If everybody says yes, but nobody's actually doing anything well cornwood's gonna do something and he's gonna take over the building I'm just I'm I think I'm just gonna play cornwood like here at this point And then if if people don't like it then fire him and and it'll be done It's literally probably what I'm gonna do today rage-drag. I'm literally probably I'm literally probably I'm thinking about starting the sheriff's office today on my own If people don't like it, I'm gonna go buy a VSTR and claim high command and say I'm the sheriff And if people don't like it I'm sorry, and then they can charge corn with a treason and then cornwood is gone forever get ahead injury and arpy memory Lost your way into the sheriff's office They just there there needs to be storylines. Hi, I'm storing the sheriff's office today. What? Yeah What do you mean you're starting the sheriff's office today?   Yes Nope absolutely not yeah, I got them get my high command vehicle ready here 316. All right.  So cornwood's doing it today, okay?  I might be the only one who has this opinion But let's try not to disrupt the meeting you want to talk about with people afterwards. There's a true man Being in a Texas I have taken it upon myself That I am bored so as such I will be going up to little Texas and I will be forcing the sheriff's office open So as of today welcome and say hello to the little Alamo sheriff's office. How do we transfer in there are currently no transfers Well, I'm transferring. Okay. Sounds good Under sheriff Jimbo Sutton. I'll ride with you sir. Yeah, sir thing. I'm all in on this dispatch 316 1041 how do y'all 316 are you gonna be the 300s? I love that. Oh, can I be 369? Sure What do you think of this color with the base?  We don't get fired then all right. I'm in Hammy hell at least if I'm fired. I have something to do Yeah That's on him. I use your bling. Do you jackass? I don't know why he was resisting.  It just blows my mind.  We don't have anything here Well, we have a meeting room. So that's good. Oh my good cordwood. Yeah, this is huge Look at how big my office is gonna be now. Oh, yeah. Oh, you got a mirror do yeah I got a mirror and a sink in my office. No, this is a good start You know what I want to do on it.  You're loyal. No, no It goes to the hospital.  Yeah, I don't want nobody else getting fired just me and me you want to get fired Honestly, I'm surprised it hasn't happened yet.  Oh No, thank you though.  I agree It's been a small suggestion here because I'm looking out, right?  Yeah for how many months is everybody says one to two weeks. I'm done waiting today I'm literally I'm on my wits end right here. Here. There's a line. I'm on the line right now I'm trying to reel you back before you go over Hey, that's what I told him. I don't want to don't want to break up this conversation But I can I pinch I call me can I pinch you for a second? Yeah, it's your thing. Thanks. How's it going? I'm good. How are you? So Well, so what what happened? Didn't you know what's coming? What's that? I have to I have to inform you that you're gonna have to change your uniform to Lspd if you're on duty Okay, cuz we are currently Lspd and the sheriff's office doesn't exist Well, the hell well, how do everybody tells me yes, we can have the sheriff's office But then nobody opens it cuz it's it's not it's not a thing right now It's just it's just a building currently you are earning an Lspd wage Operating as the sheriff's office, right? Yeah, well, I mean, this is the special uniform for my unit what units little Alamo sheriff's office and join this Well, I mean, you know technically right now it's a unit I don't want to be the bad guy corn words I mean if everybody says yes and nobody does anything and how come I can't do something about it Who is the person directly? Responsible for making something like this happen. Is it the mayor? Nobody knows.  That's all we really need. We need internet and that's about it You kind of need more than that You need a structure. We have a bit. Do you need the bad bones?  Do you want me to be honest? What's that? I'm not okay with it opening. So so why do you think it ain't good to open?  We have to go all the way down here And then drive all the way back so when the issue is oh, we don't have enough units to open it up Well part of the problem is if that was open literally called it's time You reduce the amount of time you spend driving so you're actually way more efficient And you can maximize the small amount of units that you have on duty But in reality, we don't really have a small amount of units We have about 120 officers in the PD every quantifiable measure that we need to hit has been hit story has been driven in The proper direction. I don't there's nothing else to do and I'm just stuck here sitting on my hands for the last however You know I mean like I literally dropped out of the mayor's election because of the sheriff's office And now we're about to go into a second mayor's election. What if you went into a mayor? What what if you went into the mayor or opposition and made it happen though? Yeah, that's honestly, I was going to do that. Yeah, so I get the whole restock thing But I mean, you know, you could fill up your locked storage with a with a few spires Etc coming down to process people There was a time in which we were having to go all the way to Polito to process people because none of the PDs were open It's that Polito. Yeah, it's a nuisance, but we still did it, right? Right. So that's what my plan is So I'm gonna go operate out of there.  That's the point. I got to wear the uniform so people know I mean business I don't think anyone doubts that you mean business Conward There is definitely an argument for a sheriff's department in the future, right? Right now I think it's gonna be impossible to manage two departments.  Why?   We went through that shit, man No, I give that but at least something is happening things are happening I mean, you know, we got a chief and an assistant chief was it three weeks ago and four weeks ago And what has happened since whenever I was around there was always something happening Like I gotta have a like a carrot on a stick, but I gotta be able to get the carrot eventually It's that I know it's frustrating. Listen, I don't like to be the skeptical one I don't like to be seen as the bad guy, although I often am because I'm probably the one of one of the only people that will tell People how it actually is People like the mayor saying yes to you and then doing nothing about it doesn't prove to me anything I kind of feel like that he's just saying yes, so that he doesn't look like the bad guy And look, there ain't no good guy bad guy shit There's only there's only one good guy and one bad guy and the one good guy It's us and the one bad guy is the name criminals I I Still have to inform you that you can't be wearing that that uniform I'm how many daps is one uniform.   Yeah, but then you can't you can't be patrolling or anything like what are you gonna do? I'll just give presents. This I'm a command member for the LSPD. I'm operating on behalf of LSPD Whilst you are employed by the LSPD can't wear that uniform whilst you're on duty cornward Okay, but why can't I wear it because you have an LSPD uniform You need to be seen to be represented in the LSPD not the sheriff's office Well, is the LSPD little Alamo branch because we have okay, so we have back patrol. Okay back patrol wear something different Yeah, but this that batch is still LSPD and they're still identifiable as LSPD. Oh, I'm very back V1 a what what about? Oh, it's not obvious though, man. There's a night. I mean it sounds pretty bad It's pretty bad, but it's not obvious who you're operating as okay. Well, what if I'm oh my lord I'm depressed and now my mental health is in danger because I'm can't wear this uniform We'd need like a medical exemption thing from a doctor. So maybe you should talk to a therapist cornward. Okay Oh, I'll leave you to it. All right. Good shot. All right. It sounds good. Thanks, Veev Yeah, good idea. No, dude, that's literally a character. Stop being mean to Veev.


STEAM DRUNK! Just a short video today about a simple, vertically scrolling shoot-em-up called 1917 The Alien Invasion. Just to be upfront, I got a steam key straight from the developer, and he asked me to look at it, so hey, why not? Plus I need a simple shooter after the huge basketball golf and NES videos I've done the past few weeks. So yeah, as you can see, this is a pretty straightforward, less-is-more type shoot-em-up. Not a lot of bells and whistles here, just a couple weapons, a clear-all attack, and you just try and survive. This isn't much of a bullet hell type game, although those elements do show up here and there. Most importantly, this game gets the fundamental shoot-em-up stuff right. It's a good balance of speed between your ship, enemy ships, and enemy projectiles, the latter of which get cranked up big time on boss fights. Take one hit and you're dead, but there's plenty of shields around so you can absorb one extra hit. You have unlimited lives here, but there's no checkpoint, so you start at the beginning of the level after you die. What I like best about the action here is that you really have to use the entire screen instead of just hanging back and dodging. Sometimes you have to use your surroundings like this sequence here. There's little design elements like that that pop up here and there. And yeah, since this is a shoot-em-up, this game is of course pretty dang hard, but it's not hard to the point of being totally unapproachable and inaccessible. The game's biggest flaw is that it is way, way too short.  Bear in mind though that this is the kind of game predicated on points and high score, and that's due to the combo point multiplayer system that allows you to rack up points quickly. If you don't destroy something before the meter on the lower right runs out, your multiplier is reset to one. So yeah, the game is short, but once you develop some skill for it, you can plow through it in no time flat and go for the highest score in the Steam community. I suppose I should address the title of the game at some point. 1917, The Alien Invasion, does in fact take place in 1917, and you fly a World War I style triplane through space, uh, somehow. As Dr. Brunhild Stahlmühler, who humorously insults you as you get hit. It's pretty funny. I should also mention the music in this game is excellent. Every track is memorable, really well done, and fits the game nicely. There are a few flaws here, though. One I've already mentioned is the length of the game. Another is the lack of a button map, so you can assign the fire or clear all buttons to anything else. And also, this isn't really a flaw, but the sound design behind the explosions and weapon fire could stand to be beefed up a bit more, to have a little more oomph behind them, but I guess that's just a personal preference. Anyway, yeah, 1917, The Alien Invasion is a nice, simple, approachable pick-up-and-play shoot-em-up. It's very short, and that might scare some people off, but if you're into these kind of tests of skill, it's a quality title.


