Last week, Marjorie Taylor Greene introduced the so-called Protecting Children's Innocence Act, which is an anti-trans bill that is explicitly eliminationist in its nature. So as Erin Reed explains via Twitter, it isn't just a bill that will forcefully medically detransition all trans teens, it also targets trans adults. It includes a ban on healthcare and forced detransition for trans teens, bans insurance coverage for adult trans healthcare, bans higher education slash accreditation from teaching trans healthcare. Its aim is to detransition as many people, including adults as possible, by removing insurance coverage targeting people who teach how to care for trans people and more. This would even bar psychology programs from teaching to recommend gender-affirming care to trans people. It would bar EMTs from learning how to care for us. It would bar therapists from recommending the best medical care. It would bar medical schools from teaching surgeons and endocrinologists. So the goal of this bill is to legally and forcefully eliminate trans people out of American existence. This is the definition of an eliminationist piece of legislation, and it should horrify everyone. So what this will do is it would make gender-affirming care to trans youth a classy felony for doctors with a penalty of 25 years in prison. Now Marjorie Greene designed this legislation this way because we saw the way that criminalizing certain procedures has had an effect on doctors in states like Texas where abortion is banned. So doctors in the state are so terrified to perform abortions or procedures that would remove the fetus even if the woman has already miscarried because they don't want to be accused of doing an abortion on a live fetus. So the same thing would be true here. So that way, even if they were to recommend social transition for a child, they'd be so terrified to do that that they'd lose their medical license and face penalties of 25 years in prison that they would just not offer the care altogether. That's the goal of this bill. Now at the time that I record this, it has 14 co-sponsors. It started out with five, but that number is growing, and we'll talk about why it's growing in a moment here. But just imagine what it would be like if some lawmaker with no medical degree decided to create legislation that would ban doctors from treating diabetic patients with insulin while I say that it's illegal to treat somebody with insulin. It's preposterous, right? But that's similar to what this bill would do because the recommended treatment for trans youth is gender affirming care. Now what they try to do is fear mongering claim that that means castration or bottom surgery, but that's not actually happening. Oftentimes it just means social transition. And when they reach older ages, if they show persistent gender dysphoria with years of counseling, they can get access to puberty blockers and eventually hormone replacement therapy. But bottom surgery is not performed on minors. In some instances, some doctors will do top surgery on trans boys, but it's when they're in their late teens, they're almost adults, and it's relatively rare. But really what we talk about when it comes to trans youth and the treatment for gender dysphoria has approved by medical professionals and the consensus of physicians and pediatricians is social transition and HR team. But they're saying, no, you can't treat these children in the way that it's recommended by doctors and professionals. You have to treat them in the way that we say. And that means ignored.  When it comes to treating COVID-19, she's against vaccines. So this imbecile is trying to impose her will on doctors when she knows nothing about this. Like imagine you're a teenager. You're going to school and you've been able to socially transition. And all of a sudden you're forced to detransition where clothing that you don't feel comfortable with identify as the gender that you don't feel comfortable with. I mean, this isn't a one-to-one comparison. But if I were forced to shave off my beard, grow out my hair, wear feminine clothing, dresses, I would not feel comfortable in my own skin. But this is effectively what they are forcing young people to go through. Yeah, it's despicable. It's gross. And this will lead to an increase in deaths of trans youth by suicide in the event this were ever to pass. Now, the reason why the bill is gaining a little bit of popularity and went from five co-sponsors off the bat to 14 is because she went on the most popular news show in the country to promote it. Tucker Carlson, take a look. But when it comes to gender affirming care, which is really child abuse, this is actually an assault and it's child abuse. And this this practice should never happen.  And it's an embarrassment to our country. You see, I'm one of those that believes a Republican party is only worth being a true party, worth deserving of the people's votes. If we are willing to stand up and stop horrific things like child abuse and like so-called gender affirming care, which is really genital mutilation. It's puberty blockers that cause chemical castration.  Teenage boys being castrated. This needs to be illegal. And I'm introducing a bill called Protect Children's Innocence Act. And it would create a law that would cause it to be a classy felony for any person involved in so-called gender affirming care. That means general mutilation surgery. That means hormones. That means puberty blockers. Anything involving any youth under the age of 18. Because these kids are too young to make these awful decisions that will affect them and will be permanent for the rest of their lives.   You should be in prison. So, I mean, I assume you're going to get every Republican vote for this.  But unfortunately, I don't yet. I only have five cosponsors. I'm talking to all of my colleagues and urging them. Because Tucker, let me tell you something. This is a referendum on the Republican party. When we take back the majority, if the American people elect us, we have a lot of work to do. Not only do we need to impeach Merrick Garland. We need to clean out the corrupt FBI. We need to hold the Democrats accountable and defund all of their climate garbage. 87,000 IRS Army. But there's one thing that we have got to do. And we're not even a party worth deserving of the American people's votes. If we cannot protect children from this horrific child abuse and create this to be a felony, because this practice has to end, it's the kind of things nightmares are made of. And these are monsters under kids' beds that are doing these horrific things to them. Yeah. Well, you can't sexually mutilate children. Sorry. And I hope you will send us a list of every Republican who's too cowardly to affirm the obvious. Once again, you can't sexually mutilate children. Period. So thank you, Congresswoman, for what you're doing on this. We appreciate it. Thank you, Tucker. So first of all, it should horrify everyone that they want to turn this into a litmus test.  