You know being a donor, it's so special, you know It's one of the the most beautiful things that I think anybody can can do in their their lifetime Philanthropy is so important to support our mission at Hackensack Meridian Health Which is to transform health care and to be the leader of positive change, but without philanthropy It's going to become more and more difficult for that growth and that trajectory to continue to to move forward Thank you from the bottom of my heart to all of you for your gifts. We are so grateful Hackensack Meridian gifts, they really do. They're always looking to upgrade from from better to best. Jersey Shore, it's a magnificent hospital. Literally the miracles they can perform is incredible. We have a summer home in Seaside Park. Everybody who needs a doctor goes to Ocean. To help all the senior citizens that are down in that area We wanted to bring something good to them. I had problems probably about maybe eight years ago, and I went to see Dr. Shah Now Dr. Shah is a cardiologist at Bayshore. He Examined me and he said I think we need to do further Testings. Well when he said further testings, I had myself dead and buried. Either he knew it, but he said to me Don't worry.  And It was just like a breath of fresh air So that's what attracted me a lot to Bayshore because he was a Bayshore doctor. How about I always gave? Somebody needs somebody's husband, somebody's child, Somebody to stay alive and you're giving them that gift. No matter what he'd go out of his way to help somebody and the man literally Absolutely worked day and night. It feels like he's still alive when I see his name. I know he's looking at it It's it's exciting to know that he will always be there. In the emergency room It's up to date. They want the best and they got it. Whether it be machinery Whether it be a box of Band-Aids, whether it be anything People need it. And if you needed it, you wouldn't you want someone to help you? Our mission statement as a company with Jersey Mike subs is to give and make a difference in someone's life right from the start in 1975 when I took over the original store that was our Mantra, our creed. So part of our giving always has to be with our local hospitals Our civic duty is to be involved. Our hospitals are Intracle to being part of our community. I never forget walking through the emergency room and Watching the nurses the doctors and what they do and it just really compels you kind of moves you towards giving Oftentimes we've seen coaches teachers or people in our life that push and it just doesn't work So for me, I like to be pulling people along to tell them about what I'm doing and most times That's what really works. That's what happens is when you pull people along you show them What's happening and they want to be involved you cannot push or force them and There's no need to when they really see what's happening with the hospital system They really want to be involved Everyone loves giving and being part of what they're involved with You know, we have to raise up together and that's what I think they need to do It really started just with a visit to a hospital and you see a child that's going through chemo Therapy they're going through radiation. They're not feeling well. So you want to do something to try to lift their spirits Maybe they're a football fan. Maybe they don't know anything about football You could still have someone in there to try to give them the laugh try to lift their spirits in some way And I think when you come to play quarterback for an organization You're not just in the face of that organization But you want to be a face in the community and a part of that community and give back and serve ways And it's only my responsibility to help some of those people if they're going through a tough time And you know, there's not a tougher time than one of your children dealing with cancer or any illness you want to just be there for them and if you can just sit with them for a little bit and Just make sure that child knows that there's there's people out there that care for them that are rooting for them Just makes it feel like you're doing your part a little bit. I've seen the benefits of what patent kids cancer has done I've met some of those children seven years ago that were dealing with cancer and I've seen so many of them Smiling and back in school and healthy and doing great things Unfortunately, I've seen the bad side of that also and you're inspired by the great work that they're doing because you see the Success rates, but you also know that the more work needs to be done because not every child is surviving cancer There is more room for growth and there's more need for growth and to expand this whole network in New Jersey You want an unbelievable healthcare system here It is our job to help and you know, I hope that we can continue to grow that into a force that is making a difference all over New Jersey and set the example for other Pediatric centers and hospitals across the country. My husband and I actually got bitten by the giving bug a long time ago We learned that when you give you get and I don't mean you get something in return But you actually get a sense of satisfaction. I also give because I see What the impact is what I really want to say to the donors who are highlighted in this report is Thank you. Thank you so very much and what you do for your communities and for yourselves and For the HMH network all these things really come together to make sure that you're all having The best health care possible in our state and beyond Thank you so much in the bottom of our hearts for all that you do and we look forward to furthering our partnership in the future Thank you


Very first lecture on single cell transcriptomics, so in this lecture I will just give you a brief overview of what single cell transcriptomics is all about. We will focus on single cell transcriptomics only, so of course there are also other applications you can have at the single cell. For example, single cell A-texture, we have to make some choices during this course, so we will not be treating single cell A-texture, but a lot of concepts that we teach during this course can also be applied to other methods you can do at the single cell level. I am sure many of you have seen the comparison between bulk RNA-seq and a smoothie and single cell RNA-seq as the editor of foods. I am a bit lazy, I will use that comparison over here as well because it is a pretty good one of course, so once you can compare single cell RNA-seq to bulk RNA-seq with, again it is a smoothie and an individual fruit, so the bulk RNA-seq would be a smoothie, so what you do is of course you take a certain tissue, you grind it, extract RNA, so everything from the tissue is together in a big smoothie of RNA, so you have no idea where that RNA actually came from, what the individual cell types actually were, and what most of you probably know very well, probably better than me, is that the cell diversity inside the tissue can be huge and all of these cells can have different functions and some genes are much more relevant to be expressed in some cell types than in others, so what we see during bulk RNA-seq, if you see a difference in expression, of course that's real, that's completely going on in the tissue, but we do not really have a good idea where that is coming from. With single cell RNA-seq, what we can do is know what kind of genes were expressed in the individual cells, so let's say the individual fruit, and we can actually investigate on the level of the cell type, on the level of the fruit let's say, instead of having everything together in this one big smoothie. That means that you can answer biological questions usually in a much more specific way. So why then single cell RNA-seq? Well, I hope I already explained a little bit with the comparison between the fruit and the smoothie, but cells are of course essential for biology, so they are the basic structural often and functional unit of life and they can be very diverse, they're very diverse functions, and because of that of course for many applications it's very interesting to actually do research at the cell level and not on the tissue level as we are used to with bulk RNA-seq. What you can do with single cell RNA-seq is first of all anode-detailed, meaning I have a certain expression profile and I know it came from one particular cell and based on the expression pattern I can already tell what kind of cell type it probably was just by looking at the expression pattern, and that's already very valuable I would say because then you have an idea of what kind of cell you have in your tissue. In addition to that of course you can also compare certain treatments within the cell type, so you know what kind of cells you've had in a certain tissue and then you do a certain treatment, for example you give it a certain drug or whatever or you compare certain tissues and then you can also even say something about state of the cell or the differential gene expression between and within cell types. So those are two very important things you can do with single cell tracheotomic. They are very abstract what I'm saying now but they find their applications in very in very different ways depending on of course the research question you have. So how would you usually generate single cell RNA-seq data and is also again a very broad overview of typically them. So let's say you have a tissue, let's say you have fixed tissue meaning not for example blood, liquid tissue but you have whatever let's say a tumor for example, you have to differentiate the tissue into individual cells, obviously. And then we need to isolate the single cell in order to actually sequence all the transcripts that are in the single cell and that can be done in many different ways. We will talk about the most frequently used methods to actually do the single cell isolation after that sequence. Then there is some capture of RNA where you have a lot of different molecules in a single cell and of course we are now interested because we're looking at transcriptomics, we're interested in the RNA so we have to get it out somehow very often that is done by making use of polyadenylation. So this polyethyl messenger RNA typically has and then the reverse transcription so we can actually make it into DNA so cDNA and then sequence it. After the reverse transcription there is the library preparation in order to be able to sequence the cDNA so we're going to add some adapters there and then the actual sequencing takes place and by doing the sequencing you generate our Q-fels usually and we use these Q-fels in order and with our further downstream analysis. So then we have a quality control at least at the read level and then go on with downstream analysis. And a lot of most of the part of this course especially the exercises will be focused on this downstream analysis. We will be saying quite a bit on this first part so up from the dissociation to the sequencing in lectures today but day two and three at least and also the afternoon of day one we will be focusing on the quality control and the downstream analysis. So there are many technologies to create single cell transcriptomics data. So it started relatively recently I would say in 2009 where people actually were able to sequence the RNA of individual individual cells so then the library preparation really took place at the individual cell level where you have a single cell in a single well and during time both the throughput or mainly the throughput, the number of cells you could sequence in a single study, strongly increased and a lot of different research groups and companies tried to create methods that would enable you to actually do that. There are four main methods that are used nowadays to generate single cell transcriptomics data. I think the most basic one and also very quickly used one is just where you separate your individual cell with a packed machine and then you