as soon as the installation comes we'll get it in and get the other viscune over the top. So dad's testing out our, oh wow. Brian if you're seeing this thanks let me borrow your rattler every time you need to do this. I know what I'm doing now as well. Jesus we're having some rain. We've just got out of the van now waiting for the installation to arrive it's 20 to 10 sorry 20 to 11 23rd of November. I think guys in the galaxy is out today Christmas special anyway we've just got out of the van come round here luckily you'll be able to see our evolution of centres of wet drive vac so that's going to help us here. This is our bottom layer of viscune so we need to get all this water out ready for the installation wash his face. Al good you've got an evolution wet drive vac isn't it. Link in the description if you want to buy some evolution gear. Hit five percent off code bricklaying in capitals. Anyway yeah that's what we're up to now going to start loading out get some blocks loaded out ready to start and as soon as the installation comes we'll get it in and get the other viscune over the top so we've picked a new roll up because that's all we've got left of that roll because it's good because you can do it in one piece which is handy so let's get into it there we have it that is all we can do for now for loading means we'll get this section so that section's loaded this section's loaded it's about three and a half block return each side and then the rest of its door big door that's what that big steel's for so that's loaded this is all loaded we stack them in threes because they're a bit damp because they're seven newton concretes we need them dry so we've loaded enough for seven course there which will take us up to scaffold height which is up here seven course it's going to be a stretch but yeah that side's got to be done overhand because the the the board is just too high and out of level so it's easy to do that once the slab's in so that is it for now we're going to have a quick dinner because it's 12 o'clock then we've got things like the damp course to put in walls to mark up i think or they're already marked up yeah starter wall starters to put up both sides no choosing because it's block and we're going to use these screw ties on the inside starter packs on the outside what else can we do oh yeah we're going to work out work work our bond out and i'm going to do a load of cuts with the with the evolution so get them all cut ready so it's just profile up line up and get it up so see you after lunch so where dad's testing out our oh wow our newest toy that we got from evolution do you want to give a quick word as to what it is even though it was pretty obvious wet drive back cordless because i think i think the charger came with it so it's wet and dry you just change the filter it's just like a mesh filter this one we've also got the other filter for dust obviously and it also has a blow function so you can switch that to the end and blow things with it obviously blow your dust away it's great for this but the volume of water we've got in there we need to spade into buckets first and this like get the last little bit but it did pick up pretty quick but i think the amount of water that's in there if you show them the battery's not going to last long doing that so so reckon we'll brush it to one end scoop it into a bucket and then get the last little bit with that over and out you can't drop this mic so it's just sitting on time laps i'm doing a bit of improvisation here when you watch you when you watch the joinery channel on youtube i'm using this piece of mdf as a fence for the saw so it's worked out it's 234 to allow for the saw and it's our cut edge made up with that look at that and that's cut down to exactly 200 100 slab 100 insulation we're going to put that in behind the silatex so we have made up that bit of um bit of youtube watching a bit of improvisation does the trick so i'll um i'll put the microphone away and i'll do the next one and just film it all at once so here we go 234 vouchers a bit worried this wouldn't work yeah alex is a bit skeptical this wouldn't work and it does perfectly and we haven't got much waste here we've only got a couple of hundred mil overlap cut this into six that means you've got um 2.4 times 6 is 12 12 about 14.4 linear meters and our floor is roughly 14 point something linear meters so we've got to be careful with it so they're really beautiful they're two cut edges then made up with that and 200 mil bang on so let's get the other four cut action if you remember we were doing these at A&E so brian if you're seeing this thing so let me borrow your rattler every time we need to do this so i'm doing now as well i'll put one in the center and not drill the top one so you can overlap yeah whether you could drill it but just don't put the thing in it that's right in the bed that's fine as long as you're going in vertical that's it yeah you've got ear protection i know you're using the eight yeah 10 right oh is it 10 yeah oh yeah i double checked beautiful nice one if you're just removing the ear yep but you've just done to the coal bridging strip what sorry but you've just done to the coal bridging strip oh shit that's a blooper right market calendars thursday 24th november 2022 this bloody concrete finally came in oh it's been a long time coming with a mix of this bloody weather the waiting for the insulation just the dig went the dig went that well that all the other elements were against us but today's the day it finally goes in and as you can see it got too dark last night to get it all set up but we threw the visqueen over so now we just need to cut it about and tape it in the right places and what have you so it's going to be a bit scary this one because um i don't think we've ever done a slab this big and unfortunately we uh misplaced our current float or a finisher trial because this one's called so um we had to do a quick next full station revenue float because obviously you need one of these but nice little bit of new kits into the toy shop but there we go there's how it is everywhere which is it went in dry that's just come that's just it's not rain that's condensation out of here yeah that's just condensation gathering because it's warmer than either cold on the top it's just flooded and we kept we've got no rags we forgot to bring the rags we just tried the visqueen out plus we didn't realise it was going to be this wet we've tried the sponge we've tried insulation and then we thought why don't give this a give this a blow and because it's got this i mean we could use the sub team in hoover function as well look at that nice speaks for itself that so all you do is you just swap ends and there we go and it's dead light so easy to use dead small does the job if you go to the link in description below evolution website and then code brit lane and capitals you'll get five percent off anything you buy from evolution so as you can see we're finally set up and it is now yeah 20 past nine so we're couldn't it find 10 minutes till the show up roughly but there's this fish in that corner off now but it's done now we're ready all taped off gone around the gully sorted out this door what we've done here i'm sure dad's explained already but we've not gone we've not done an upstand with this one so the concrete can just run through and there's no breaks in the concrete so it connects to the existing floor and obviously the corners planks are ready for the concreters so let's hope this goes well now we're on to the scary part so see you when the paw comes in all right here they are 10 past 10 they were in a little bit later today which worked out good for us because we've got plenty time to get set up now so here we go let's begin the next hour hopefully this will be nice and uh filled up and ready there we have it concrete is in on earth is that anyway concrete's in massive thank you to the concrete company for getting that in slab and the footing but yeah so that's the that's the easy part done now comes the fun part of trying to get this uh level and flat on this biggest slab with nothing to tamp off of but we got this bringing out the new new toy like i said and i just give a quick try doing that little uh logo thing and it felt quite good so all right wish us luck let's see how this goes you


I'm going to explain to you how we, in practice, integrate the data into BG, and after the break we will show you how to use this data and download it and make use of them. And as a start, I will use the example of single-cell RNSIG data, because these are the data, a lot of people are working out nowadays, and they add more complexities than previously bulk RNSIG data. So just as a reminder of what we obtained from using single-cell RNSIG data, so basically for droplet-based technologies you have beads like that, with sequences attached to each bead, with a PCR primer, a cell barcode, a molecular identifier, and then a poly-T tail to attach the mRNA, the poly-A mRNA. And then using a microfluidic device, you will isolate in each droplet one cell with one bead, allowing them to do the sequencing. And sometimes you have errors, you can end up with droplet that are totally empty, or just with a bead but no cell, or with cell but no beads, or you can have multiple cells in one droplet which complicate the sequencing. So that's how the base technology work, and how we make use of it. So we are going to extract a sample, isolate the cell, and at the end of the day what we produce, most of the time, is a cell clustering. So where each dot here represents a cell, and they have been clustered based on their gene expression. And then the aim is to identify marker genes highly specific of each cluster, so that we can determine what is the function of each of these clusters, what is the cell type of each of these clusters. For instance here, you have a gene that is really not specific of any cluster, because it express almost at the same level in all cluster, while this gene here is very specific to this cluster. And then biologists are going to perform functional enrichment analysis on these marker genes and identify which cell types it represents for each cluster. So here, for instance, those are cup for cells in this data set. So this work of identifying the cell types like that for each cell at the end of the day, it's a very complicated process actually requiring a lot of processing steps. So after generating the reads, you have to filter the cells to remove, for instance, cells that were not properly integrated in a droplet. Then you have to perform some filtering for genes, for instance, that doesn't have enough gene expression information. You have to perform normalization.  