Because that's not even happening there. But there is arguable mutilation of children's genitalia happening in the United States frequently. In fact, between 1980 and 2010, nearly 60% of newborn infants have been circumcised, which is a procedure where the baby's foreskin is surgically removed, which is an irreversible procedure still common in most areas of the country, excluding the West Coast. Where's the outrage for this? Does this not qualify as genital mutilation of children? Well, there's no outrage for this because Tucker Carlson actually defended this procedure and just pay close attention to the reasoning as to why he supports circumcision. I can't even believe that he said this after making the argument we just watched him make. But take a look. You're saying to me that there are opponents to female circumcision. Look, as you know, there's a lot of research. I don't want to get into the circumcision debate on men. But there's research. OK, well then there's research that shows there are profound medical advantages in that. There's no research that shows there's any medical advantage to female genital mutilation. First of all, that research is contested. So he supports circumcision because there is profound medical advantages to circumcision. Oh, OK, so he's trusting the science here, right? Well, here's what the experts say about gender-affirming care for trans youth. Doctors Matuk and Wald of Columbia University say gender-affirming care not only saves lives, but denying this medically necessary treatment to trans youth endangers their health and well-being. And this is the position supported by the vast majority of experts by the medical consensus, the American Academy of Pediatrics, the American Medical Association. So all of a sudden, when it comes to trans people, Tucker doesn't support the profound medical advantages. Why?  Sure, I think that he knows that, but it's because this isn't about science or genital mutilation. This is about hate. He hates trans people, and he wants to forcibly detransition all trans people. He wants to eliminate them from society. This is what that's about. Absolutely make no mistake about it. This is why he wants to make this bill a litmus test for all Republicans. And when we're talking about genital mutilation, it is not an accurate comparison to gender-affirming care for trans youth, because again, what we're talking about here, oftentimes with really young trans people, is social transition. Let them live the way that they want to live, wear the clothing that they want. But when they're older, perhaps they can qualify for puberty blockers. Perhaps they can qualify for HRT. And by the way, puberty blockers, these are given to cis girls to stop them from developing too soon as well. So would this bill also ban puberty blockers for young girls? It's just, it's such a hateful, cruel bill. The fact that they want to make this litmus test tells you how dangerous this party and their ideology is. Now there's other things that Marjorie Green said that I want to point out here. She called gender-affirming care child abuse, and she called it genital mutilation. Okay, but again, what about circumcision? She claimed that teenage boys are being cash-rated. That's not actually true. She hasn't provided us with any evidence that this is happening because she's ignorant. She doesn't necessarily have to give you the evidence because they've been sufficiently fear mongering about gender-affirming care to trans-youth for long enough that the viewers on Fox News, they automatically assume that what she's saying by default is true. When in actuality, it's ignorant. It's medically illiterate. And what she's saying, like her anti-vax views, it's all not supported by the overwhelming majority of the scientific and medical community. Now let's go back to the claim that Tucker Carlson made where he suggests that every Republican who's against this isn't a real Republican. This is the most popular news show in the program. So if it's not necessarily the most popular bill right now, wait for it because he's going to use his platform to generate support for this type of legislation. So that way in the event Republicans ever were to take back control of all branches of governments, this is what they'd want to do if they take back the House. I wouldn't be surprised if this bill passed within the next couple of years. We don't have to worry about this in the short term because this bill wouldn't get through the Senate. If Democrats held the Senate, this wouldn't be something that Joe Biden were to sign into law. But what this tells you is the trajectory that this party is on by trying to force other Republicans to go along with this eliminationist and frankly, genocidal bill against trans people. And it should horrify everyone. Even if you're not trans, this should horrify you that the rhetoric has reached this level, where they're trying to eliminate a portion of the population from society forcibly, forcing them to not live the way that they want to. Whatever happened to freedom? Shouldn't we be allowed to dress the way that we want to, live the way that we want to? They make it seem as if trans youth come out on Monday and then by Friday, they're getting bottom surgery.  It is incredibly cost prohibitive. We have a broken healthcare system where healthcare is not free at the point of service. So it's not even an easy procedure for trans adults to get, but yet they're pretending as if this is something that's a common phenomenon and children are getting castrated. That's just not true, but it's fear mongering that they're using to drive an anti-trans agenda. And it's gotten to the point where it is very, very dangerous. We are in genocidal territory here and people need to pay attention to this because this is only the beginning and it's going to get a lot worse within the next couple of years. Mark my words.


