li aluwa wa zasawa tumdarao banaakia atama kwa. Atama toa kizaliwa, toto amuizi yule. So utukama wale indosi siwa wa fatlizi ya sanu tu na pato aneishi maisha shida wa naji, onatumia drugs. So numatu kwa kudienawa kwa maisha protekte fucha. So mi no suagadi, ni kama mbi ya avenye, nili kuchu kwaivi, so wacha tu move o naikitu. Tu skueni wepe kiako, skupe oengine piya nao piya one. M'aimuizi mera, m'aimuzi mera, m'aimuzi yule. Tu mera kwa. So utukama kwa kwa kwa industry, mera kwa kwa kwa mabili, wili kwa waka skueni, nao mini ni kwa masika kwa kwa kwa kwa kospa, kwa nani nao nani haja. Kwa kwa kwa kwa kwa kwa, kwa kwa kwa kwa kwa kwa, We faced this many challenges But We felt like there are so many difficulties in the situation of unilike that we are facing because my mama said we needed to react to that song so I felt like that song was the best I love it but it's a very interesting song I felt it So Kost leverage and I felt it so good so Kost, if you order areas wanda between.... so holds goes up and from life circumstances yako ahma wana tu mea? The people used... Sama na zagu k follow up in a life me dungun a+, w said to life is special ac punishment will be like more naku na dekumera hi kring mandori us wild heart naka Master to me sika Prens Jawabu. Kwa kataku LES. Tohima tohima ni suong wakizakan ina manisha kipon tryin. Oh yua tema? Chema tema. Ya, Kipon tryin. Kwa suna pata sezin gina ya watu na give up in life ay manishi saswa ki kosa le, wabadake hi Shukutoma kisha kufu nkukusa. Ita baki waotas. Witu badili kwa? Yur hitu la zina zita badili kwa. Ta wabi woda ini kai. and then bado ni wambia waatu Okipata kitu Godam ni kublesu siya irigasa ana Na sumplen ni mefa ninaji tia an An kikutina wa ni hiya mimi An aduwa rumonda beg an Mi nataka ni kuhu mimi Yani, ista kiki Wei, lafiyaku Goda kikublesu yun ni hako sawa Temi yung gita fika na mba dun tabari kiyotu Ya ya ya So any Ok, basically to saymentu your music ni kunspire other people and just it's more of You sell positive vibes through your music Ya ya Through itu ni me pitiya na feel like maybe kuna muntuna pitiya chene ni kuna pitiya So kama ni kuna You often wa dogu enya ni foti liya Maybe ni wopatiela zima tu wa watu willo atu atu wa taku wa Atleast wana Mi nataka, mi nataka wana that we inspire me to wu kwanje So sigui, sigui Like I feel like wuna fa We perform one of the songs because sigui ni gani Achetwone, achetwone Samu atu atu pera gani Tema, tema Atami nataka yo ya keeping to try Keep trying Tema, tema, are you ready for us? So achet wu pera flow bas Ok