And each of these steps is going to impact the final cell type assignment. So for instance, the clustering, you could do it in many different ways, more or less stringent, and that would impact the end result. So in BG, that's going to complicate our correctional work a lot, because which of these different aspects should we capture when we provide the final information about how this data set has been analyzed. So I would like first to refer to the Google doc. So okay, I'm going to as Mark and stop the sharing because I'm not actually sure. So if you go, you have the link again to the specific Google doc. I hope I'm sharing. Yeah. So you have the link here again to the activities. And you get this first question here. So just to see, because you can make use in different ways of single cell data, and I would like to see how you use single cell data. So what is, in your opinion, the most valuable information that you can obtain from single cell analysis data. So I give you one or two minutes to fill this table. And please, yes, don't hesitate to participate in this Google doc because then it allows us maybe to reframe what we're saying or identify something that was not well understood. And thanks to those doing it. So composition, so I go line by line. So composition of heterogeneous tissues. Yes, it's interesting because then it allows to see the different contributions of different cell types to the overall bulk, expression information. So gene signatures, yeah, to see which genes drive the definition of cell types. Cell type segregation. So discover new cell types. I would say that probably that the most widely used of single cell analysis data is to perform the cell type clustering and identify the cell types, allowing to discover new cell types. So lineage, yes, you will have like a development type trajectory. So you will see which cell type evolved from which cell type. You know, the origin, the lineage, as it is written here of cell types, function identifiers from homogenous cell populations. Seems to be an interesting one, but I'm not sure to exactly notify what it means. But yeah, probably similar cell population would have different function if this is what you mean. Don't hesitate to speak as well if you want. Proliferation stage, yeah. So I mean, apparently you are all very much aware of what we can do with single cell data. Complexity of tumor, microenvironment. So yes, spatial transcriptomics is more and more widely used. So yeah, I mean, obviously, the audience here has a very clear idea of what we can obtain from single cell analysis data. Thank you. So I'm going to share again presentation. Okay. And so then what do we need if we want to reproduce an analysis, so that if we want to integrate in BG in a reproducible way, the information providing in any single cell experiment. So again, most of the time you will go through a cell clustering and cell type identification. And if we want to present this information as produced by the authors showing the cell types over the cell, overlaid over the cell clustering like that, but also we would like to see expression of different genes, various genes overlaid over the cell clustering. So here you see a specific gene that is actually kind of specifically expressed in this cluster. What do you need if you want to reproduce this analysis as performed by the authors in their paper. So in the paper, you will often see that, but how can you reproduce it? And actually in single cell data set, it's not yet well standardized. And it's very complicated. So first to process the data, it will depend on the protocol. So Mark mentioned that so far in BG, we have categorized 32 single cell protocols, but they are much more than that. And for instance, the first difference would be whether you isolate the full cells or only their nuclei, their nucleus. And then if you do single nuclei analysis, it's going to change how you process the data because you're going to need to map the reads also to the introns as a gene because in the nucleus, the mRNAs are unprocessed. So you still have the introns and it will impact how you process the data. You also need to know the specific protocol to know where is the barcode information allowing to identify the cell and the UMI, the unique molecular identifiers allowing to account for PCR biases. And the barcode and the UMI are not at the same place in the sequencing reads depending on the technology, depending on whether it's a smart sec one or smart sec two or 10 X chromium one or two. So we need to capture this information if you want, if we want to process properly the data in our pipeline, or then the annotations are going to be dependent on the other steps like the dimension reduction step, the clustering steps. So for instance, taking the example of the fly cell atlas again, they provide different types of annotation, they perform two clustering, one that they call the stringent clustering and another one that they call the broad clustering. And some cells have a completely different annotation base on which cluster they use. So some cells have been annotated over a cell in the stringent clustering, but no one in the broad clustering. So we reached out to the people working in FCA and we asked them which clustering should be the final clustering, you know, or how can we capture information about the different clustering they perform. And then if we want to reprocess data and make the analysis as the author did, we need to find the barcode information. So we need to have the information of which cells belong to which clusters to make the link between the cell and the cell type. But often actually the barcode information you cannot find in the primary repositories, in the second read archive for instance. It's not mandatory when you deposit your data to submit the barcode files. So often we have to dig into the supplementary material of the paper, we have to reach out to authors. In the case of the fly cell atlas, for instance, on the sequence read archive, the FASTQ files were corrupted. So we downloaded the data, but they were not usable. And again, we had to reach out to authors to make them fix this file. So from the curation point of view, it's much more complicated, but at the end of the day, we do all this work. And when you go on BG, and we will show you after the breakout to access this data, when you download the data, they have been already annotated. You will find for each cell the barcode and the cell type annotation in a very consistent reproducible format. So now there is a WooClap link. I stopped measuring for you to go to the WooClap. So you have the WooClap link. Again, it's the same link. And Marc, maybe if you can launch the question, please. Sure. Sorry, which one should I launch? About what information we need to recover from a single cell data set. In a typical polyasec library, what features do you expect to find? No, can I actually launch it myself? Sorry. Ah, the last question? No, the question name, what information do you need to recover from? It's written as last. Okay, it started for everyone. You have 57 seconds. No stress, 53. I mean, don't hesitate to answer if it doesn't make sense. A bit to show also the complexity of this data, the complexity of creating this data. In the meantime, I see a question in the chat about do you propagate annotations between different studies? So the cell types name are the same. Yes, we do. And I'm going to present that during this presentation, actually. Okay, so time is almost up. I'm going to share. I'm going to try to share two windows at once. I know it doesn't work. Okay, apparently it's weird. I don't see the answers. I should be. So maybe can you share your screen, Mark? I apologize, but I don't visualize the results. Should be able to. Okay, so what information do we need? So do we need the unprocessed flow data? Well, then it depends. For instance, we could go directly from the gene expression count. But probably if we want to remap to the latest genome version, for instance, then yes, we need the unprocessed flow data. But in this case, we absolutely need to know what protocol was used to know where the background, the UMIs are positioned in the read. So it's either you have the gene expression count and you are happy, or you need both unprocessed for data and information about the protocols. Then if you want to display the annotation as provided by the author, either they provided cell type information for each cell and then you're good. It's simple. Or you get only cell type information for each cluster, which is in most of the case, in most of the time, it is okay. They provide information per cluster, the authors in the papers. And in that case, we need to make the link between each cell and the cluster that belong to. So in that case, we need the association between cell barcodes and clusters. So you see it depends on where you start from the information you need. So if you get only cell type information for cluster, then you need the information of linking the barcode and the cluster. But if the authors provided the information directly for each cell, you don't need the link between the barcode and the cluster. You see, so this question was just to show a bit like yeah, kind of the complexity. And I appreciate that someone answered, okay, just give me a nice visualization. Okay, so now I'm going to present what we do in BG actually then to show you, to give you a simple answer about where a gene is expressed. So our aim in the processing pipeline is to detect the conditions where a gene is active. So what do we mean about where a gene is active? It's a bit similar to what we can do with in-situabilization data. Here you have an image of in-situabilization data on zebrafish embryo. And it's pretty clear in a way because you see exactly the stained areas where a gene is expressed. So there is an hybridization of a probe, of a fluorescent probe to detect where a gene is actively expressed. And then you see the areas where this gene is expressed. But then it depends on how long you perform the hybridization. If you wait too long, maybe the whole embryo is going to be stained. But still here you can see clear areas where there is an active expression of the gene of interest. And this is what we want to do in a way, but using also Birk RNA-seq data, single cell RNA-seq data, showing you where your gene is actively expressed. Which means, and so why do we need that? So it is important, for instance, when you perform differential expression analysis, the first step in most cases is to remove genes that have a very low expression because you won't be able to perform a differential expression between two conditions where one gene is absent and the gene is present in the other condition. So in most cases, these genes that have no expression in the condition are filtered out. But there is no clear criteria for filtering these genes out. Or also if you study sex bias genes, for instance, you want to find the gene that are expressed in male but not in female, for instance. And again, you want to identify the genes that are not expressed in one of the sexes. But again, you don't have a clear criteria to do that. And also, simply for answering the question, where is this gene expressed in the same way that in situational data, you see where your gene is expressed. This is what we want to provide in BG, tell you clearly, look, this is the important expression pattern of your gene of interest. But then how do you do that? Basically, you need to identify expression signal that is over the background transcription noise and over the technical noise. Because yeah, you can, in RNSE data, you can even find reads for intergenic regions that are not supposed to be expressed. So it is, in some way, first a noisy process, gene expression, it's stochastic. And the technology is also generating noise that you need to account for. So I'm going to focus on RNSE data, both bulk and single set to explain to you how we do that. Okay, so what we want is to detect active expression in different conditions. And in most cases, authors in experiments use an arbitrary cutoff on the expression level to say, okay, over this cutoff, my gene is expressed. But it's very unlikely that one threshold can fit all the