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All right. So after we have done some clustering, we found that some cells are quite similar among each other and that they form a distinct group. And now we want to actually know what these cell types are. So we will start looking at cell type annotation. And before doing that, we ask ourselves, what is a cell type? So it's considered to be the fundamental unit of life. And at the beginning, it was defined in terms of function, location, tissue type or cell morphology. So here we have an example of an image of a C elegans where the nuclei of all of the cells have been stained. And we can see that the organism has different organs. And of course, within each organ, the different cells will do different functions. And based on their location in the body and their function, we can define different cell types. So initial definitions were in terms of function and location, for example, but you can of course extend them to other features such as the presence and absence of cell surface markers. So for example, if we have one cell type, that is, for example, immune cells that flow in the blood, we can have within this immune cell type several other subtypes depending on the presence and absence of surface markers. Then you can extend this definition to the gene expression. So based on a full molecular profile within a type of cell, you can define additional subtypes based on their gene expression pattern, for example. So for the moment, a cell type is really defined within a biological system. And the different biological systems will define cell types differently. You can define them based on cell cycle phase, for example, or if they're differentiating or not. If they're migrating or not, for example, if you have tumor tissue, some cells in the tumor will rather stay at the tumor and other will mutate, for example, and start to spread and metastasize. So you have even different cell types within a tumor cell type. So the question to why we should identify cell types, of course, is because samples are heterogeneous. So remember that we had this image of a smoothie at the beginning, where a tissue is a mix of different cell types. And so we want to see first if we process a data set through single cell RNA-seq, which cell types were we able to recover and what these are. So we want to find the different cell types in our heterogeneous sample. Another question could be to profile cells from healthy and tumor samples and see how much they differ from normal cell types. So if you have tumor cells, what is their different gene expression pattern? So you have to identify these different cell types. Then the one of the goal of identifying cell types would be to find new cell types that you haven't analyzed before. So maybe if some of you are familiar with immunology, where cells are analyzed a lot with flow cytometry using surface markers as proteins, basically, when you analyze with flow cytometry, you're limited to a small number of surface markers. And so it is possible that a cell type that you have in your sample is missed because you don't actually stain for that particular surface marker that is expressed in that cell type. While with single cell RNA-seq, you process the whole mix of cells and then you will have the presence of this missed cell type. It allows you also to follow cell fate and determine cell differentiation mechanisms. So for example, if you have a timeline experiment in each time point, you want to re-identify the same cell types and see how it evolved. So this is why it's important to identify where your cells are. Finally, it's quite fancy now that using single cell RNA-seq data, you can determine how cells communicate with each other. So we will see probably in tomorrow's lecture some ideas about cell-cell communication tools where we determine which cells express, for example, ligands and which other cells express a receptor. And these two cell types might be able to communicate. Finally, we can of course try to compare the abundance of cell types in different conditions, etc., etc. So probably all of you attending this course have a good idea of your biological systems and probably know which cell types you want to identify. So here is an example of changing abundances in two different conditions. So here we have some lymphatic endothelial cells that come from wild-type mice. And then after knocking out a transcription factor, we see this green population of cells that is appearing that is nearly absent in the wild-type cells. So basically the idea here of using tools for cell type annotation is to identify what this new population is. I would like to give a warning about analyzing differences in abundance in single cell RNA-seq data. So it's not always the case that the frequencies that you have in your dataset of the different cell types is really a good image of what you had in your actual sample. So here in this experiment, we can see that the differences are quite striking where one population disappears and one appears. And using biological replicates, three mice per condition, these differences were conserved across all mice. So we sort of trusted the differences in abundances. Plus, it was validated with flow cytometry, for example. So I would give a word of caution about interpreting differences in abundance in single cell RNA-seq data. You always have to validate if your differences are actually true. So a small note about surface markers. So they're often considered to be the gold standard, especially in immunology. And these cells are actually proteins that you can encounter on the surface of the cells. And you can tag them with antibodies and try to identify which cells have these surface markers and which don't. And depending on the presence and absence of these surface markers, you classify your cells as belonging to different cell types. So here we have an example where we have, for example, the CD45 surface markers expressed in all cells. So we have probably all cells that are belonging to the immune cell type. But within this CD45 population, we have, in addition, different surface markers, which will define new sub-populations and so on and so forth. It is important to know that in mRNA sequencing, so single cell RNA sequencing, it can happen that surface marker protein expression is not reflected by the level of mRNA that you have in your single cells. So the dynamics between the level of mRNA and the protein at the surface is not always correlated. And here we have an example of the CD4 surface marker, where we have a single cell RNA-seq data set. Some cells are high for this. And at the same time, for this data set, the surface proteins were tagged. So remember in Khart's presentation yesterday, he showed you, for example, this hashing method where you can tag a surface protein with an antibody. And basically this is what was done here. So for every cell, you have the mRNA level of your gene of interest and also the surface protein that is measured. And here we see, even though these cells have the surface protein that is expressed, that is present, at the mRNA level, the CD4 is rather low. So to identify cell types based on surface markers, it is important to not only take one, for example, and call it a day, it's maybe good to use several genes. And so one example could be to use a method, so we will see this tomorrow, to find markers of different clusters and then use a list of all of the genes that are significantly more expressed in like, for example, here or here, and see if they belong to your cell type of interest or not. You could also use a combination of more genes. For example, if you know that the CD4 secretes some proteins, if they're both expressed here and here, then you can more confidently say that