have your individual cells in an individual well and they do a library preparation on those individual cells in a well. Typically the method that is used to do that is called smart seek. Then we have droplet-based method and a very famous example for that is the 10x platform when you have your cells in individual droplets and that's how you separate the cells from each other. Then we have combinatorial indexing and that's where you basically add barcodes to your cell, shuffle them, add new barcodes and with that you have some kind of combinatorial index and you can figure out which origin came from which cell, more about that later in other slides and there's also Michael well-based where you have a Michael well-played so a played with very tiny, tiny wells that can fit basically a beat and a cell together and by that you individual life, the individual cell, or you separate the individual cells from each other. I will introduce those four frequently used technologies in the coming four slides. First of all there's smart seek with smart seek and you use FOC to get a single cell in a single well. We do the library preparation for a cell in an individual well and that has quite a bit of advantages because for example you can sequence the entire gene. It's very typical for single cell transphytomics that you only sequence a small part of the gene in order to estimate expression but for smart seek you can sequence the entire gene as you might be used to with bulk RNA where you also typically sequence the entire gene. Then there's Tenex genomics, more about that in a complete separate presentation later on so that will be after this presentation. So what do you do with Tenex genomics? You isolate your single cell in a GEM, a gel bead in emulsion so you have your cells inside an oil that in those oil you have an emulsion with your cell in there and the cell is combined with an individual gel bead and because a gel bead has specific properties you know which read origin you came from with cell. A bit of broad explanation about it actually does more about it in the presentation after this. So with Tenex other than smart seek what you do is you sequence your gene only from the 3 prime end so only the 3 prime end of your gene or of your transcript get a sequence meaning that you do not have any idea of other parts of the gene so you cannot do for example differential ideal form expression so you only measure gene expression with this typically frequently used Tenex platform. So just to explain a little bit more about this gel bead so we have in a solution we have gel beads with oligos attached to it and those oligos contain an adapter so those which is needed for the sequence thing then there is the barcode that is unique for the bead so let's say this bead has the light green barcode well this big has the pink barcode this one the red one and so on so each bead has an individual barcode and then there's a UMI more about the later on and then there's a poly BT bill and that is of course you to actually capture the messenger RNA that is poly adenylated so that's poly A. So what you will have is you move these beads and cells through some kind of a cell disorder and because you have less cells than beads just by chance you have a big chance that a single bead occurs with a single cell at some point in this oil and because of that you have a single cell associated with a single bead and because you have a unique barcode for each bead you know that everything you capture on that bead comes from a single cell from an individual cell because you have these barcodes you are going to sequence them later you know which beads all came from the same cell. So if you then look at what you are actually sequencing in if you're using the 10x 3 prime platform compared to for example SmartSeq well with SmartSeq obviously you often you sequence the entire gene so if you have the gene body over here so let's say if you take all genes together and you look what part of the of all the genes are sequenced so for 10x for the 3 prime method you mainly sequence the 3 prime end of a gene well for SmartSeq you sequence the entire gene so you have much better coverage over the entire gene if that is important to you then it is relevant to take it into consideration of course. Usually with the 10x platform you have a much higher throughput in terms of number of cells so you have you can sequence way more cells compared to SmartSeq because with SmartSeq you sequence the individual cells in an individual well and at some point these places become too big so you can go only up to what you can handle in let's say a 384 well plate for example so typically people do not use more than more than four plates for 384 well plates in a single experiment so therefore you sequence often way more cells with a 10x platform but with Smart SmartSeq you do not only sequence the entire gene but you also usually detect more genes per cell just because you can more specifically generate libraries for a single cell if they can optimize it more and therefore you have a higher library complexity per cell so you can see there's more type more genes and therefore you have a much better representation of the whole transcriptome of your single cell so if you compare the two so SmartSeq and 10x genomics so both of them are based on sequencing messenger RNAs a fully adenylated RNA with 10x genomics there's a strong bias towards the pre-primand SmartSeq you can sequence the entire gene for 10x genomics you only can use it for expression analysis maybe differential polyadenylation if you would be interested in that but that's a very specific research topic I would say for SmartSeq you can both measure expression but also either form analysis if you would be interested in that so you have a low number of transfers per cell you can measure with 10x genomic at some point you can just kind of the beef that be used for 10x genomics can just kind of capture more RNA that is already in there for SmartSeq you can have a much higher library complexity per cell and therefore you capture you actually sequence more transcript per cell for 10x genomics you use this cell sorter which is quite an investment for SmartSeq you only need a vaccine machine which is in many that already available for a drop that base or 10x base you go up to 100k cells per run for SmartSeq and you usually do not go up to more than a thousand uh cell per run um a bit of a downside of 10x genomics can be that you have to run all itself at one through this cell sorter which means that a single sample is a single library is a single run can be there are ways to to do to do with that but by default you only have one sample as one library which means that the sample is always compounded with the library for SmartSeq you can use more complex or experimental designs so that means that one cell is one library and can for example have plot design with that so isolated by droplet so you can have quite a bit of doublet in there meaning that a single beat is associated with not one cell but just by chance with two cells and by having that you of course do not estimate the expression profile of one cell but of two cells at the same time can be a bit of an issue also for example if you load too many cells on there you have a lot of doublet for facts it can you can have some bias towards the outside for example it's easier apparently to have bigger cells involved with with facts than smaller cells so you might actually have a lower representation of smaller cells in there first cell of course 10x genomics is way cheaper because the throughput is much higher compared to to SmartSeq so it very much depends on your application whether you want to use 10x genomics or for SmartSeq I have a question about that now I'll ask it now that's most convenient so let's say we are well we're having comparing SmartSeq with 10x my question is by having this kind of information which method would you use if you are mainly interested in rare cell types so cell types that do not occur very frequently in tissue and which one would you choose if you would be interested in mainly low reactivate genes the genes that are not very highly expressed in your cell this is them great so most of you have just correctly I would say so droplet based for rare cell types and SmartSeq for low reactivate gene the droplet based you segment a lot of cells you can sequence a lot of cells in a single rung meaning that you are also cells that are relatively rare so that do not occur very frequently are probably also in your sample or in the number of cells you are sequencing so you have some representation there that you might miss those for SmartSeq because you are just limited to a certain number of cells so usually up to a thousand let's say but with SmartSeq you have much more complex more complex libraries because you can just sequence more transcripts within a cell and therefore you sequence also these lowly expressed genes there's a question of Tui yeah I would like to ask that if the cells are too rare and then if you use the um how to say if you use droplet based and then you have another population that is so dominant it's going to be at the end it's going to be kind of overridden by this big population no is it the better way maybe to sort out this rare population for SmartSeq yeah definitely definitely yeah so if you can of course enrich your sample for these rare cell types that's always a good idea to do that and they could even lose smart things to actually sequence those if you have it enriched enough but there you can of course also imagine that there are situations where you are not pretty sure what kind of cells they are and therefore you can really sort them out or maybe you are just very you're you're the very exploratory exploratory phase so you are just looking for a method to sequence a mark a diversity of cells instead of a diversity of transcripts within the cell and that's where you would use 10x if you are interested in these well these big libraries for cells that go for smart thanks good question yeah and so cell sorting is always an option of course definitely okay so what we have done now is compared the droplet-based methods and then maybe the 10x platform with platform that is based on individual cells and individual wells which is often SmartSeq the SmartSeq method there are two relatively new methods that have quite high potential and not super new but they are immersing let's say they are used more and more frequent so that's why I wanted to also discuss them over here one of them is combinatorial indexing and there is a company that actually commercialized it which means that you can actually buy kits which is usually very nice therefore much usually much easier to apply much easier to use so it is a completely different way of being able to individualize the single cell so what you do is you have a cell your cells in an emulsion let's say and you fix it with formaldehyde and after that you do a ligation with barcodes to your RNA but this RNA remains inside those cells after that you split your cells into multiple pools and you add a new barcode then combine those again and split them again at a new barcode and then just by chance you do that four times just by chance you have a unique set of barcodes and unique set of combination of barcodes per cell meaning that just by sequencing this unique set of barcodes you know by a certain by certain chance that all of those reads you have you have been sequencing originated from a single cell so by doing four rounds it is very unlikely that you end up with depending on the number of cells to put in of course that you end up with cells two different cells having the same combination of indexes and therefore you have individual reads per cell or reads per individual cell you can trace them back to the individual cell so the nice thing about this is that it's quite flexible so you're working with fixed cells and there are no specific