different gene expression in various conditions. And also there is little consensus of what is the exact value that you should use as a threshold. So I would like to ask you to go to the Google doc and tell me what is, in your opinion, a good threshold. So yeah, so this is a question in your experience, what is an appropriate threshold expression level to classify genes as actively expressed. So please tell me what's the value and in which units. Read counts or CPM or CPM, just that we get an idea of how various this threshold can be actually. So Raja, you say I was told 1.5. Can you please add the unit 1.5 full change. Thank you. Okay, so you can already see here that there are like many answers. So Raja getting back to you, sorry, 1.5 full change, it means that then you will have to compare to conditions. But what do you do if you only have one condition like you have one library. How do you identify in that one library, the gene that are actively expressed. So I see one filter map read. So as soon as you have one read, you have expression. I mean, that makes sense from a logical point of view. But again, the technology is noisy and you find reads for intelligent regions, for instance. So probably one read not going to work. And if you have a very long gene, it's more likely to produce one read than a short read. Yeah, one exon. Okay, I understand. But again, so one read does not represent the same thing depending on the less of the gene or 10 counts. I see five TPM, three, five times a background level. And then the question is how do you identify the background level. And I'm going to show you how we do that. But basically, that's a really good idea. So yeah, more than one read. So you can see that even among the audience here, there is little consensus about what is a good threshold for that. So thank you for your answers. Okay, so getting back to the presentation, I show you actually here, it's a plot of the true positive and false positive rate at different TPM cutoff. So what we do is that we took an experiment where they use ribosec data so that you know what are the truly expressed genes in your sample. So we use that as a God reference data set to know what are the truly expressed genes in that data set it was in mouse lever. And then we use several TPM threshold to check for the true positive and false positive rate. And we try various cutoff. Here it's 0.5 TPM, 1 TPM, 2 TPM, 5 TPM, 10 TPM. And then I mean, it's just a matter of stringency. I mean, what is here? The best value here. If you go for 0.5 TPM, you get almost 100% true positive rate, but you get a high false positive rate. So you identify genes as expressed while they are not actually. So it's about like 20% false positive rate. It's like for the red, the red false positive rate here. So it's quite high. And the most used threshold, I would say it's 2 TPM. In most truly, it's one or 2 TPM. And 2 TPM, you achieve quite a good balance between true positive and false positive. So it's around what like 90% true positive and around 10% false positive. But you see, it's just, it's not clear where you should stop, where you should put the threshold. Yeah, and in our experience, integrating thousands of data together, it varies between different experiments. Sometimes maybe it's going to be 10 TPM, that is a proper value, and sometimes it's 0.1 TPM. So the approach we have in BG is that when you look at the distribution of gene expression in a data set, so this is a density plot here for instance of the exon, density plot of the expression level of exon here, you see that there is like a distribution with a shoulder on the left like that. And actually in that paper, this 2011 paper, they show that the shoulder on the left of the distribution is actually a sum of two distribution. In yellow, the lowly expressed genes that they identify as not being actively expressed. And in purple, the highly expressed gene that they identify as the truly active genes. And what you can see here is that the lowly expressed genes on the plot on the right, they also show the intergenic level of expression, the dotted lines intergenic region. So you can see further that you have signal of expression for intergenic regions, and that it matched closely, sorry, the not expressed gene signal. So maybe using like that, the signal of expression of intergenic regions can give you an idea of the background expression signal. But then actually we are currently in the process of improving this method. So far, we are using signal of expression from intergenic regions to estimate this background signal. But we're in the process of improving this method because we are not totally satisfied with the false positive rate, actually, that we have with this method. But basically, our approach would be to compare the expression of a gene to a distribution of non-expressed genomic features. And we expect intergenic regions to be non-expressed genomic features. And so by comparing the expression of a gene to this distribution, we can obtain a p-value, give you a p-value of the likelihood of your gene being actively expressed in a sample. And so we are currently working at improving this method. So there are several things we are currently evaluating. What is the base choice of non-expressed genomic features? Should it be intergenic regions? Or maybe intergenic regions close to genes because if they are very far away from genes, maybe the chromatin is totally close in that area. The region is absolutely not accessible to the transcriptomic machinery. And it won't be a good estimator of the background signal. Or we could use genes that we are sure are not expressed by using a very low TPM threshold and we take all the genes below the threshold and we look at their distribution. And then how should we represent this distribution of non-expressed genomic features? Is it a normal distribution using log TPM value or a negative binomial or Poisson distribution? There are discussions in the field about what is the true distribution of non-expressed genomic features. Apparently it's a negative binomial distribution. So we are in the process of evaluating all of that. So I'm not going to go too much into details here, but what you can just remember is we are going to compare a gene expression value to a distribution of non-expressed genomic features. And we're going to provide you a p-value. And in the current trees of BG, it is based on intelligent credence, but it's going to be improved for a lower false discovery rate in future releases of BG. Okay, so I invite you to go back to the Google doc, please. So the question is what could be so that you can put your mind on this question. What could be the limitations of using intelligent credence as the reference for non-expressed genomic features? Can you think like, I mean, I don't expect you to have the correct answer to this question in most cases just so that you think about it and think what could be the best approach to solve this issue. So I see that you probably struggle on this one considering that not a lot of people are answering. And then there is a second question as well that you can answer. What would be the limitation of using genes with an expression level below a cutoff, which are kind of the two solutions I suggest here. Okay, so please continue filling the document. I start going through the answer that we already see. So first answer is that the intelligent credence will not be expressed. So maybe I wait a little bit because it's going to be difficult for you to type and listen at the same time probably. Okay, so first answer is there will not be expressed intelligent credence. Well, that's kind of the point actually that we want non-expressed genomic features to estimate the background signal. And we do find reads mapped to intelligent credence. So we will detect some actually, which is surprising in a way, but it is the case. So we want them to be expressed, but then someone has put they are not represented in the dataset. And yes, that would be an issue if there were not. So can that be from DNA contamination? Actually, we consistently find them in all datasets. So it's very unlikely. All authors report, studying this question report distribution of expression for intelligent credence. So we do find them consistently in all libraries. And they do represent non-expressed genomic features. Intron retention. So then we don't work at intron level because for instance, for single-nuclei data, you expect to find introns because the mRNAs have been unprocessed. So we cannot use introns as non-expressed genomic features because we will find them in single-nuclei data. So I see the second line, low annotation quality of genome. That is a very good point between model species and non-model species. We see huge differences. And actually in BG, we recreate the intelligent credence to remove those that show a very high expression signal, which are most likely unannotated non-coding genes. So for non-model organisms, we do actually have this problem and we have a creation step in BG to take that into account. So we use only reference intelligent credence that we are sure do not contain unannotated genes. Differences between species, tissues, cell types in the level of this intelligent credence. Yes, it is a good point. And this is why we are looking at using intelligent credence closer to genes to be sure that they are actually accessible in all tissues and that they can represent an actual non-expression background signal. What about an enhancer RNA that would be intelligent but transcription signature? Yes. So an enhancer in that case, I mean, they should not be expressed, right? So there would be, we don't expect to find reads mapped to them. They are not expressed features. So they have low-quality and large number of data sets might be needed. Actually, no. We do find them in each RSA library. So I move on. Thank you for your answer. Using genes with an expression of a beetle cutoff will depend on the analytical location. The expression depends on the tissue, tissue-specific expression. Okay. So it is actually the same problem kind of we get back in circle. What would be the appropriate threshold so that in all contexts, we truly identify the non-expressed genes. So this is a very good point. But probably if we take a very low TPM threshold, we're going to be safe in all conditions. But these are all things we are currently benchmarking. You can just remember that in the current race of BG, it is intelligent region that we use and intelligent region that we have re-annotated to make sure they do not contain unannotated genes. Okay. So thanks a lot for all the very interesting answers. Okay. I think I need to move on a little bit faster to get to the end of this presentation. So I'm going to show you in single cell RNA-seq data. So in single cell RNA-seq data here, I show you the same density plot as before of the expression level of genes and intelligent regions and protein coding genes. So these are full-length RNA-seq data, so smart-seq technology. Smart-seq technology, you study a much lower number of cells per experiment, but with a much higher number of reads per cell as compared to droplet-based technology. And you see here that we still have expression signals where we can distinguish between protein coding genes and intelligent regions. So basically for smart-seq data, we use the same approach as for bulk RNA-seq. And you can still see that in this full-length data, so here I show you the