these cells are CD4, even though they don't have the CD4 mRNA. So about possible ways to annotate cell types, you have two different ways to do that. You have the manual way to annotate the cells and the automatic way. So the manual annotation is used very commonly because people know very well their biological setting, what genes the cells should be expressing or not. So they have a list of marker genes of the cell types they expect in their sample. And they will just look at where these genes are expressed, if one gene is expressed in cluster 0 and not in cluster 1, then I can say my cluster 0 is this cell type. The drawback is that it can be time consuming. You have to check many genes. And of course, you require expert knowledge about your system. And it's not always easy to find exactly which cells should express what. So sometimes it can be a bit subjective and inaccurate if you work in a biological setting that you don't know 100%. Then there is the automatic way. And here you require actually a reference. So what I mean by reference is that you have an expectation of the cell types that you have in your sample. And so, for example, in the literature, you find the publication of purified cell types that were profiled, for example, with bulk RNAseq or that will profile with microarray. And then you use this list of reference cell types and their expression profile to use as a reference to annotate your unknown cell types in your data set. So this means that you need a reference. And this reference can, as I just said, either be a published bulk RNAseq data set of sorted cells, or it can be also even a single cell data set that has been published, where I don't know, it could be cells coming from a big organ, but you're only interested in a subset of these cells to use as a reference. You could manually select these cells in the published single cell RNAseq data and use as a reference. Again, the drawback, then there is one, is that it can miss some cell types if in your sample you have some cells that are not present in the reference. So always make sure that the reference is quite broad and includes all possible cells that you have in your single cell experiment. So the method is that for every unknown cell in your data set, you will receive a label for that cell. So this can be either this assignation of cell type can be either done per cell or per cluster. So this we will see with the method that we use in the exercises with single RNA. You have to, you have the choice to predict a cell type label either at the cell level or at the cluster level. And the way it works is that it uses a correlation of the different single cell expression profiles to the profile of your reference. So let's first explore a little bit how manual annotation can work using marker genes. So this is a single cell RNA seek data set from one patient suffering from a brain glioblastoma. Actually this is a data set that is available on the 10x genomics data website. So 10x genomics has several data sets that you can use to play. So if you want to, after you do this course, if you want to test your code on another data set than the one we propose, feel free to go to the 10x data set website. And you can actually download the three files that you get after cell rangers. So the filtered barcode matrix, the barcodes and the features files. And you can just follow the Surat pipeline. So here what we have is different cell types from the brain. For example, we have microglia where we know that CCL4 should be expressed and we color our cells according to the expression of this gene. And it seems that this group of cells express this gene and the rest don't. And here we have another marker, for example, GFAP, which is usually expressed in astrocytes. And here we have another group of cells that is colored. So we can start to see which potential cell types we have in our data set and start to annotate our cells. So of course, maybe if you use manual annotation, you will need a list of marker genes for every cell type that you expect in your data set. And sometimes it's difficult to find easily if you don't have previous knowledge. So here are a few databases with cell type markers that you can browse. There is PanglaoDB, for example, which is a collection of created single cell RNA seek data from mouse and human. And they actually compile lists of markers for different cell types. So maybe we can have a look at this website, which is here. So for example, if we would like to have a list of markers, if we go back to the brain. So here is the home page and we can go to data sets, cell type markers. And we can browse a list of different tissues. And for each tissue, you can have a list of different cell types. So let's have a look at astrocytes, for example. So here we have a table of gene expression markers that were compiled from a collection of public single cell RNA seek data. And it's nice because it lists each gene, whether it's a marker in both species, so mouse and human. So in this case, we see that this is a marker for astrocytes in both species mostly. But sometimes for other cell types, you will have either it's for human or for mouse. Then you also have some specific specificity measures, which could be interesting to have. So for example, the specificity will tell you the frequency that this marker is not expressed in the other cells. So any other cells that is not an astrocyte, what is the proportion of these cells that express that marker? And for example, for GFAP, it seems to be quite low. So it seems to be a marker that's quite specific to astrocytes. So this can help you and maybe select which are the cleanest. So, for example, ALDOC could be also highly specific for for human, but maybe a bit less for mouse, et cetera. So then you can compile a list of marker genes like that. Then there is a cell marker is also a database for mouse and human. And it's also contains a list. I think I have it here. Yes, perfect. So basically when you're on the cell marker website, maybe I see if I can open another link here. Yes, you have, again, mouse and human. And then you can select your different organs and then if I can zoom in, no, it's quite small. But basically, again, if we go back to the brain, we can select astrocytes, for example. And the website is a bit slow, but if you wait patiently, you should be able to see like a cloud of genes. And the size of these genes will tell you how specific this gene is for this cell type. So again, we see that we have many genes, but GFAP is quite a good marker and you can be quite confident with that one, but you could add and look at others also. So this is I like when this websites are sort of visual like that. Then we have other possibilities to find cell type markers or even like reference data sets. So for example, single R. Single R is the package for a cell type annotation that we will use in the