devices needed so you did buy a kit which is not super expensive and you do this combinatorial indexing just by yourself you can do it by yourself in the lab and then do the sequence thing a bit of a disadvantage disadvantage of it is that it's quite laborious so of course by doing the splitting and the pooling again that requires quite a few minimal steps but still it's a good alternative to to droplet-based sequencing like the Tonex platform for example because you do not need one of those cell disorders and in terms of throughput and everything you are at a similar level so you can also go up to more than 10,000 cells per experiment for pool then another alternative is based on micro wells commercialized by VD it's called VD-Repsidy so what you do there in a way it's kind of similar to smart sync but what you do is you do not use fuck who actually have an individual cell in an individual well so what you do is you have a micro well you have beats in there and you sparsely load cells on the micro well and because it's sparse meaning that you have very few cells in there just by chance it's very likely that you only have a single cell in a single micro well so many of the micro wells do not receive a cell and a few of those they do and because of that you it's very likely that you have a single cell in a single well within that well you lyse your cell hybridize your messenger RNA to a beat again in a way similar to this droplet-based and you'll message your RNA hybridizes to that beat and then you continue with the sequencing like Tonex genomics and also like VD-Repsidy by the way the only sequence the three prime and of a gene one advantage of this is that you can actually see your doublet in there so if you have two cells associated with a single beat you actually know that that is happening so you can estimate your the percentage of doublets you would expect in your dataset you cannot trade it back to the individual barcode by the way so you only know that there are doublets in there but you do not know which barcodes are associated with doublets or which beats associated with doublets okay so this was the question I just asked so then a few words about experimental design in a way experimental design is not different from any other biological experiment so that means also for single cell test with Tonex you require replication you require randomization and you require blocking if if needed which means that well if you are comparing a drug for example so treated with uncreated for example you need replicates of treated with uncreated and if you have for example cages with mice make sure you include those cases in the block design also with experimental design like any other biological experiment be aware of confounding factors so confounding factors are factors that confound with your treatment so that are not your treatment but are associated with the same sample that receives the treatment or did not receive the treatment which means that for example make sure that if you have multiple people handling the the different samples make sure for example that not one person is doing all the treated ones and the other person is doing all the untreated ones that's what that's what confound is also this can happen with reagents or for example the time of processing do you process one of them at one day one treatment at one day one treatment at the other day for example that's also confounding and can even be the sequence itself or the lane or the library so lane of the or mainly the lane can also have an effect on expressing your measures so if you have for example all of your treated samples on one lane all of your own treated samples on another lane you could just be measuring lane effects if there are any of those factors and it's they pop up during processing for example realize that they are there try to have it randomized as much possible have it blocked as possible as much as possible and if there are any factors that could affect the expression make sure to record it because if it is nicely blocked they can actually correct for it later on. There are quite a few papers on experimental design in single-cell transcriptomics and there are two links to those at this slide. So just another example of a confounded design let's say you are doing smarttik and what you can of course do what would be the most easy would be to have let's say we have the light gray ones are untreated and the brownish ones brownish mice they are treated and principle what you can do is having all the cells of the individual mice on a separate plate process these plates for example you processing takes quite a bit of time so maybe you can do two plates in a day process these plates individually and then have them also individually on sequencing lanes. Of course what you get then is that you get an orient effect of the plate and if you cannot process all the plates on a single day they already have an effect of the day and then you also get an effect of the sequencer lanes and they're all confounded within the plate and you can imagine that these effects can become pretty big at some point they just build up. A better design there would be a more balanced design where you actually divide your different mice over the plates maybe would even sometimes be better they're completely randomized but within those places can be quite of a challenge but then let's say you have all your in all your plates you have your different mice represented so your plates are in the blocks and then you have these different plates divided over different sequencing lanes so you nice when you have a block and you know which plate well of course which mice is in with individual wealth or your late one can even correct for example the plate if you're doing differential gene expression for example uh question for you there you go for the late two experiment design so let's say you want to compare two groups of mice three in an untreated to this rock and this preparation is labor attentive as is very often the case obviously so you ask a good colleague and what will be your plan there so your three possibilities here okay once we answer so stop there we go nice very good so the message came across obviously uh that's very nice so of course well the third one is is just not not correct uh i process all the treated and processes all the untreated so we don't get mixed up so that's where you start having confounding effects because your colleague might do the this preparation slightly different from you than you or completely different from you and then you're just not measuring the treatment of the drug no you can be measuring the actual different ways of how to prepare this issue and usually you're not interested in that you're interested in the effect of the drug and you cannot separate them anymore because they are confounding uh asking a colleague is a bad idea i will take multiple days process which means that you will get a day effect of course at some points you just sometimes you just have to have multiple days or very often you just have to have multiple days then you try to process of course not all treated samples you meet one and all untreated samples maybe two now you try to um you try to randomize that but of course if we can process everything in a single day um and not too long time span we randomly assign the mice from both reasons of each of it so we can and we write it down so we can correct for it later on so then a few um related methods uh to single cell transcriptomics what you can do in addition to measuring um gene expression um is to quantify proteins so what you do there and that's the method that is uh very frequently used in combination with the 10x platform with um sci-thick for example it's where you have an antibody uh get me any antibody and attached to that antibody you have your adaptive sequence that is required for sequencing an index that is specific for the antibody and then let's say for example a poly-A till and um if you make sure that these antibodies you can let them bind to to yourself you wash away the unbound antibodies and then you proceed to the sequencing and because it has the poly-A till also these sequences they are captured by the 10x platform and because you have a specific barcode associated with an antibody you know that that antibody actually attached uh to yourself nowadays um this is this has become more evolved this is not a poly-A till anymore but a specific capture sequence so you can actually know you have uh those um antibody associated uh oligos they do not compete with the messenger RNA to a B um so what you can do is therefore quantify um uh proteins together with messenger RNA which can be super powerful of course and you can have quite a few I think you cannot have up to three to four hundred maybe even more maybe even five hundred uh different antibodies and have a very uh good idea of the entire uh proteome of the cell in combination with the entire transcriptome this same method a very similar method uh you can use to actually combine multiple samples together in a single run for 10x or drop of patient method so what you do there is you have an antibody that uh binds to any cell and uh you have again an index uh on your uh attached to your antibody and of course an poly-A till where it needs a capture sequence but that index is not specific for certain antibody or certain protein no it's specific for a certain sample so you make sure these antibodies they attach to yourselves before pooling so for the individual samples you hybridizing with these antibodies then you pull yourself together and then you proceed with uh regular uh 10x protocol so you make sure they are associated with individual uh beats with individual indexes and then you do the sequencing but you also then are therefore sequence for each cell anything all the uh and the the barcode sequences of the antibodies that were associated with the cell meaning that you figure out where that cell cell origin came from from with sample it came so you be multiplex it but also if you have multiple barcodes within a single cell let's say you also know that it was a um a duplex so a doublet I could say a doublet so multiple cells within a single well so that can be quite powerful and also can reduce cost quite a bit I have another question for you okay so of course related to what I just said so if you're using antibodies to quantify proteins and I just gave the answer in the in the presentation which sequence do you use in your downstream analysis for quantification so you want to quantify the protein right so the more antibody binds to your cell surface proteins more like the the more proteins there likely work right okay I think I will give you the answer or at least show what what you have entered okay very good okay so most of you have really understood what we have been talking about it's great so it's indeed the barcode link to the antibody tagging the protein of interest so you have all these antibodies uh bind to your cell surface protein and the more of these antibodies bind the more of the barcode you will have in uh in your individual cell let's say an integral thing that was captured by the by the oil and is together with the same uh gel bead and therefore you have more of the barcode the barcode specific for the antibody and the more of the barcode you have the more of the protein was in there just to see an image so the more of those guys binds the more of that index you have in there and the more you see okay last slide um so we have been talking about single cell sequencing a lot and we will be mainly focusing on that of course but there's also something called single nucleus sequencing it's kind of an alternative to single cell RNA so you get very similar data out of that you also at some point get counts for cell but then is for nucleus actually um can be very interesting for example for tissue that are difficult to dissociate so that are difficult where to actually uh get the individual cells out but usually it's more easy to get the individual nuclei out of there um uh an advantage of that is that because you extract the individual nuclei you also get rid of all the ribosomes which means there is no translation of transcription factors anymore that can affect them the transcriptome again during processing so you kind of stop this whole uh translation process meaning that you might have a better representation of the actual messenger RNA that was in the nucleus in the tissue itself um uh a downside of that is of course you miss old messenger RNA uh that is not in in the nucleus and i'm not very sure whether it's because of that but at least what people have found is they found a lower representation of immune cells and surf proteins um and when you do single nucleus RNA so that's a bit of a downside meaning that if you are mainly interested in those kind of genes then single nucleus RNA think might not be a very good idea or maybe you might not if make me do