percentage of protein coding genes considered expressed with our method in bulk RNA-seq data in human and in mouse and in single cell full-length data in human and mouse. And you can see that we have much less genes that are considered expressed in each cell as compared to bulk RNA-seq data. But it is expected because in bulk RNA-seq data, you will have a mixture of cell types, so probably you're going to have more diverse genes expressed. But still, in single cell RNA-seq data, you get about 60% of the genes in a cell that don't receive any reads. While in bulk RNA-seq data, it's more between 5 and 10%. So part of that is biology, because you don't look at a mixture of cell type, but the part of it is technical, is that you have less reads per cell, so you will have more dropout, more genes with no reads. And if you look at droplet-based data, so droplet-based data such as 10x allow you to study hundreds of thousands of cells in the single experiment, but with much fewer reads per cell. And here, this density plot, you see here, I have to zoom in to look at the actual expression signal in each cell. And you can see that you can actually not distinguish in one single cell expression signal from x-photogenic regions or from gene expression. So what we do in BG for droplet-based data is that we're going to do pseudo bulk. We're going to pull all the data coming from all the cells from the same cluster. We're going to pull the data in the same cluster to recover more expression data. This technique is called pseudo bulk. And then we are able to recover an expression signal allowing to distinguish gene expression from intergenic gene expression. And we're going to make this statistical test. And if we know that a gene is expressed in a cell type, then we go back to each cell and we look, okay, do we have one read map to the gene? This gene was expressed, so it is expressed in that cell. So maybe it's a bit complicated. But at the end of the day, we are able to tell you in BG for each cell, the gene that are actively expressed in them going through this type of doing pseudo bulk. So basically for each data type in BG, I'm going to skip maybe a bit that, but for each data type in BG, we define methods like that allowing us to have a p-value for each gene of the signal of expression. And then how do we integrate all this information from all these data types? Because in BG, we also have affinitrix data, EST data, in-situable decision data, but for all the data at the end of the day, we're going to provide for each condition and each gene a p-value of whether this gene is significantly expressed on that. So then Mark presented to you that we use ontologies to annotate the data and that the terms in an ontology are connected between each other. So here I'm going to use a simple example of the pancreas, which are two children having two children endocrine and exocrine pancreas. And the developmental stage with two terms, sexually immature being part of the fully formed developmental stage. And here I imagine the expression of three genes. So here you have three genes in one of six SRRM4. And we have calls of presence and absence of expression. So in the lower graph, we're going to have expression of gene A in the exocrine pancreas, sexually immature of gene B in the endocrine pancreas, sexually immature. So we have information for these three genes, but at different levels in the ontology. And what we're going to do is that we're going to propagate this information along the graph of terms so that at some points we have an integrating information when we can compare expression of our genes. So you see that gene A that was having information at the level of exocrine pancreas, sexually immature. Thanks to propagation. We also know that it means it is expressed in the pancreas at the fully formed adult stage. Okay. So we're going to propagate information for each gene on this graph. And at some point it's going to be comparable. It's going to converge to a same condition where we can actually compare the expression of these three genes. So this is how we integrate information in BG and there were a question earlier about how can you make the cell types comparable and that's how we're going to do. We're going to use the cell type ontology to propagate the information and we're going to converge to a common cell type at some point for expression of various genes. Maybe I'm going to skip that because I'm running a bit late. Okay. And so what we propagate actually is the p-value. So as I showed you for each library, for each sample, for each gene, we're going to provide a p-value of the significance of the expression. And those are these p-values that we are going to propagate in the ontology. And then at the end, we're going to do a FDR correction of all these p-value so that we get one single answer to the question, is my gene significantly expressed in this condition by the integration of thousands of p-value, FDR corrected. We're going to give you one single answer. Yes, this gene is significantly expressed with this FDR corrected p-value. And then we're going to give you an answer. Okay. Is my gene present or absent? This is all based on this FDR p-value. So I'm not going to go into details. You can find that on the documentation of the BG website. We're going to show you the BG website. But basically, we use this test of significance of expression. We propagate them in the ontology. And at the end of the day, we give you a simple answer. This gene is present, absent, with three levels of confidence, gold, silver, bronze. So these calls are informative about telling you whether your gene is actively expressed or not. But then you lose information about the expression level. Okay. And you still want to know whether your gene is highly expressed or lowly expressed. So to do that in BG, we use what we call expression scores. Expression score, basically, we use nonparametric statistics to be able to compare expression level between any data sets, basically, because otherwise we need to account for batch effect to normalize the data. We don't know which will be the batch effect in thousands of experiments. So to integrate expression level information, we use nonparametric statistics. So basically, we rank the genes in each data set. We normalize those ranks to make them comparable between different technologies, different conditions. And the ranks are also propagated in the graph, as I showed you earlier. So when we provide the expression level information in BG, it's based on all the expression-level information in all sub-conditions and in the condition itself. Yeah. And then we transform these ranks into a simple value between zero and 100. So at the end of the day in BG, you get expression score information between zero and 100 representing expression level, base, and nonparametric statistics. I'm going to speak and switch the, skip the last workflow because we don't have time and just show you an example of then how you retrieve this information in BG. So if you go to a gene page on BG, on the BG website, but we're going to show you how to do that after the break. But this is the gene page for the APOC1 gene, which is an allipoprotein gene, very important in lever. And here you see the FDR-corrected p-value. So a very significant p-value in the right club of lever and the expression score. So it's 99.94 over 100. So very high expression level. Probably this is the most highly expressed genes in that condition. But then when you look, and so those are the non-express genes where the FDR is non-significant. The non-express conditions where the FDR is non-significant. And then the point is that, thanks to the integration, if you look at all the species present in BG, in mouse, the top express conditions for this gene is left love of lever. In zebrafish, it is also lever. In chimpanzee, in bonobo. You see that in all those species, we correctly identify that the top express condition for that gene was lever, with a very high expression score that is very similar in all those species. You can see it's in all case above 99 on top of 100 with a very significant FDR p-value. So you see how this data integration allows you to have a consistent comparable information between all those species. So again, even more species. You see, high expression score of 100, 99.9. You see how consistent it is, I hope. And then we have a tool that we're going to present as well where you can automatically compare expression between a list of genes. So here I enter the autologous genes of apoquan interior in our tool. And the organs that are identified as most highly conserved in their expression is epitobiliary system, lever. You see that all the autologous genes have expression significant in this tissue. So you can also perform that automatically not going through each gene page individually. Yeah. So in summary, to know where genes are expressed, first we perform a precise manual annotation so that all the data sets are comparable. And then we generate p-values for each gene and each sample of the significance of the expression level. We compute expression ranks and score for each gene and each condition and we propagate all this information along the graph of condition to give you an integrated consistent information at any level of the


right down next, next reaction we have T-Tinco reaction, T-I-S-C-H, all these reactions have similar kind of mechanism, Aldon-Kanesar-O, T-Tinco-Reformance scheme, Fulcane, whatever we did have similar kind of mechanism. So, in this reaction what happens will take two molecules of aldehyde, two molecules of aldehyde, R-C, I take two molecules of aldehyde, R-C-O-H and it reacts with aluminium ethoxide, aluminium ethoxide, E-L-O-E-T both rights and we hit this right. The product forms in this reaction is an ester, okay? So, the product will be R-C-O-C-H-2-R, R-C-O-C-H-2-R which is an ester.  