exercises. And via single R, you have access to another package, which is called cell decks, which is like a database of microarray data from sorted pre-fight populations. So this you can actually, when you need to use single R, you have your unknown cell types in your single cell RNA-seq data set. And you need to have a reference to do the annotation. So you have direct access to these references. Then you have the human cell atlas. So this is a consortium that aims to create a cellular map, a reference map of like the whole human body. I think they also have some mouse data. So here again, you have this option to use the human cell atlas as a reference. And finally, the single cell portal. So this is an interesting online tool because it contains published single cell data sets. And actually you have the option to visualize the data. So you can create UMAPs. So you can view UMAPs for the published single cell RNA-seq data. And you can also download the normalized matrices for the data sets. And you could actually use this downloaded data as a reference in your single cell annotation. And the good thing is that authors that submit data to the single cell portal actually provide their cell type annotation that they did before. So if you find one that is nice and nicely annotated, you could download it and use it as a reference. Again, if after this course, you want to further play and exercise all of the scripts that we did, the single cell portal is a nice source of data to play with because you can download the matrices and just import them into Surat. So here is the concept of module score. So we have seen, for example, in PanglaoDB that you can have several marker genes of astrocytes. So instead of going and checking each of these genes, like for example, if you have 200 genes that should be specific to a cell type, you don't want to go and see where each of these genes are expressed. So you could calculate a score for your list of 200 genes. And in Surat, you have this add a module score function that allows you to calculate this score for a set of genes. And what it does is that it compares the expression level of the genes that belong to your list of genes.  It could be a list of path of genes involved in a pathway also if you want. So you compare the expression level of your genes in your signature to some randomly selected genes that are called the control genes that are randomly selected. But they have a similar expression level across all cells compared to your signature genes. And then the score is just the deviation of the expression level of the genes in your signature compared to the expression level of your control genes. And so for every cell, you get a score which will be added to the metadata of your Surat object. And then you can color your UMAP according to the score. So here we see, I think this was a pathway activity. So genes link to a pathway. And we have some cells that are very high, some that are intermediate and some that are rather low. But you could use this for marker genes for any list of gene that is of interest to you. There is an additional package. So as you saw from Mahasha's presentation for clustering, you have hundreds of methods for dimensionality reduction. You have hundreds of methods. And again, for scoring of list of genes, you have several methods available. So I just show you the add model score of Surat. But of course, there is a development of these kind of methods. Here is one that's called UCELL that will also do signature scoring. And one of the advantage of this is that you could specify which marker genes should be absent. So for example, if you know that one cell type expresses one gene, but absolutely does not express another gene, you can provide this list of genes to be part of your signature. While with the Surat model score, you cannot have negative scoring, so negative genes. So in this case, you can include this and it can be maybe a bit more precise to get a more precise score, including the genes that should be absent. All right, so let's take a bit into single R. This is the tool that we will use in the exercise. So as I just said, it can have easy access to reference data. So there is the human primary cell atlas. It's manually annotated. It contains it's every cell type that is included there has several annotation one like main or broad cell type like immune cell, for example. And then for each of these samples in the reference, you can have even further subtypes. So you can have both levels of annotation. It contains many samples. Then you can also have access to the data from the blueprint and encode. In this case, it includes reference samples from bulk RNA-seq and has several broad types and also subtypes. And if you're working on mouse, you have the in gen, oh, sorry. You have access to the immunological cell types through in gen and also a data set of mouse RNA-seq that's linked to brain, for example. So again, here we are a bit biased towards human and mouse. But you can, of course, use single R for a non-model organism, for example, if you have a reference. So that's always the important part. You could generate your own reference and then use it to annotate your single cells. So you can classify each unknown cell in your data set, either to main type or subtype, and do the labeling either for each cell or for each cluster. How does single R work? So here we have an example of a single cell. It is unknown. It has to be annotated. This is its barcode or cell name. And here we have an example of our reference data set. So this is, for example, several cell types that were pre-fied and profiled using bulk RNA-seq. And so each dot represents one sample that corresponds to that reference cell type in our reference. So for example, for neutrophils, we had several samples included in the reference. But for dendritic cells, for example, we had just one. So if you're really interested in annotating dendritic cells in your data set, maybe this reference would not be the best choice. So again, it's always good to really carefully choose your reference. But it can give you a first idea if you don't have another reference to see what type of cells you have. So we have several different types. And what it will do is like first step in single R is to select a list of genes that are differently expressed across your reference cell types. So because it will calculate a correlation for every single cell, the computation can be quite long. So it will only calculate the correlation for like the top differentially expressed genes across the reference. And there are several methods to select these genes, but typically it will select the genes that have a median expression in your reference that is higher compared to the median expression in all other cell types. So it will take the gene expression of your unknown cells and correlate to the genes expressed in each cell