That's simple. Reach higher consciousness by going to the toilet 10 times. All right guys, welcome back to the channel. If you're new, my name is Bobby. Guys, you wanted me to react to Raw Vegan Rising with this video, five benefits of a fruit based diet. I am aware of Raw Vegan Rising. He has been a Raw Vegan for a while now. However, I haven't seen this particular video. Let's have a look. That's what I want. I want to be Raw Vegan. Let's go. Hello, amazing, beautiful people and soul family from around the world. How are you guys doing? Fantastic. Welcome to Raw Vegan Rising. My name is Shane Sterling. We're going to talk about the five benefits of a fruit based diet. And oh my goodness, there's hundreds of benefits of a fruit based diet. But in this video, I'm going to stick to the most critical, most crucial ones that you truly need to understand. This is life changing information. Now, all healing starts in the mind. The mind dictates what the body does. Now, if we fuel our body and we start to activate our cells and get our cells oxygenated and empowered with the electrical activity. Empowered cells. Yeah, I agree that everything starts in the mind. However, if you don't put in proper nutrition, nothing will happen. Electrons in raw living foods, it actually starts to activate our mind even more, gives us more mental power to see the broad bigger perspective, which gives us more true as well. There are plenty of studies on vegans and vegetarians where you can see 20% less brain weight. So therefore, I absolutely agree with you, you need proper nutrition to have a healthy brain ability to stay in a healing modality. So it's like a feedback loop. Okay, we use the mind to empower the body and then the body back empowers the mind. And we spiral upwards and upwards and upwards to higher consciousness and connection with spirit and a broad perspective of how we can contribute to humanity. Very fascinating. You hear this over and over and over again in the raw vegan slash fruitarian community. However, you see no results. I said it before, if you would experience spiritual ascension through diet alone, freely durian rider, raw bliss, etc, etc, would be the most spiritual people, they would be gurus. However, you see nothing of those sorts. Salvation does not come from diet for generations to come. This is the healing that we're all looking for. Okay, and it comes from a fruit based diet. If you end up on this channel, you probably look for that type of healing. However, most people that are still somewhat sane do not look for healing. But right now I'm going to get into the top five benefits to just help you understand how this works and how I thrive as a raw vegan. Let's jump in. Number one, optimal digestion. Okay, it's all about digestion. We got to move it through, move it through fast, move it through really fast. The faster your food moves through your digestive system, the healthier you're going to be and the more accurate. Exactly right. This is something that John Rose preached as well.  You can't make this stuff up. This is really raw vegan ideology. Those people believe that if the fruit comes out of you just the way that it entered, then you are healthy. Man, this is a perfect sign of you not digesting anything.  If you eat plants, 80 to 90 percent gets excreted. Yes, it happens fast. Most of the time you get diarrhea, explosive, diarrhea through veganism.  Obviously not much will come out. Your body will be nourished. No, this is not a competition who can run faster to the toilet. You are going to be bottom line.  Your mind isn't going to work very well. You're not going to have that activation of the mind to take you to a higher place. What you're talking about is not the activation to a higher place, but a sugar rush. And to help heal. That's it. Okay, so we've got to make sure the food we're eating moves through the digestive system extremely fast. You know what the other benefit of that is? Tell me. Is that we can, it cleans the digestive system very well so we can absorb and utilize nutrition better. Are you aware that fiber actually rots in your colon and it converts to saturated fats to butyrate? That is a fact. However, meat does not sit within your colon. Yes, it digests a bit slower, but with it, it gives you sustainable energy. You get more nutrition from less food and less digestive effort. What kind of nutrition do you find in fruits? Please explain. Where's the protein? Where's the saturated fat? Where's vitamin D? Where's vitamin B12? You have none of it. It activates us. It activates the healing awareness that we have with it. It activates your insulin secretion. Because we are all healers. That's what it does. Okay. And it starts with optimal digestion, the root system of our body. Okay. Primary, primary healing source of our body is our gut. How about you would eat raw meat, which digests perfectly fine. So you would have no digestive effort and you wouldn't even need any healing. Hmm. All right. Number two, optimal elimination. Elimination is the key to all healing. What we're not eliminating, we're accumulating. That is ridiculous as well because it presupposes that you're putting something into your body that needs to be eliminated.  Your food is already toxic. This is why you want to flush it out fast. The cooked vegans are even worse than you raw vegans. I give you that. However, still you want to flush it out. Sickness is not only an accumulation, but it is a symptom of malnutrition. The symptom of waste accumulating in the body. Now, the mainstream medical narrative says that all the different symptoms are a different disease. And we have different pharmaceutical drugs for the different diseases when they're all just symptoms of one disease. The accumulation of waste, which creates inflammation. And that means that the only disease that we suffer from is poor elimination. Again, this is false as well. As I said, most people nowadays suffer from malnutrition, not because they don't get enough calories. No, no, no. They get too much calories, but they don't get enough micronutrients. That is crucial. However, you know me, I'm always honest on this channel and I do agree with your stance on Western medicine. Poor elimination is the root cause of all of your suffering. Wrong. Mentally, physically, spiritually and emotionally. If we can eat fast digesting foods, clean out the digestive system and have optimal elimination, it's going to activate. It is a false God. This is what you see here again. They worship nutrition. Don't get me wrong. This channel is about nutrition as well. However, once you eat your species specific diet, you don't worry about it anymore.  That doesn't matter. Then you can really focus on the things that are important in life. For example, building a family and providing for that family or truly aspiring to finding God within. You don't need any fancy nutrition tips in order to reach God. But of course, this is not what they are interested in. They're interested in spiritual ascension, which is a pipe dream that they chase and never reach. On a very high level, we cannot have disease in our body if we have incredibly good elimination. Period. Bottom line. We have to eat food that supports immaculate and exceptional elimination for an immaculate and exceptional connection to our consciousness. It's that simple. That simple. Reach higher consciousness by going to the toilet 10 times. Number three is optimal microbiome. The microbiome is all the microorganisms that create our body, the DNA and the molecules that make up our body mostly. The microbiome doesn't create the DNA. Microorganisms. If we feed the microbiome fruit, which is high in fiber and high in water and high in enzymes and high in vitamins and minerals, we are going to empower those microorganisms to activate our body, activate our cells. Also, the enzymes in fruit bring electrons into the mitochondria of the cell. If we want to feel exceptional energy and vitality on raw food, we just simply need to eat high enzymatic food, which is fruit. It's so simple. The microbiome is the whole. Are you aware that all the fruit that you're eating is not found in our natural environment, especially not as white people? We never had access to that fruit until the industrial revolution. Do you get this? On top of that, those fruits are modified. They do not occur like that in nature. Look into wild bananas, wild berries. There's very, very little flesh on it.  All of those fruits that you see in the supermarket are modified. They are created by us. So how can it be that we need a man-made created fruit in order to ascend to higher consciousness? Key to our well-being and our consciousness and we share this body with ancient microbes and we cannot underestimate. Again, if there are ancient microbes, why do they need modern-day fruit? What we feed those microbes. If we feed those microbes the wrong foods, dark energy foods, meat and dairy and cooked foods and starches and processed foods, we are creating a dark, heavy rain cloud over us. It's very hard to escape when the microbes demand that food. But how do you explain that pretty much all ex-vegans feel better, lighter mood, they feel happier, they're not hangry any longer. It seems like this dark cloud has been lifted after they go ex-vegan. It's strange. It's not even us. We want light and happiness and consciousness and joy and freedom. That's what we want. But the microbes keep us down. So be very careful when you feed those microbes. Fruit will heal them, okay? Fruit will read. You can tell me what you want. You are starving.   How do I know? I did it. I did it myself. I was a raw vegan myself. I was a fruitarian myself. I was a regular vegan myself. I was starving constantly. And after years you would think that those microbes would adjust to the input. But they never did because guess what? I am a human and everybody feels the same way. I talked to hundreds and hundreds of ex-vegans, ex-fruitarians, even raw vegans that have been raw vegan for a decade. They all admitted after they snapped out of it that they've been malnourished. They were constantly starving. This beautiful day where you ingest flesh for the first time again after starving yourself for so long is a memory that you will never lose in your life. It is beautiful. I will never forget my first piece of sushi after being vegan for four years. All of a sudden you will realize that you've been eating only side dishes. Structure and rebalance your microbiome. Number four, optimal energy, okay? Impossible. Energy is the key and not just energy. Energy that comes from within the cell. Energy that feeds our mind. What is real energy? You are aware that within the cell you have ATP storage as well which requires creatine which you do not have in your diet. Again, you're just running on sugar. You do not have optimal energy that feeds our mind.  