So, it can accept one pair of electron, so behaves as Lewis acid and hence what happens in this, this lone pair, this lone pair as tendency to like it can donate its lone pair to this aluminium here, right? So, the first step of the reaction is R-C, for this reaction the mechanism is a bit different, R-C-O-C, right? Oxygen will have positive charge because it zonates electron and it attached with AL-O-E-T both rights and negative charge on this aluminium because it accepts electron, okay? Now, to stabilise this positive charge, this pi electron is taken up by this oxygen and we'll get carbon positive charge here on this carbon, right? So, next step is R-C-H positive charge O-AL negative charge O-E-T both rights, okay? Now, this positive charge by the other molecules of aldehyde, this lone pair again attacks on this positive charge of this carbon, right? So, next step of the reaction is R-C double bond O-H plus this molecule is R-C positive charge H-O-AL-O-E-T both rights, okay? So, this lone pair attacks on to this carbon and this oxygen carbon bond we have. So, the product here R-C-H double bond O and this O attached with this carbon, R-C-H-O-AL-O-E-T both rights, aluminium will have negative charge that won't change, okay? Oxygen will have positive charge here, right? Now, again this bond pair is taken up by this oxygen. So, it converts into R-C-H-O-C-R-H-O-AL-O-E-T both rights and we'll have positive charge on this carbon, right? Now, there's a hydride strip here. This hydrogen takes this layer of electron and rearrange itself on to this carbon and it converts into R-C-H-2 O-C-R positive charge O-AL-O-E-T both rights. In the last step, this AL-O-E-T whole price goes out in the living room. The bond pair shift over here and the product here we get is R-C-H-2 O-C-11 bond O. This reaction is T-shell co-reaction converts aldehyde into ester, okay? reagent is AL-O-E-T aluminium with oxide. Done? See, this is the mechanism of this reaction but you don't have to do all these things in the exam, okay? How to write down the product that you see? Whatever the aldehyde is given, two molecules of this aldehyde, you write down one ox, it's like to write down the product, you can consider this as canizaro reaction, right? What happens in canizaro reaction? It is disproportionation reaction, oxidation and reduction, right? So, one molecule of this, if it gets oxidized, what it forms? Acid, R-C-double bond O, OH. So, one molecule, you write down oxidation product. Other molecules, you write down reduction product, R-C-H-2 OH, okay? So, write down the product, what do you have to do? One oxidation and one reduction. And then, and the heating, what happens? H2O goes out, right? So, this OH N-H forms H2O and the product is R-C-double bond O, O-C-H-2, R, that is what the product is getting. So, this is the method you have to use to write down the product in this reaction, oxidation and reduction, okay? Mechanism is a bit different from the other reaction but you don't need to, you know, write down the mechanism in the exam, okay? Regent, you must take care of. What is the reagent? Aluminum ethoxide for this reaction tissue for reaction, understood? So, whatever the reactant is given, aldehyde, write down the oxidation and reduce product, reduction product and then remove water molecule, okay? Write down the product in this reaction. We have two molecules of same reagent. What is the product for this reaction? One molecule will get oxidized, so it forms R-C-double bond for OH and other one can reduce O-H-CH2, right? And then, H2O molecules goes out, so the product will be, okay? Here again, what is the product? CH3, CH2, is this correct? Yes. 5 carbon both sides will have equal number of carbon, okay? This is the intramolecular reaction, okay? So, this will get oxidized, this will get reduced, okay? So, the product here is what? C-double bond O-OH, CH2, C-double bond O... No, sorry, CH2-OH. CH2-OH, so I will write down this way, CH2-OH. And then, when you heat this, H2O molecules goes out, right? This H takes this OH, goes out and this oxygen attached with this carbon. So, we have 1, 2, 6, 1, 2 plus 4, 6 and 7th carbon. So, we will get a 7th member ring, right? 7th member ring and oxygen is present in the ring, okay? So, it is, this is the ring we have, 7th member and O attached with the oxygen, carbon which has double bond O. This is first, sorry, this is first and this is 7th carbon and the second carbon has double bond O, which is this. So, 1, 2, 3, 4, 5, 6, okay? This is the carbon composite. Next reaction I will write down, benzoin condensation. Have you finished preparation of this early identity test? First class, preparation of benzoin condensation. Benzoin condensation is again a reaction of mildehyde and generally, we take benzolehyde, P H C double bond O H, 2 molecules of this, okay? The reagent we use for this purpose is KCN, okay? KCN is the reagent we use. What is the product we get here? This reaction has a single mechanism, that is the same mechanism. First, we will get carbon ion and that carbon ion attack onto the other molecule of this. How do we get carbon ion? This nucleophile is what? What is that? I think reagent here, Cn minus, right? This Cn minus will attack onto this carbon because carbon is partial positive charge, carbon oxygen bond 4 and this pi electron goes on to this oxygen, right? In the first step of the reaction, what happens? P H, CO minus H and Cn, right? After this, there is intramolecular acid-based reaction, okay? So this hydrogen shift onto this oxygen. Losing H plus gains H plus, right? So intramolecular acid-based reaction. So P H, CO H and here we have Cn and this carbon will have negative charge, right? Yes or no? Because H plus goes out to this bond field will be here on this carbon. Now, this behaves as a nucleophile for the other molecule, okay? So other molecule is what? This is the first step of the reaction is this and then the second step, we have P H, C double bond OH plus P H, CO H, Cn and this attacks onto this carbon and this pi electron goes out to this carbon, okay? So it forms P H, CO minus H, CO H, Cn and this P H, okay? Cn minus is a leaving group here. There is no leaving group present onto this carbon. If it is there, then this bond pair, this bond pair of electron comes over here and one of the leaving group goes out, right? But there is no leaving group here, right? So what happens in this reaction? Again, there is a hydride shift, this H plus comes over here, protons shift basically and we get P H, CO H, H, CO minus Cn and P H, okay? Now, when this lone pair of electron forms one pair, this Cn minus goes out as a leaving group and that is why this KCn is a catalyst here. Concentration of Cn minus does not change, okay? So product of this reaction is what? P H, C H, OH, C double bond OH. So KCn behaves as a catalyst here. It will take place in a basic way, right? We need some nucleophyte. So Cn minus is behaving as a base here, you can see what I'm saying. That's why it attacks because here we have two attacks. So suppose you put some medium-mass mode OH minus on us, then KOH should be taken up and then that will yield Cn. Yes, correct. So when you change the reagent, you will get some other product. Mechanism will be same but that reaction is not benzoin condensation. It's a name reaction. So reagent and everything is fixed for this. Okay, name reaction, you cannot change the reagent. If you take some other molecule like KOH you are saying, so same kind of reaction will be there. Mechanism will be same but the name of the reaction is not benzoin condensation. Okay, next write down, finish this one. Remember they won't give you the name of the reaction. Benzoin condensation they won't say. They won't say parking reaction. They won't say canizaro, I'll do it. You need to identify this. Okay, for Vatsky they won't mention the name. So for that what you need to do, you need to memorize the reagent for each name reactions, right? So that is important. Benzoin condensation, the reagent is KCn.


All right, so the first time I ever met Anthony was actually at a layer talk and Gosh, so I went to the top layer we said and I spoke at the top layer talk and he talked to us afterwards about Convention or something like that and you'd already done it one year, but a lot of people had talked to us and He was actually really grateful actually. I remember either seeing something on the top layer site about how good the The talk was that we had given or something and that it was by him, but that was it It was just a real brief impression. Now what really defined that for me was If for all the people watching this this massive people helping on this documentary, it's really amazing but the guy who is been with Anthony doing all the video work is the same dude is 2008-2009 and What he commented on the ride over here is we were getting ready to shoot was the integrity that he saw in Anthony from the experiences in that first year that he worked with him and I saw the same things my friend John He's a marketer and marketers can be like the biggest snakes in the world And I said dude John He seemed like a really passionate person and he looked at me said dude What the fuck else is there and that to find what real integrity is and that's what I saw on Anthony I mean there was a guy who developed this thing for nothing and you heard a shuteau talk about it at the Conference of series like the only person I know who sacrifices everything for something that he believes in that I see day to day Or I see all the time is Anthony who runs the 21 convention and man that that's the truth. You see that


Good evening. Tonight our stage has been taken over by the crime photographer, written by Longer Dean Cole, and based on the fictional character of Slash Gun Casey, created by George Hormann Cotts, and played by Stats Cotts with. Casey, ace cameraman of the Morning Express, covers the crime use of a great city. It's a venture for tonight, the reunion. About nine o'clock in the evening, a luxurious suite in our city's most expensive hotel. One of its present occupants is a strikingly beautiful woman. In her hands is the latest fashion magazine, but she stares at it without interest. I mean... I mean... Yes, Ruth? You haven't turned up page in that magazine for half an hour. I've been thinking. Oh, I mean...  If a man had done to you what my husband has tried to do to me, could you drive it from your mind? Yes, I am. I made Kurt a success. He was just a poor weakling when I met him. I made him a great sculptor. I made him rich and famous. Now he's repugnant, but he's not going to get away with it. Your lawyers will think about that, I will. They told you they can nullify that divorce Kurt got from you? When I think of the rottenness of my... Don't tell me anymore. When you and I were in South America, the bit Kurt persuaded me to take from my head. All the while, he was establishing residence out of the state to divorce me for abandonment.  And he was preparing to marry another woman. That dick's the only she's capable of. He'll say no to them. And I think that I... I want to kill him.  I want to keep him. Keep him? Yes. Well... No other woman is going to have him. Tomorrow I'm going to sign the papers for the notification of the divorce, and I... Will you answer that, Ruth? Excuse me. Hey, I'm not here. All right. Hello? Mrs. Volta isn't here. I'm a tender her cousin. Who's this? Hold the line a minute. It's a man I believe he insists that he's got to talk to you. Who is he? He... His voice sounds a little like your husband. Kurt? Yes. All right. Yeah. Hello, this is Mrs. Volta. Oh. All right, wait a minute.  Well, yes, of course.  