type. And you will obtain a Spearman correlation score against each sample in your reference. So you can see sort of the distribution of the correlation score to each reference. So here we see that our unknown cell has overall low correlation score to neutrophils. So it's probably not a neutrophil. And here it was just sorted according to a median correlation score. And we see that very good correlation score or I don't know how good it is, but the highest correlation score was obtained for CD8 and CD4T cells. So the score are very similar. So probably this unknown cell is a T cell, but it's maybe hard to say if it's CD8 or CD4. So single R will include a step that is called fine tuning. It will reselect the genes that are specifically different between this sort of close matches between CD8 and CD4 and re-correlate and compute the score and then have like a final cell type label decision. You can do some annotation diagnostics once you assign a label to each of your single cells. And here are some of the plots that you can generate on a single R object. So one is called a plot score heat map. It will take these correlation score of every single cell. So in the columns, we have the unknown single cells in our data set. And in the row, we have the different reference cell type labels. So here we color according to the correlation score obtained for each single cell against each reference cell type. So here we see a group of cells that has very high score to duct cells, for example. And at the top, you can see the final assigned label for each unknown single cell. So it took the one that had the highest score and it's the label is indeed associated with duct cells and the same for the others. So this is nice to to to have a look at because you can see if some cells have like a sort of high scores for several cell types, then you may need to go and maybe choose another list of reference samples to better define your cell types or to manually look and see if you can discriminate and make a final decision based on marker genes, for example. Another plot that you can generate with single R is called plot delta distribution, also on your single R object. And what it will do is to calculate the deviation of your score for that label against the median of the scores obtained across all cell types for each cell. So here every dot is one cell and you see that the deltas for the cells assigned as being as inner cells are quite high. But for a few cells, this deviation is rather low. So maybe you would have to manually check and see if these are really as inner cells or if there's something else. So try these during the practical exercises. So as I was just saying with single R, you have access to references directly through the cell dex package, for example. But in the case you have, for example, a non-model organism or if your reference samples are not included anywhere, you can actually use your own reference. So here's the example where we have some cells. So again, this is a mouse, but it's just the example to show that you can use any reference to annotate your cells. So these were immune cells exposed to different strains of LCM virus. And we wanted to know how well these clusters correlated with bulk RNA-seq of different populations that were available in the lab. So it's really like custom reference that we used. And when you use this with single R, the reference just is like a matrix of normalized gene expression for each one of your samples included in your reference. And of course, the cell type name or label that belongs to each sample in the reference. And then you can see which clusters correlate most with each reference. So here we have naive, etc. So this is typically what you can do with single R and using your own custom reference. Here is a paper that sort of evaluates different methods for cell type annotation. And in terms of performance, I think that in the paper, they concluded that the tools that are called SVM, SCPRED, and single cell net were the best ones. So they evaluated Python methods and R methods. And they also checked whether you need prior knowledge of the cells or not and also whether there is a rejection option. This rejection option is the fact that if you have a cell that has a low score, for example, for one of the references, do you force a label on that cell or do you just leave it as unannotated or undetermined cell type? So we see, for example, with single R, which is R-based. Here you can see that all the different underlying methods that are used. So we just discussed that this is correlation against a reference set. You don't need any prior knowledge and there is no rejection option. So you have to keep in mind that when you use single R, all of your cells will receive a label, even though these cells are not part of the reference, for example. But you have these diagnostic plots to help you in deciding more or less manually if some cells are wrongly assigned or not. So this is something to keep in mind. And other tools, they do have this rejection option. So single R was not the top performing method, but it was among the best. And because it's quite easy to use, we often use it day-to-day in our analysis. Other options for cell type annotation that are based on Surat functions. One is the cell type label transfer. So you have seen today with the integration that you can actually use this exact same method as the integration to actually annotate unknown cells using another single cell reference. And this is what was done here, for example. So the commands are quite short again. First, you find the anchors and then you combine and put a cell type label using the transfer data function. So here, the reference is a single cell RNA seek data from annotated cell types. So we have four cell types. And then you can have a single cell attack seek data, for example, and see which cell types you have in this data. And you have used this cell type label transfer for this prediction. So the cells that you want to predict don't have to be attack seek. They could be another single cell RNA seek data that you want to annotate. So using this label transfer is also an option. And here, I think there is no rejection of the cells, so all cells will get an ID. The authors of the Surat package also developed azimuth. So you have two options to use azimuth. And basically, I think it's just based on label transfer, but they offer a list of references of cells in different tissues. So you have PBMCs or cells from fetal development or human lung, etc. So you could actually just install that package. So here you have the vignette. If you want to run it locally, just install that package. And then it's quite easy. You use run azimuth on your query with the reference that you want that is included in that package. And then you'll be able to do cell type annotation. And here, the screenshot is actually from a web app