You want to calm your mind. Energy is freedom from the body being burdened so the mind can be activated. And when the mind is activated it can see the healing pathways before us. Healing our feelings. No, yet again you want to calm your mind, not activate your mind. Your mind just chatters and tells you all kinds of things. Sometimes positive, sometimes negative. It's all BS. It's always a lie. You want to calm your mind so you can truly see. Physical body, yes of disease and ailments but also healing ourselves, healing the future, healing our potential and our purpose because you know why we came here to this planet? Tell me. To contribute to the energy, to contribute to the fabric of reality around us. When we eat. I came here to serve God. Fruit and we have very fast digestion and we get more nutrition and more energy from less food and less work. We have freed ourselves up to contribute energetically to the fabric of reality.  Tension of love and joy and peace into all existence and the future. We need to be thinking many generations ahead. Eating fruit serves the future because it serves us in this present moment where we can put our purpose forth. Right here we can put our purpose into the present moment. Well, all by eating fruits. That is amazing. I really get into the present moment when I eat a steak. Better way to do that than to eat a fruit based diet. What? Why? Okay. Number five, optimal mental health and mental health is simply having our full faculties, having our full joy, feeling good, feeling optimistic. If you're someone who struggles with anxiety and depression and feeling lack of motivation, again, if you're into studies, you can check it out and find that most vegetarians and vegans are depressed. You're gonna say, well, they're not raw vegans. Most raw vegans and fruitarians are super anxious. They're nervous messes. Why is that so? Because they're not getting any B vitamins on their diet. No proper vitamin B12. Again, no vitamin D3, no DHAEP, no cholesterol, colon, no creatin, nothing. And therefore they become very unhappy, very nervous, very depressed. Knowing where your purpose is and maybe not even having a drive to figure out where your purpose is, you need to eat fruit. Fruit gives us the highest level of mental health because it gives us that energy which feeds the higher mind and that brings us into our higher self. Absolutely.  Then you need a couple of transport drivers that eat meat and bring those fruits to the supermarket. Then you need a couple of cashiers and employees within that supermarket to sell you those fruits. Maybe you drive to the supermarket in your car that was built by meat eaters. But don't worry about it. You're living up to your highest potential. Then we can see the bird's eye perspective. We know exactly where to go and what to do. It feels good and there's nothing more rewarding than feeling a sense of contribution and purpose. That's what heals mental illness. That's what heals apathy and depression and anxiety. When you have purpose, I agree. When people look up to you, do you know when I went raw vegan for the first time, I created my group Raw Vegan Heroes. This is my group coaching program because I knew if I had people around me that I was coaching, hundreds of people in my group, that I would be accountable to them.  All these people are counting on me. We've all been there. I was a raw vegan slash a vegan YouTuber and I felt the same responsibility. But in the end, you understand that it is your life and you cannot malnourish yourself for your vegan audience. But of course, this will keep you trapped longer. You created your own prison. Have to understand that accountability to other people on this healing lifestyle is actually how we go from where we are to where we want to go. That's why I created the Raw Vegan Heroes community. It's all ego, of course. He wants to be a raw vegan healer. This is his persona. He believes he's super spiritual, but in reality is super attached to his persona. And now it serves hundreds and hundreds of people from all over the world. The Raw Vegan Heroes community is a coaching program that you can join. And when you join, you get support from me in weekly live streams. I have a 14 day meal plan filled with transitional cooked vegan, high raw and fully raw recipes over 35 people. People really need a plan for eating fruits only recipes that you can learn about how to be a fruit based raw vegan, how to be an undenatured raw vegan. It's undenatured raw vegan by eating denatured unnatural seedless fruits. All right, I saw enough. All right guys, and this is it for this video long enough as it is. If you liked it, leave it a thumbs up if you haven't subscribed already. Guys, please do so. And if you want to support this channel, all the links are in the description box below. Thank you very much. All right, may God bless you all much love and peace.


President of the United States. Well first, I want to welcome you and thank you for taking time from your busy schedules to come to Washington. These remarks are also being videotaped for the Williamsburg Conference for International Youth Exchange, so to all of you attending that conference, let me extend a welcome. This is such a distinguished group, both here and in Williamsburg. Perhaps I shouldn't tell the story of the mama mouse who was trying to teach her offspring the ways of the world and found herself one day and her family face to face with a great big cat.  And the cat, of course, turned tail and headed for where it came from, turned to her little ones and she mama mouse said, now you see, that's the importance of a second language.  Well we do need a second language, a language of understanding. Two years ago today I took the oath of office and in my remarks I said that peace is the highest aspiration of the American people. We will negotiate for it, sacrifice for it. I still believe deeply in those words and the best way, the only way to that peace is through understanding among nations. Some of you may remember what Winston Churchill said of the United States following World War II, what other nation in history when it became supremely powerful has had no thought of territorial aggrandizement, no ambition but to use its resources for the good of the world. I think that was one of the finest chapters in our history and we have every reason to be proud. But today almost 40 years later there are many in the world, especially young people who have no personal memory of that period, who do not understand America and what she represents. They do not know that America still possesses. That spirit that Churchill described, a lack of understanding is a serious problem for our future and that's where you, the members of the President's Council for International Youth Exchange enter the picture. You and I strongly believe in the American ideal. We must trust our system and our values enough to know that young people from other countries, if they have a chance to visit us and live among us, will come to understand the American experience and there's another side to this idea. While we receive guests from other countries, we will also send our own young people to experience other cultures and to carry the American values to their host countries. To help promote the exchange, last May I propose the International Youth Exchange Initiative, which was endorsed for the leaders of the six other countries at the Versailles Economic Summit and I'm pleased that representatives of our Versailles partners could be here today. I want to thank the members of the President's Inaugural Trust for the donation of a million dollars to the Youth Exchange Program and I understand that almost another million has been, what, it's four million, a four million, all right, sold to the gentleman in the corner chair and I understand that another million has been pledged by Equitable Life, Atlantic Richfield, NVF, Phillips Industries, Time, Westinghouse, and Archer Daniels Midland. Now how much does that make it? That's the million. Well, I'm confident that these are the first of many donations from the private sector to reach our goal of ten million dollars over the next three years. The Council's work underscores our strong belief in the private sector's role in building the bridges of understanding. Understanding cannot be measured, but our reward will come from those first awkward introductions when a young person meets his or her host family and just months later when those teary-eyed goodbyes as friends part company. So I thank you all for being part of this transformation of nations into individuals. And just within the last few days, if I could add a little experience that I was not present to see, but that others of our administration were while we've been entertaining Prime Minister Nakasone of Japan. And at a dinner just recently, he and his wife and lovely daughter, he in a toast revealed that his daughter had been in such an exchange to the United States. And before he finished telling about what this experience had meant, his daughter was in tears and he was crying. And he also related and the young man who had been the exchange or the family and that he had seen them and that they too had shed tears and their happy memories of having their daughter in this country. And they felt the same way about the young man that had been in their home on the exchange. And I just thought it was very convenient that this little experience that happened right now while we're meeting here. But again, I thank you all very much. And what this exchange does is carries out something I've long believed. The world will be all right if we all start talking to each other instead of about each other. Thank you very much. Mr. President, we thank you. And as chairman of your council for International Youth Exchange, I can assure you we share your conviction that the exchange of young people undergirds the cause of peace and broadens international understanding importantly. And we want you to know, Mr. President, that we are indeed pleased to have this opportunity to serve our nation through private sector cooperation with government. To you, sir, we pledge our very best performance in support of your initiative with the intent to carry through to success. The million dollar contribution from the inaugural trust represented here today by Charlie Wick, Bob Gray, Bill Fitzgerald and Ambassador Mittendorf is a big first step that puts us on our way. But to you, sir, we promise to stay the course all the way until we have raised the entire 10 million dollars. Mr. President, we thank you for being with us today. It's getting recognition. They had tried repeatedly as we all know they get off the ground with their new flying contraption. And this story fits. They wired their sister captured. We have actually flown 120 feet. We'll be home for Christmas. She received a wire of their news and ran all the way to the local newspaper office, handed the wire to the city after a local favor. He looked at the wire and said, well, isn't it nice they're going to be home for Christmas? Because we are making problems in building for years without being addressed and they will be solved. So while I'm at it, I wonder if I might ask Drew Lewis to take Schmike to stand so we can salute them for the dedication and excellence of which they serve their country. And three, this is Jean Gilpatrick is not only a member of the cabinet, but the main United States is stronger than either the peace, freedom, progress abroad, and through it all, to renew our faith in the God who is what is our life. And he has to face the awesome progress and see only two of our housing models under numerous consumers in the galaxy that's ever reducing the is your mind. You earn it together. We'll stop the case. And gradually more and more of the way and by facing up to a different we will ensure that the lifeline of socials we're restoring dignity on the uniform and I think we believe in the moral power of Western civilization. And in America, it is to keep in your channel.