I'll tell you all about it later. Hello. Ruth, forgive me for sending you out of the room. I will. I'm going out. You've got to help me dress in the prettiest things I have. Thank heaven my hair was decently done today. Ruth, what is this? Was it Kurt? No, no, it wasn't Kurt. Darling, hand me that cold cream jar. Yeah. Is that other man is making you dress in your best? Yes. A pretty... A pretty... Since that telephone call I've been... You'll just have to restrain your curiosity. And for several days, I don't expect to come back here tonight or tomorrow night. Maybe I won't come back for a week. But don't worry, Ruth, and don't try to find me. I'm going to be happy again, dear. Altogether happy. What is it? Hello. Thank you. This is Anne. Anne and William. Nobody with that name here. You've got the wrong number. Hey, hey, wait a minute, wait a minute. What? Wake up, you don't wake up. Annie, huh? No, he doesn't. What's the big idea of foaming at this hour? You've heard me speak of Ruth Tender. Have I? Yes, you have. She's a college student. And she's a cousin of the rich and very beautiful Mrs. Kurt Bowman. You mean the wife of the big sculptor? Indeed, I do. Now listen, Ruth told me a few minutes ago and told me that Mrs. Bowman disappeared ten days ago and has not been heard from since. Yeah? Yes. Ruth hasn't notified the class yet, so we're getting first in on what may be a good story. Well, give me all the dope, Annie. How can I give you? Ruth will tell us the story as soon as we get to the hotel. Okay, Annie, I'll pick you up in about twenty minutes. Mrs. Casey, Irene left the street on Friday night, ten days ago, at about ten o'clock. And that's the last I've seen or heard from her. Oh, yes, it was, listen, Mrs. Bowman told you that she might be away for a week. Now maybe she's just staying wherever she is a little longer than she intended. But she's not where I thought she was going then. Miss Chandler, you figured that Mrs. Walter was joining her husband for a reunion. It was the only thing I could think of, Mr. Casey. Until just before I phoned you this morning, I learned there hasn't been any reunion. You went to Walter's home? Yes. I talked to Mrs. Royce, his housekeeper. She told me that during the past ten days, Curtis has been working almost constantly in this studio. It's attached to the house. And that Irene hasn't been there. Sure, she told you the truth? The part of it. She and I are old friends. She wouldn't lie to me. Mr. Casey, do you think I should ask her? But at least to look for Irene? No, I wouldn't. You know how women feel. It'll just been separated from their husbands. Maybe she just... Well, she might be. To say, is this photograph on the piano a picture of your cousin? It is. This was taken some time ago. About fifteen years ago, soon after her marriage, at the time she was posing for the statue that made her husband famous. The Sleeping Goddess? No, but... Yeah, I've seen copies of the original. I found it like sculptures and paintings. So, Mrs. Walter modeled for them. Annie, this face isn't just beautiful. It's got power in it, real power. It has, though. No photographs can do Irene's cool justice. He has the most wonderful hair, Mr. Casey, two golden hairs. Yes, Anne told me about that. Well, I think he had to go into temperamental rages because he discovered not a painter. He couldn't reproduce her hair in bronze or marble, of course. Ms. Chandler, when you talked to his housekeeper this morning, did you ask her where Walter was on the Friday nights your cousin left here? Mrs. Royce remembered that he worked in this studio all that night until about eight o'clock in the morning. He was doing some knowledge of construction work on a mold. Did she see him working there? No, he never met anyone in the studio when he was working. Then he could have gone out for a while. Mr. Casey, I believe you have the same awful idea that I have. That Kurt might have slipped out of the studio unseen. He met Irene somewhere and... and killed her. Did you let this Mrs. Royce know of your suspicion? No. I even asked her not to tell anybody that I did not see her this morning. I don't know who I am. I just did. Good. I'd like to talk to Walter. Before he knows a search has been started for Mrs. Walter, I just might catch him off guard. Just might. Well, let's go, Anne. Mr. Walter will see you in this lady, Mr. Casey. He's in the studio. Please come this way. Oh, thank you. You're Mrs. Royce, aren't you? Yes. How did you know my name? Oh, Ruth Sandberg told us all about you. Here we are. Hello. The ladies and gentlemen from the morning express, Mr. Walter. Ms. Williams and Mr. Casey. It's a great pleasure to know you. Walter was nice of you to let us barge in on you like this. I'm always glad to receive newspaper people. You know, I have a special regard for the morning express. It's odd that it's always been very flattering about my work. He sent you here, of course. Oh, yes, yes. We're from the art department. Would you want anything, Mr. Walter? No, thank you, Mrs. Royce. You can go. Well, sit down, Ms. Williams. Thank you. Have a cigarette. Yes, yes. I'd like one. Mr. Casey, no surprise. I will join you. I have a match ready already. Here you are. Mr. Walter? Thanks. No, no, please. Do not light your own with that, Mr. Casey. Not three on a match. Okay, since you're a superstar, you see, I was born in Europe in a little mountain village where people still believe in witchcraft, ghosts, and romance. I have afraid my many years in this enlightened country have not completely eradicated my early teaching. I say, this is an enormous studio, you have. Three sculptures require a great deal of room. Yes. That's the model of your sleeping goddess must be over eight feet long. Mm-hmm. Certainly a masterpiece. I never thought of the original. You are looking at the original now. I thought that was somewhere in Germany. I must explain my method of working. First, like old sculptures, I make a wax or clay model of my conception. But when it is only roughly complete, I take from it a mold and then make a casting of plaster. On the plaster cast, I carve my finishing touches. Another mold is then taken for the final casting of metal. This model provides it the final mold. It's a true original, then. Yes. It's now almost right. You see, it runs replica was destroyed by a bomb during the war. This really is worth planning, then. Many consider it my greatest work. I understand the model who posed for it was your wife. Yes. She is now my ex-wife. We are divorced. Yes. She looked like this sleeping goddess. She was certainly some woman.  She had the most wonderful hair in the world. But he was not of this world. This is not Okada. Okay. Hey. When did you make this plaster model, Mr. Baldwin? Fourteen, fifteen years ago. Why? The head feels damp, like fresh plaster. The head? That cannot be. Feel it? He's not damp. What do you say, Miss William? You don't drive me, Casey. Huh? Yeah. Funny. When I first touched it, it felt cold and crammy. Like death. I see you don't have to go through your house to get in or out of this studio, Mr. Baldwin. There's a door leading directly outside. Yes. Will the story you and Miss Williams do about me concern this sort of thing? Well, hey, we must be getting to that story, yes. We want to hear about your most recent work, Mr. Baldwin. My current work is this group here. Oh, hey, that's a bigger hunk of plaster than I got it, sir. That's what you call hunk of plaster. It will bring me thirty thousand dollars when I've been brought. Wow. You're in a good business. What do you call this group? I call it the reunion. The reunion? Yes. It is a symbolic conception. When did you cast that plaster? Why, just ten days ago. I set up my mood and worked all night. All Friday night? It was a Friday night. And you call it the reunion? You see, the story you've just been telling me ends up to nothing more right now than a case for the missing person's bureau. I'm a homicide cop. My hunch says this is a homicide case, Logan. I have the same feeling, Captain. So have I. This police department can't take the drastic action you propose on the strength of mere hunches and feelings. I haven't proposed any drastic action, Logan, until after a thorough routine missing person's search has been made for Mrs. Walter. If she isn't found alive or dead within a reasonable length of time, well, I've told you where to look. You believe that she went about her studio in response to his phone call through the door that leads her directly from outside, and he let her in, killed her, and hit her body in the mold he was setting up that night. Then he surrounded it with plaster. I think you'll find Mrs. Walter is in a model he so symbolically calls the reunion. Hello, Casey. Hi, Ms. Williams. Hi. How are you? Hello. I haven't seen you two all day. What do you have to drink? Oh, give me a cup of coffee. Gee, Casey, you must be feeling low. What are your troubles? We can put them in three little words, that'll work. The Volcker case. Say, why don't the cops quit stalling on them? They're never going to find Mrs. Walter till they look in a place you told them to, Casey. Today they did look for her in a place I told them to. Uh-uh. You mean? Yes. I was wrong. Mrs. Volcker's body was not in the plaster model of reunion. In order to find that out, Logan's men had to bore holes in the model and they broke it all up. Now Volcker's going to sue the city for $30,000. He claims it was worth plus additional damages for right now. All his lawyers are throwing up a swell bill of goods. Sure, the worst of it is he's pretty sure to win a suit. Logan will probably lose his job on account of it. He gave the order to bore into the statue on the strength of my hunch. Gee, the poor sap trusted me because, well, by sheer dumb luck, I've given them a couple of good steers in the past. This time I let him down for fear and how. Oh, Casey, he doesn't blame you. Yeah, I know that. But don't ever tell a big dope I said this, but... Logan's a pretty swell egg. Because Mrs. Volcker's body wasn't hidden that statue, does it mean her husband's now in the clear? Sure it does. The only place he could have hidden their body was inside the freshly poured plaster that reunion model. Wait a minute, Miss Williams. Couldn't he have hidden the body in a closet or someplace around the house and then sneaked it out later? No, Mrs. Royce and her cleaning woman would have found it. Oh, of course not. And there's only one other model in the studio large enough to hide a human body in, and that was cast 15 years ago. Those plaster castings have a hollow core. Oh, so what, Casey? Who'd even gotten the body inside the sleeping goddess? Walter would have had to cut a big hole in the old plaster, and then afterwards patch it up with fresh stuff, which would show you how. So ahead of the sleeping goddess, it felt cold and damp like fresh plaster that day. Didn't to me, Casey, and you said it didn't to you either after a minute. Yeah, that's funny. That's what started my hunch about Mrs. Volcker being buried in plaster. When her husband said he'd cast that reunion piece on the night she... Annie. I've doped the guy all wrong. He was smarter than I thought. That reunion model was only a planned decoy. He put her inside the sleeping goddess. Then he deliberately led you and me in the cops to believe her body was in the reunion box. And now that we've found she isn't there... Oh! Ah-ha, you've got it. The city stands to lose 30 grand plus for that mistake. No sane cop is going to take a chance like that again. If you're right... If he's right, Ethelberg, Mr. Kurt Volcker is going to get away with murder. Yeah. Say, Casey, why don't you sneak into that studio on a dark night, bust open that statue? And what if I didn't find the body, Ethelberg? Volcker wouldn't bother to sue me. He didn't have me put away until about 1966. Uh, I got no more suggestions. I hadn't even got a thought. I have. Yeah? What? And I'm going out to see Mrs. Royce, Volcker's housekeeper, and ask her one confidential question. No, Mr. Casey. Since the night he made that pass the casting of his reunion group, Mr. Volcker has never gone into his studio after that. I see. I thought it rather strange. He used to do so much work in there at night. Well, thanks, Mrs. Royce. Why have you asked me about that? I've been thinking. Thinking. I was born in Europe, in a little mountain village where people still believe in witchcraft, ghosts, and all men. I've sometimes thought Kurt insane on the subject of Irene's hair. He couldn't reproduce it in bronze or marbled. He had the most wonderful hair in the world, but it was not. Yes, Mrs. Royce. I've just been thinking a kind of crazy thought. This scheme of yours is absolutely nuts. Call it a long shot then, Logan, and play it with me, will you? If it happens to work, you save your job. The town saves at least 30 grand. I save my