version of this package where you could upload some raw data. And it will do all of the processing and annotation online. So these are two ways that you can use this package. Finally, I think this is the last example is, again, another method for a cell type annotation. And here, the idea of the authors of this package was to take published single cell data from several sources. And so by combining, so you have each of these cell types that is present in each of these sources, but you have a small number in each. So by combining this data set, you have a bigger data set that you can use as a reference and maybe annotate more easily. And then here, what you can do is if you have your unknown single cell data set, you could actually project your unknown single cell data set on top of this exact same representation. And then you have a density plot that will show you I have a lot of CD8TX and a few CD8 naive. So basically, it will project your unknown cells onto this dimension. And then you can see where your cells fall. And so in the end, sort of annotate your cells. You don't have to use it that way. We don't have to project it, but you can also just simply use it to annotate your cells in the end and then show it on your own UMAP after this annotation. So this is another way to annotate cells. And you can use the algorithm also using your own reference. So if you don't work on immune cells, you don't have to use their own reference. You can use your own. And here in this case, there is a rejection method. So if a cell does not belong to one of the reference types, it will not be annotated. I think that's it. So we haven't mentioned the Oscar book, I think, yet. So the Oscar book is Orchestrating Single Cell Analysis. It's an online book created by people at Bioconductor. So it's mostly based on Bioconductor packages. So no Surat will be involved in there. But what I like about this book is that it has, for each section, it has explanations and some a bit of background. And so you can have, even though you don't use these tools, I think reading about the theory is always interesting. Then if you work with multimodal single cell data, so that means you measure like RNA plus a taxic or other sort of combined modalities on your single cells. There is a paper here that can help you in dealing with it. And finally, there is a bit of a more recent cell annotation review than the one that I showed that you can also have a look at. Nice. So the question is, which cell annotation method will you choose for your own dataset? Manual annotation, automated, or I will try both and maybe compare. So there is no wrong or right answer. It's just to see what is your gut feeling there. All right, we have 25 people, 25 answered. Nice. So it's good that you're brave and trying several methods and and see which could give the best annotation. So I'm happy to see that it all depends on your biological system. It's it may be possible that if you work on a non-model organism, only the manual will be feasible because there is no reference available. But if you work like on human and mouse, there is so much published reference that automated is just doing the perfect job. So it all depends.


Within recent months, the Atomic Energy Commission and the Department of Defense have conducted important tests of an experimental device based on the thermal nuclear principle leading to the development of a very large-gilled weapon. In addition, a proof test was made of a large-gilled fission weapon. These constituted Operation IV. The report of these accomplishments is about to be presented in film form. We will show you new techniques and new scientific developments. We will minimize the portrayal of normal military service support since you are familiar with this aspect of test operation. As commander of Joint Task Force 132, I invite you to observe Operation IV, carried out on our Pacific Proving Ground, and if we talk at all. We wouldn't be out here to open sea if it wasn't a beacon. Captain, we're in position. Recommend we do speed, sir. Very well, Mr. Adams. All engines ahead one third, sir. Engine room con ahead one third. Engine room reports making turns for four knots, sir. Very well. How are the wind and tide affecting us? As predicted, sir. Good. It's important that we stay within that area so we remain within the television column. Yes, sir. Welcome aboard the USS Estes. As you may or may not know, the Estes here is the command ship of Joint Task Force 132. We have minutes to go before the first-class mic shot of Operation IV. Um, 59 minutes now, to be exact. We've been here since daybreak. Let we talk last night during the early morning hours. Now, as you can imagine, feeling is running pretty high about now, and there's reason for it. If everything goes according to plan, we'll soon see the largest explosion ever set off on the face of the earth. That is the largest that we know of. 8,000 men will view the event from this point. By the way, the carrier over there is the Rendova. She's primarily set up as a base for fighter security and helicopter re-entry. And that's the Curtis over here, the weapons assembly ship. In the time between now and HR, I'd like to show you around, if I may, and introduce you to some of the people connected with this operation. And in general, piece together the events which have brought us to this point. To start off, I'd like to show you something over here. You realize there are many miles of ocean between us and any we talk at all. To know what's going on back at the at all, these antennas are receiving televised signals and are giving our men here a second-by-second account of what's happening on shot island. The television receivers are in here, in the control room. But before going inside, let's take a look at a chart. It may give you a better idea of our location. This is our position, 10 miles south of any we talk island, or about 30 miles south of Ugalad, the shot island. We must keep a very accurate position here because of the televised signal. It's a very narrow beam, and the ship must stay within that beam. Now, perhaps you're wondering why we're out here in ships. Well, the answer is very simple. It's too dangerous on land. We're expecting a yield of 4,000 to 10,000 kilotons. That's equal to between 4 and 10 million tons of high explosives. Considering all possible effects from a bang of this size, the best observation point is at sea, on a mobile platform at a known safe distance from zero point. Okay, let's go inside now. Well, this is it. This is the control room. I'd like to have you meet Mr. Stan Burris, the commander of the scientific task group. Oh, Stan, I wonder if you could tell us something about the operations that go on in this room. Sure, I'd be glad to.  