The call to order the Monday, May 16th, 2016, and the Ireland Community Development Board recorded by ACMI. First in our agenda this evening is Certificate of Completion for the Bright View Assisted Living Project, and Joshua Davis is here to make that request. Good evening. This relates to a special permit that you all issued in 2012 for the assisted living facility at the old Sims Hospital site. And my client neglected to ask for a certificate of completion at the same time that they got the final certificate of occupancy. This is sort of a, as some of you may recall, this is a parallel process because of the land disposition agreement and the urban renewal, the fact that it was an urban renewal project. So the certificate is simply indicating that the improvements have been constructed. It's not saying that everything else under the land disposition agreement. But a lot of the requirements have been fulfilled. Those are mostly ongoing. So the request tonight is to ask the board to have the chairman sign it, notarize it, so that we can record it and satisfy Bright View's lender that everything is OK. I think that answered your question. I think it was just for the board. Wait, so that's done? Well, I believe we're a chat to make sure that. Did you do a drive-by? I had a reception. Did you do a drive-by? About a year ago. Well, I will just say that I did speak to both in special services and the ten engineers' office, and both of them said that there were no outstanding issues related to the project. Excellent. Yeah, we closed down the input plan quite a while ago. I had nothing better to do, so I drafted a motion if you want it. We're at it. Without Bruce here, we need some of the details. Let me just read it first, and then I'll make sure I agree. Yep, OK. I move that we vote to approve and issue a certificate of completion under the land disposition agreement dated as of August 25, 2004, between the board and Sims Redevelopment Associates LLC as amended to write you, Arlington LLC, as owner of the assisted living land described in the agreement, in the form submitted and to authorize the chair to execute a knowledge before a notary and deliver the certificate on behalf of the board. And actually, I would say the land disposition agreement dated as of August 25, 2004, as amended between. Oh, I'm sorry. There it is. Never mind. Second? Motion. All in favor? All right, all right. Thank you very much. Thank you, Josh. Enjoy town meeting. Those of you who are not. I'll give you a call in a couple of days. Yeah, I'll let you know as soon as I get back. All right, great. Thank you very much. You're welcome. Moving on, recap of town meeting and next steps. Jenny. So I thought we could spend a little bit of time just sort of reviewing everything that happened at town meeting with the zoning warrant articles the last time you met. You had a brief discussion or celebration of articles six and seven. And so after that point in time was then all of the residential zoning warrant articles. So I wanted to just give you some air time to talk about that and if you wanted to. And also then talk about what are the next steps for moving forward, which include responding to the article 11 motion. And along those lines, what I wanted to share with you is beginning work with Adam Chatelain, the town manager, to devise what will be sort of a recodification committee that would be in relation to the master plan implementation committee and then also a subcommittee that would be looking at residential issues. That's the sort of beginning thinking. Further fleshed out once we get to the master plan implementation committee meeting on May 26. That's kind of just the timeline of things, but we're still working out details and understanding a little bit more about what that article 11 motion says, making sure we're responsive to it, and also making sure that we're responsive to the recommendations that were in the master plan, which included a zoning audit and some very clear steps that we need to take in terms of sort of looking at the zoning, cleaning it up, and then also addressing if we can some policy issues to align the master plan with the zoning with the master plan. So I wanted to just put this on the agenda so you had time to talk about it before the master plan implementation committee meeting. Yeah, so I think it was, I certainly felt that it was a lot of confusion and that type of thing. And I think part of it was a little bit of, well, two-fold. Number one is, is I think we were nibbling at the edges a little bit on what it was that we were trying to do and trying to explain it for one reason rather than the other type of thing. And I think then we need to be kind of transparent about, and I think we always were. We always said that the gradation of the driveway would push back in that type of thing. But I think we kind of got to lay it all out a little bit. If that's the approach we want to take, then we need to lay it out a little more clearly. But the other part that I guess I kind of am curious about is what folks in our position kind of old land use type zoning and that type of thing. How other communities similar in nature to Arlington have gone about this kind of process and how, whether they've done it in a piecemeal fashion or whether they just kind of tried to rip off the Band-Aid with some rather large recodification. So I guess at this point, I don't have enough knowledge of it to understand which way other communities have gone, what the pros and cons are of that type of thing. I think what we're trying to do was make some changes that could help move us along. But part of me thinks maybe we're better off just kind of looking at it holistically and trying to figure out everything that we may want to do and how we may want to do it. And I guess I'm just not into reinventing the wheel. There's got to be other communities that have been in kind of a position with 40-year-old zoning, 40-plus-year zoning that when they did bring it up, maybe it's form-based, maybe it's something else, some other ideas. And then finally, just kind of doing that soon so that we can kind of get in front of it if we do want to do it for next year. Sadly, it's just not as much time as you think in that whole time. Anyway, so those are my thoughts after that whole time. I can wait to respond if you all want to continue or respond to it. Yeah, no, I mean. So just one little tip it is, we did allocate $50,000 in community development block grant funds towards recodification of our master planning. So we will have that to work with. Because that was always part of the problem is we didn't have those funds. How many funds? So that'll be active July 1st. And so the beginning of the process, and Laura, you can fill in the blanks if you want to see anything, but the beginning of the process is kind of scoping it out. What exactly do we want to do? What's feasible? What can some of our staff be able to possibly work on or advance versus what do we need a consultant to help us with? Which I think we definitely will need a consultant to help us with this if we go down the path of wanting to recodify completely. And then part of that is also kind of having a conversation about what are our goals. And I don't know that we've actually had that conversation. Maybe you had that conversation during the master planning process to an extent, but I think now it's time to revisit that conversation and understand what we must do and then versus the things we might have to live with. Which is somewhat contingent upon funding, somewhat is contingent upon the timeline, and possibly other factors as well. Because I think a big part of confusion and the fact zoning is hard and everything else is the way it's currently set up, right? Like I said, we're kind of nipping at the edges here and no one understands if we change this, then this has to happen. Well, what does this actually mean? I live on a hill. But there isn't this kind of, it's all, to me, it would almost be easier to explain a recodification completely, you know, completely because you don't end up with this Swiss cheese that we have over here. You know, of this parcel is this, this parcel is that. Yeah, the master plan when RKG was doing the zoning audit, my understanding is based upon the conclusion of the audit was to, you know, the town had a couple of choices. One is to completely recodify, address the numerous districts that were in place and clean it up because there's a lot of language that's either outdated or does not align with, you know, sort of current best practices or, you know, do bits and pieces sort of along the way, which many communities do that because it takes, it's quite a process to recodify. But there are communities who if they have the foresight and the will, you know, organize a group of people like a larger committee of people, which includes, you know, cross-section of staff, various different boards and committees that deal with land use and zoning and policy issues and, you know, may include other, you know, residents or business leaders, et cetera, depending upon what your, again, what the goals are of that process. And, you know, you can get at it in the big way, cleaning everything up, get at it in little pieces and then maybe you can also get to the policy issues. And that means taking the visions and the goals and the strategies that are enumerated in the master plan and saying, well, how do we need to re-zoning in order to achieve these goals? Because a lot of them actually do require, you know, zoning initiatives in order to advance them. So right now the zoning doesn't match the master plan in a lot of ways. And I think that that's, you know, taken out in various strategies. Yeah, I remember as we were passing the master plan a year ago or talking about it, you know, one of the big things was to do that kind of, okay, if we went that direction here or the changes that it would make, but we didn't have the money to do that. So, you know, I'm wondering with this CDBG, how far it does against, do you remember that as well? Or yeah. Yeah, it may not get us all the way through. Right, I'm sure it wouldn't, but it'd be nice to understand where it would start and how far it gets us. Because I think that's actually, once again, I think that's more easily explainable than kind of, you know, they're picking the opposite. I think that comes back to kind of what are the goals of the project? You know, what do we ultimately want to achieve at the end? And then what can, what's the scope of work to achieve that goal? And then once we get the scope, we can figure out what the, what we think are some of the cost estimates to move forward. And then obviously go up to our, have an RFP to bring somebody on. The other kind of cost factor would be community engagement, which I think is, to