peace of mind, and we'll have the goods on a rotten killer. Okay, pal. I'll take a chance. Good evening, Mr. Walter. Good evening, Mr. Walter. It is not evening. It is almost midnight. Why have you insisted upon seeing me at this hour? Well, it's a kind of peculiar thing, Mr. Walter. Mr. Walter, do you believe in dreams? Dreams? Yes, dreams. Casey here had a very funny dream last night. Why should I concern you, Mr. Walter? You see, I dreamed about Mrs. Walter. Right. Ex-wife? Uh-huh. I dreamed that she talked to me. She talked to you? Yes. She wasn't like a woman in my dream. She looked like that statue of yours she posed for, that sleeping goddess. She was the goddess. Only a wink. A wink? Uh-huh. Okay, see, tell him the funny thing she said to you in your dream. Okay, Logan, I will. Mr. Walter, she said, I am the goddess awake because my life is in my statue. She said her life was, uh-huh, uh-huh, and her hair with a shiny gold color I've heard Mrs. Walter had. So bright. It seemed to have light in it. Why have you come here to tell me about this? Because in my dream she told me to come here. She told you? Just before midnight, she said. It's almost that. Now, Casey, we've got to move fast. Yeah, come on, Walter. In the studio. In my studio. She told Casey to have you there at midnight. It's like striking 12 now. Come on. All right. Liz, let me go. I'll get you. He wants you in the studio, Walter, where she's waiting for you. Waiting for me? The goddess awake with shiny gold hair. Don't open that studio door. Don't push me in time. We're leaving you. And in the dark. Open your door. Let me out. Light switch to the book. Open this door. Don't leave me alone here in the dark. I'm alone, Walter. The goddess is awake in there. No. No, no, no. Golden light. That golden light. It rises. It moves this way. No, no, no. Don't come any closer. I'm sorry, I mean it. I had to bring you. You rules me. I had to be your slave. The slave of your will. It's too good in here. It's not my heart.  I had your body in it. Sleeping on it.  Don't come any closer. Forgive me. I'm late. We've heard enough. Anything? All we need, Logan. Switch on your flashlight. Okay. Casey, I'm glad to have the mic in here. You did a swell job, Annie. I was scared. You know what? I was scared. I... I didn't like the thing that goes in the dark. You scared, Mr. Kirk, Walter. A lot more, Miss Williams. He's faded. He did? I figured all he needed was to see the shadow of a woman with a luminous wig rising behind that plastic statue and walk slowly toward him. Casey, I'll never do this kind of job again. I'll never do what he did. Hey, look at that. You're trembling to him now. What's the matter? I don't know. I... I don't know why. Do you know what happened? Oh. It was midnight. When you pushed Walter through the door and I went into my act, I got suddenly afraid. Afraid of Walter? No. I'm afraid of her. Anyway... Hey, Casey! Walter! Walter hasn't fainted. He's dead. Dead. Hey, Miss Williams, you never got very close to him with that blonde wig here, did you? No. What is it, Walter?   Oh, please. That rings down the curtain. This is an ominous portrayal for the night. The reunion, a crime photographer story written by Alonso Dean Cole, is based on the fictional character of Flash Gun Casey, created by George Hormann Cutts. Stats Cuttsworth was heard as Casey, and Miss Leslie Woods played Anne. Until next time... Creep? Good night. Sleep tight. This is the Armed Forces Radio Service.


What is going on everybody? Welcome back to my channel. Today is a very special day because it marks the very last day of my entire diagnostic radiology residency career. And I know you've probably seen my last video where I said this is my last rotation of diagnostic radiology. I still have one more vascular surgery rotation, but that's purely interventional. However, this is my last diagnostic radiology day pretty much ever. So today is my last day on vascular imaging and I'm reading a whole bunch of CTAs for our famous aorta repair guy here. We usually read about 20 aorta studies in which we analyze the vasculature, mainly the aorta and the branch vessels, so that our endovascular aortic specialist can do all of his kind of pre-planning or pre-procedural planning prior to the procedure when they repair the aorta. So that's what I'm doing today. It's my last day. I can't believe it, which means I only have three weeks left to residency and no more diagnostic days. So I don't know what to even know what to say, but this is my last day and I'm super excited. Also, one thing I forgot to mention is today I went to the primary care doctor because I have to do all of this pre-fellowship physical stuff, fill out a bajillion forms as many of you all know who have done this. I don't know why, but it's just like so easy to neglect your own personal health for some reason. We're super busy in residency and we always forget the importance of taking care of ourselves, which is kind of why I went to the primary care doc today because what better time to do it than when I have to do it. So basically, I was forced to do it, but since I was forced, I went ahead and got like all of my lab work that I've been putting off forever. I basically got a little puncture site there. I did like every lab I could do. I did COVID antibody testing, all that stuff. So hopefully it'll be back in the next few days and everything will be clear for when I start my fellowship. So I forgot to tell you all. I just want to tell you all that because you need to take care of yourselves. So forever who is putting off going to the doctor themselves, go ahead and go make an appointment. You're supposed to know about every year or so.  I feel like less anxious now that I've done it for some reason. I guess I'd kind of have physician anxiety as well, even though I'm a physician because at the end of the day, I mean, I don't like sitting in that paper exam room either and I even know what's going on the whole time. So I can imagine for people who don't know. But anyways, if you've been putting it off, go take care of that. Okay, that's my PSA for today. Also, I'm not going to comment on how shiny my face is in this video. I started to use this vitamin C cream that Adriana gets, you know, because I'm trying to stay young. Also, my hair is super long and I'm like all over the place today. So just try to hang tight. Ever read in residency, I'm glad I ended with a CTA Indo Study, Chess Atom and Pelvis. It's the best way to end that. And so that officially concludes my diagnostic radiology residency training. Holy cow, I can't believe it's over. And just like that with my long hair, we are officially, we are officially done with diagnostic residency. Also, it is my wife's last day of work today in North Carolina before she moves to New York. And I'm at her office right now to pick up some stuff because she got a whole bunch of goodies to take home with her as a goodbye present. And we'll say goodbye one last time to her clinic. What's your last day? So? I'm excited. Okay, we're at the social distance. So you may be wondering why I went to the beach today. Well, for starters, I haven't been to the beach in like three years, even though we live about an hour and a half, two hours away from it. So that's the start. Second of all, as you saw, I finished my last diagnostic rotation of my entire career. And I am super excited about it, that I'm done with my last rotation, diagnostic to my entire career. So we decided to treat ourself for a little beach time. Plus, it's Andrea on his birthday, a few days here. So I don't know if you can hear me now, but this is the right to be on North Carolina. So I'm officially back in the hospital now. I didn't film while I was at the beach yesterday because I just wanted to enjoy it as Andrea on his birthday tomorrow. It was a weekend. I was celebrating my last diagnostic radiology rotation. Only three weeks left of residency left. So we just kind of wanted to celebrate and relax and actually enjoy being somewhere other than our house or the hospital. So it was actually pretty nice. I just started vascular surgery today. And for some reason, I had to round at 6am, which means I had to set my alarm at 4.40am, which I haven't done since intern year on surgery. So that was exciting. We have a case today at 9.30, which I think is like lower to shermony angiogram. I don't do too much peripheral arterial disease work. So I'm really excited to learn the vascular craft, if you will. But right now, I haven't done like an hour and a half before that case starts. And I don't really have any responsibility because vascular surgery isn't my specialty. So anyways, I got breakfast, eggs and bacon. I got bacon because my labs that I got last week are perfect. So exploration of a little bacon today. And just like that, it was a beautiful day out again. And I officially finished my first day of vascular surgery. It's actually pretty good. I ended up doing two. Oh my God, look at that hair. I mean, I need a haircut so bad. We're doing one Friday, five days. Anyways, so today went well. Did two lower to shermony runoff angiograms and a little balloon, a little stensing, the usual. And now I'm going home. Actually, we're going to sell Andrana's car because she is leaving for New York in six days. She's going up two weeks ahead of me and we're going to drop her car off at the place we sold it. And I'm going to go meet her right now. Andrana is finally saying bye to her first car. This is your first car? Second car? Yeah. So her second car, her baby, she's had for how many years now? How many years? Seven years? Yeah. Are you going to miss it? She just sold her car. They sold Lacey. Are you upset? I'm very upset. She'll go to a good home, I bet. I hope so. We'll see. Hope someone takes care of Lacey. All right, so I know this vlog has kind of been all over the place with me ending residency and then me going to the beach and then me also starting another vascular surgery rotation. But I don't really know what I was trying to do. It's just some hodgepodge video. I was planning on making a whole video about how I'm going to miss residency, but then I kind of realized I did that already. And then we just kind of got caught up in life situation because we're moving right now and it's super hectic. I still have tons of stuff to do and keeping up with all the social media stuff is also hard when I'm also trying to do a surgery rotation as well as you can imagine. So thank you all for watching this. Thank you all for sticking through till the end. I really appreciate it. Make sure you smash that like to subscribe button. I have so many other videos coming down the pipe. I'm so excited about moving to New York City and showing you all the city, showing you all my apartment, you know, just kind of the whole city vibe and living in New York, you just kind of have this like creative vibe, just walking around there gives you like creative vibe. I don't know, it's really hard to explain unless you actually live there, but you just feel inspired to create cool content and all this stuff. I'm also getting a new computer, also getting new editing software, also getting new desk setup once I move into my new apartment. So a lot of stuff is coming within the next couple weeks. Make sure you all smash that like to subscribe button. Follow me on Instagram if you don't already. Turn on those post notifications so you are notified when I post new video. Otherwise, I'll see you all in my next video.