me, the most illuminating part of town meeting is the need for a lot more community engagement and across the town. And that we want to be very deliberate about how we do that. And we want to be, we need to be engaging very broadly because it's both an educational process as well as, you know, a process to tell people, you know, the way that we do the zoning and the way that the process works in town. So I think that that's, and we want their input, you know, there's a piece, the master plan is completed, yes, but that doesn't mean that we never ask for it, but it can on what we want, you know, what is the vision of the town, and what do we want, how do we want to see things moving forward? So I think that's part of that process as well as engaging people in that vision to get to some other endpoint. I think one of the toughest parts with that community engagement is sort of two of the toughest parts. One is we see the same folks a lot, which is great. I mean, they're very active and everything else, but to get that next level is something else. Now, we've got enough press that maybe it does bring, but the other part of zoning too is you get a very individualistic view of it, right? How is this going to affect me and my property? Which is totally understandable, but, you know, the civic engagement we're looking for is maybe a little bit broader. And I think that's where I think it is a challenge to try to figure out how you navigate that. I think it's gonna be really difficult if we're gonna go and do a holistic zoning change and do it within a year. And with the funds we have set aside, I see it as sort of three parts right now, how could be relevant, but the first part is like you said, understanding what the mission is and what we're trying to do. And then establishing how we go up through that. I mean, with who, with the outreach, community, everybody, business, the business people, the contrary, everybody. And then third thing is just drafting it so everybody understands it. And putting it in such a way where it's, I just can't see that happening that soon. Yeah, it's pronounced. And so we have to ask ourselves a question is do we say that we're gonna do a big approach and then we should let everybody know that with this big approach. It's gonna be more than a year. Or are we gonna say no, we're gonna keep to our word saying we're gonna look at this over the next year and bring something up for the next town meeting. And that's something that I think we have sort of decided right now. Or at least think about it. Well, I think it is a couple of things. And I can talk about this with the master plan implementation committee next week on that committee as well. But given the conversations that we had, given the turnover in town, given the turnover in town, this might be a good time to start to take a look at the master plan and begin that first review of where we are. Even though it was only approved a year ago, it really started, and I don't remember how long it started, Laura. Is it five, two and a half years? Before, yeah. Between the first, the World Cafe. Yeah, I remember that. To adoption, it's two and a half years. And so now it's three and a half years. So it's probably time to start this summer at looking at what we want to accomplish there and work with them to maybe hold a forum or to one specifically geared towards zoning to find out what direction we should head in and how we should have held a forum. Who would be, I'm not following. We start a public forum. With just 10 meetings. Something like the World Cafe. Something open to the general populace of the town to get ideas and start to head into this process. And we can put the option that there's a holistic review in doing an entire reconification. Maybe what's necessary, we can look at some other better crafted piecemeal changes. But I think that's where we probably should start. With a review.  I'm saying those are the options. Figuring out how we approach this so that we're doing it. And I don't know what the answer is. I don't know either, but we'll get there. Is the master plan implementation committee taking a look at what other communities have done in order to bring specifically, I guess zoning as much as anything else into the 21st century? Like what, you know? And it just, I can't look at this. And it just, it's not intuitive. It's not easy. It's not, you know, it gets crushed under its own weight. And maybe that's the way it needs to be, but. You don't have to need to have that many districts. Yeah, and I think for me, I think simplification in order to make sure that we understand the goals. But I think the goals were actually fairly well set up in the master plan itself. But what can be done from a simplification perspective, both from a, you know, a synthesis, you were talking about kind of going through and making sure it all gels, right? And I think that is part of the problem. And then to say, okay, and then here are the things that we're trying to accomplish with respect to maybe sizing with respect to a bunch of other things. How are communities addressing from a best practice perspective? You know, I think part of that was missing from the conversation. And, you know, and because it was missing, it led to, you know, what seemed like very technical changes that would have big impacts that no one really understood. So anyway, so. I think one of the takeaways I had from this past year is that there's certain things that there's a lot of general agreement about. It was kind of remarkably easy to change the mixed use and the parking. I think everybody would agree that there's too many zoning districts. Those won't be hard. Right. I don't know. I kind of disagree a little bit, okay? Because someone's gonna have property and those scattered all of those districts there and they're gonna want to realize how's this gonna affect my value?  And all of a sudden it changes zoning or under the guise of simplifying zoning. All of a sudden they perceive that they're losing value. That's a bad thing. But that's gonna happen no matter what. Maybe the game value. Well, that's true. The game value. But that's gonna be quite a bit of a big up there. Oh, I agree, but that's why you need to have, like as your backup, what other communities have done. Like, why is this the way to go? You know what I mean? Like, I agree with you completely. And people will be affected. But that's why it's even more, and I think we saw that with this, even in this year when we did it, right? I mean, that's what everyone said. But we couldn't really explain it well enough to say, yes, but here's why, you know, from a community as a whole, this is the right thing to do. You know, yes, people will be affected. We'll try to mitigate some of those effects either with timing or something like that. But I agree with you, but I don't think we can avoid that fact. But I think that's why we need to kind of get our ducks in a row, is because people will be affected. And so if we don't have a good explanation of why and how, and you know, these communities did it and here is the result, you know, then I think we're just gonna end up with what we had this year, which was a lot of confusion. So the point of this conversation was just to let, you have some of your time to kind of reflect on what occurred at town meeting and, you know, the next steps moving forward. And also just to kind of get your feedback for things that we can bring to, back to the master plan implementation committee. I think we put the card before the horse in the right. The way I'm hearing, I mean, you're talking about recodification, out-of-pacation, which is the master plan is articulated, things that are great about this community, things that need to be fixed, things that need to be reinforced. And they went about it, our master plan went about it by talking about neighborhoods, didn't it? It said there was a this area, there was a civic area, there was a central development area, which is also called no brook sometimes. There was other things that we wanna keep the same way they are, but it proved them. So there were goals set out by the master plan. The recodification is a tool, a potential way, to deal with those. And I completely agree with Mike's comments that you need, we need to know how that's being done in other communities. So maybe there shouldn't be a recodification. Should there be and how should it be done? But then we superimpose from out of left field one of the issues which has to do with nationalization and a bit of tiptoeing to my mind around that issue. If it's a good idea and it's part of the master plan, then own it and then say how are you going to do that? Maybe it is through recodification. Instead, we immediately got into the weeds on how you could kind of tweak zoning in order to make it kind of work to make houses small. But then we found out, well, that's too much of a broad brush approach. That's not really gonna work, so we kind of learned something, but I think we were ahead of ourselves. Obviously we learned that. So I think there are two things that are going on. What is the bigger picture of the method that we go about the zoning change? Is it, how has it done in other communities? Is it a good thing to do it that way? Do other communities do it on a piecemeal basis? Have very strong goals that are articulated? And then the second one is, okay, now we've got a kind of a way to do it. Here's an issue that apparently is very important to the master plan, which is mansionization. Let's attack it in that context. So I think we had double whammy against us because we went into detail without a clear goal being articulated. And I think that was the difference of the mixed use. I think you're absolutely right. This time on mixed use, we had our ducks lined up really well on why this was important. Right out of the master plan. Right out of the master plan, and also here's what it is. Yes, then we got into some specifics, but people, they were concerned about the specifics, but when you put them in the context of what it was we were trying to do and what the why you provided for greater height, but you had step backs and everything. Like once you could put it in the context of policy, here's what we mixed use, the goal was to develop our commercial. To improve, to make it more productive, bring people into the community, all the things that were part of them, clearly articulated. Whereas when we tried to attack mansionization, even that, didn't want to use the word. So we didn't really have a clear goal, because I think I found out that in some cases we want to let houses maybe get bigger, some houses we don't. I'm not sure now, so we kind of learned. So we have to get that goal very clear, but if, and I'm hearing two things again. Recodification is a saying where our tools aren't right for a number of things that we want to do, and we ought to articulate all those things. Sizing of dimensions, dehumanization is only one of those things. Right, definitely. Yeah, that was only, that was not everything. That is one piece, one subset of many other things. So, and just since I have a list in front of me that Laura put together, I'll read it. So the kinds of things that were talked about were reduce the number of uses that require a special permit, consolidate and refine the business zoning districts, create commercial and industrial design guidelines and incorporate them into the zoning bylaw, amend the zoning bylaw to provide redevelopment incentives in all or selected portions of the business districts, et cetera. So I mean, there's a lot of other things that are talked about in the master plan that relate to the zoning bylaw that again relate back to the master plan goals and strategies. And yes, other communities when they adopt the master plan the next step is them looking at the, what are the policies that need to change or the regulatory process? That's a typical next step in a process. And sometimes you could go about that by completely revamping the zoning bylaw or creating lots of overlay districts or patching up one piece at a time. I mean, it very much depends upon the community and the process involved in adopting zoning, which of course varies depending upon the type of community of your charter. In this community with town meeting, we have a very special, we have a unique process and it requires a lot more education. It requires a lot more outreach and buy-in and that's a little bit different, I would say, than some other places, but not incredibly so. But you still need to have that process, but the timeline is longer so we need to make it very intentional and we want people to understand what's happening to your point, the how and the why. Yep. So, and the impact and having an understanding of, why are we doing this? Are there other communities that have done something similar? What were the outcomes, et cetera? So all of that stuff would be part of that scope that I was talking about of what exactly are we doing here? What are the goals of this process? Do we want to get at addressing many of these issues? Some of these issues, sort of just cleaning it up, having the zoning bylaw makes sense and making sure that it's clearly communicated to the people who need to use it the most, which is a combination of our customers, the developers, development community, the building inspection office, the planning department, the residents, everybody. We all need to understand what it means and if we can't read it or there's contradictions in it, then we, I think that that's a clear indicator that, and that's pointed out frankly in the zoning audit and that's something we should probably address. Then everything else is kind of part two, which is do we want to get to policy issues? Do we want to get to some of the other bigger goals that we're probably very passionate about other than commercial districts and things of that nature? There are a lot of things, in other words, like Milbrook, for example, you mentioned that. So one is just the system kind of thing. This is the tool, making it work better for you for the whole approval system. For all the users, yeah. And the other is a series of goals that would be brought to bear. So I think we sort through those in a bigger picture way. Right, back to Andrew's point, I think, which is the timeline for, I can actually, you noted this, which is the timeline is very tight. And the reality is, this is my understanding, I wasn't here, but you adopted the master plan and then it took a while to appoint the master plan implementation committee. There was a gap of time, and you had probably roughly, I would guess six months, maybe less, to develop those zoning recommendations. Or even less. Maybe less, right, four months because you took a warrant, it was January, like October, January, like two months. So we don't want to start this in September or October, for that matter, we want to start soon. Although I will say, from my perspective, I'm more interested in how we want to tackle it than the timing of it. I mean, if what we were talking about was something a little bit grander, if you will, and we need money and we need time and that. I think all of that is actually fairly easily explained if you have exactly what it is that the project is, right? And I don't think, so I think that's the big question for us sooner rather than later, is how much are we gonna tackle in what order, so. And so that's the idea of the master plan implementation committee meeting on May 26th is to get at this issue and to also understand how we need to be responsive to the article 11 resolution. Central school. So I realized after putting this on here, the RFP is still open. So the bids are due actually on Monday, May 23rd. So I don't actually have much of an update. We had a walk through last two weeks ago now and actually made that and have had a number of people and organizations who are interested including town departments. So there's, it's still in process. So nothing is definitive at this point in time. But what I wanted to ask is if any one of you would want to participate in the review of the proposals because I had not asked that previously once we receive them to evaluate the proposals against the criteria and make a decision for help with the decision. Any volunteers? Yeah. I don't think I know enough about the central school. I mean, how do you know if this was your agenda? I did it last time so I can kind of look into it. I just was sensitive to your time. Yeah, exactly. Yeah. Well, if you wanna, if you need help, I would help you out. I don't know enough about it, I'll do it. You're on the feasibility committee for the senior center. Okay. So it would make sense for you to continue but if you can't be there, maybe an alternate. I can help. Okay, perfect. I just don't know enough about that. And I can help you to have to speak. Okay, yeah. Or if anybody else I'm thinking could help as well. Yeah, me and you. Having the experience of. I'll be sure. Okay. I'll do it. Okay, thank you. Okay. Meeting minutes from the point. Yeah. I didn't have an issue with the zip. I can, I don't know the motion. Yeah, I was looking at it. I would like the motion to approve by do both at the same time. Well, I think Andy wasn't at the first one and I wasn't at the second. So we probably should separate the two of them. Yeah, okay. All right. Motion to approve the list. April 25th, 2016. I'll second that. Fall in favor. Aye. I guess that's the one I abstained. Yeah, yeah. I abstained from that one. I approve the meeting minutes from May 2nd, 2016. I get, you know, there's a red word. Second. All in favor. Aye. And I'm abstaining from that one. Okay. Anything else we have not discussed? When's next meeting? June 6th, June 6th. Yes, June 6th. Two weeks, though. You're granted a break. That's right. Memorial meeting. Okay. Do we have anything on the docket for June 6th? We do. Yes. We will have a special permit hearing for the, I'm always forgetting them. It's learned to grow, but they're called the spring board schools. Yes. And there may be another one as well. And the housing production plan. And the housing production plan. So that actually could be a very important answer to the question that was, was consultants were coming. And the plan needs to be adopted by the sport, as well as the Board of Select. And you'd expect that, right? Up to you. Okay. If you're comfortable. Yes, but it doesn't have to be. Do we get a preview of that then? Yes, yes. Yeah, just this week and then it'll be final Monday night, final issue. The committee, the advisory committee is going to review it next Monday and make changes. And then sort of give one more revision. It's long. It's very long. But you know, you can focus on what interests you most, but you know, you can focus more on the strategies, the goals and strategies. The pictures. Yes. We like pictures. I'll move to adjourn and reconvene. Well, are we reconvening? Or am I the only one reconvening? And thank you. Yeah, so I don't think we're going to reconvene. I think we're only going to adjourn. So I make a, I move to adjourn. Second. All in favor. All right. Thank you.


Appalachian Wireless offers the easy way to pay with forward pay. And now it's even easier with unlimited talk text with three gigs for just $44.99. No credit check, no contract, no problem. That's today's Appalachian Wireless. Friday afternoon, police cars and firetrucks drove through the community of River in Johnson County, complete with lights and sirens. Johnson County Sheriff's Cruisers were packed full of toys. Santa and Mrs. Claus were on the back of a firetruck and members of the community came to meet the little girl who sparked this entire effort, a six-year-old girl named Dora, who stopped smiling. When I went to the school with Mrs. Claus, Dora was put in my lap and she didn't smile. She didn't climb up, she didn't hold me like the other kids. So when she got down I asked her teachers, what's wrong? And she said her mommy's sick. Four months ago, Dora's mother, Rebecca Colvin, was diagnosed with breast cancer and that was followed a month later by more bad news. And found that it was stage four breast cancer. It was in my breastbone and spine. And even though she doesn't really understand how serious that is, seeing the toll that the treatments are taking on her mother has obviously affected Dora. I don't know that six months ago it didn't seem like she knew a stranger. She was like just as quick going to somebody as she was with me. But for a little while Friday afternoon, Dora smiled. She even laughed as she opened presents with her family. Presents they would not have gotten had Dora not touched so many people. This would have been probably the worst Christmas that I'm aware of that they've ever been able to have. With the gas money and to get back and forth and everything, it's pretty strapped. Rebecca says her doctors say her cancer is responding to the treatments, but that still doesn't stop her family from worrying. I thought that my nieces may not remember her mother, especially the youngest Samantha, you know, she's two. But everyone will always remember this Christmas. When Santa and Mrs. Claus delivered presents and everyone was together. And even though Rebecca says times are tough and the treatments take a toll on her body, she's not giving up. I'm going to fight it. There's just no two ways about it. I'm not much from one just sitting down rolling over and just letting it happen. I'm going to fight it. I've got a long life to live and I'm going to leave it with my kids and my husband and my family. Reporting in River Kentucky for EKB News, I'm Shannon Deskins.