The lights indicate that the timing signals are functioning properly. The dials, this collection of dials indicates that the liquid deuterium is in the proper state for firing. You will look close. You will see that it is now 55 minutes before each hour. As time clicks off, more and more lights come into operation. This is the one-minute light, 32nd, 15, 5, 1, through firing. Since this is a thermonuclear test, and we are using a type of hydrogen in a liquid state, it is necessary to keep a close check on the condition of this fuel. The hydrogen is not in the proper state or at the proper level. It would have such a marked effect on the results that there is no point in continuing the experiment in which case we would postpone the shot. Is there any chance of that so far, Bob? No, there isn't, Stan. The cryogenic system has been operating perfectly for several days and we're not particularly worried now. That's fine. I have here a layout you may be interested in. This diagram will give you a general idea of the whole setup. Data from the sequence timer is piped over to a display panel. Likewise, cryogenics data is piped over to this display panel. This kind of display panel is new to atomic test work because of the large number of remote control and metering problems encountered in this operation. For one thing, the mass to timing and metering apparatus is located next door to the shot cab rather than being placed some 20 miles away on Parry Island as is usually done. This close view is possible, of course, because the lens of a television camera rather than human eyes is watching events. So cryogenics data piped direct from mic and information from the sequence timer is transmitted from the small building attached to the main cab and is relayed by way of a TV antenna atop a 375-foot tower to the estus. So that's the flow. From timer on through to display panel, cryogenics data to panel picked up by a television camera and relayed on out to the estus. A very ingenious arrangement. But what happens if you have to stop the firing mechanism or can you stop it? We can stop it all right if we have to. We have a radio link direct to the firing panel in the shot cab. If we have to stop the shot, we simply push this button. Just a simple flip of the wrist, huh? That's right. But a lot of work goes down the drain. You understand we don't want to stop this thing unless it's absolutely essential. No, I can understand that. Say, I was out on deck when you fell his return. Well, that is when the firing party returned. Uh, what happened out there on Shot Island? If you'll excuse me, I suggest you talk to Colonel Lunger about that. I have a timing signal coming up. All right. Then, Dick, the firing party's big job is to see the last-minute details of arming and firing and to make sure that the Shot Island is secure. That's the broad brush of it, yes. I've been a member of firing parties before, but this was different somehow. A man standing, as I stood, on the outside of the building housing the mic device couldn't help but feel to sense the importance of this moment. Inside, a handful of men were making a final check. We're arming a device which could be the key to a new era in atomic weapon-eering. I don't know just how the others felt, but I felt small when I thought of the experiment being readied inside. This one test could take us out of the realm of kilotons into the fantastic world of megatons. And then, at each minus six hours, the job was finished. The mic device was on its own and ready. We all moved to take a final look at the gadget which represents a year of intensive engineering. As we moved toward the pier, one couldn't get away from the feeling of being alone, of knowing you were the last to leave an island which might be shocked beyond human reentry. We made the run from Shot Island down to the anchorage of the Estes off Perry in a fast crash boat. Soon after, the Estes made way through the deep entrance between Perry and Japtan Islands, out to a point ten miles southeast of Perry, the rendezvous area of the task force ships. We finished our job at the cab, came over this side, and here we are, waiting on station just like everyone else. Well, we won't have to wait much longer, Dick. All right, thanks. Well, that's part of the story. A small part, actually, of this whole ball of wax. An operation like Ivy has many facets, some large, some small. Behind the present activities on this ship, behind what went on at the atoll itself, whatever preparations had to be made in the States, is the broader story of policy, philosophy of planning. Now, this deeper story, the background, so to speak, has its roots entwined in the broad international picture, in the larger aspects of the Atomic Weapons Development Program, in complex scientific conclusions. Not long ago at Los Alamos, I was talking to Dr. Alvin C. Graves, head of the Test Division there. He's on board now in Flagplot as the scientific deputy to the Task Force Command. He's one of the men who can tell us about the thinking behind this operation. We're not really sure what progress the Russians have made in this business of nuclear research. And so the only safe assumption to make is that they're interested in producing a fission bomb and to use it as some sort of trigger mechanism for a hydrogen bomb. It's obvious we don't want them to have a hydrogen bomb before we do. And so time is urgent. Time is the thing we have to beat. Dr. Graves, the historical side of this operation is particularly important. I wonder if you'd mind speaking right to our camera. Not at all. First, let's go over this business of the hydrogen bomb. Why use hydrogen? Hydrogen permits us to have an inexhaustible source of energy. It's plentiful. We don't have to worry about the critical mass limitation. Second, we can get hydrogen very cheaply. If we can use deuterium, we can distill it from ordinary seawater. We're floating on it right now. That's very interesting, Dr. But what is deuterium? I was coming to that. First, let's consider ordinary hydrogen, such as is used commercially. An atom of this kind of hydrogen has the simplest nucleus known. It has one proton. But hydrogen exists in two other isotopic forms. Deuterium, which has a neutron in addition to the proton, and tritium, which has two neutrons in one proton. Ordinary hydrogen is not considered a good bomb fuel. And tritium, because of its expense and rarity, can only be used in limited quantities. Well, that's part of the background of Ivy leading up to the present. In one minute, it will be H minus 45 minutes. H minus 45 minutes. It's down the deck for a bit. All right, fine, Dr. Quite a bit. Hi, Jerry. Where about Mike? It's too late to worry about that now. The shot island is about over there? That's right. It's generally north and west of us. For the past half hour, the ship has been headed directly toward the shot island, and will continue to do so until shot time. You know, there's one thing I can't quite put together. That's this business of success or failure. I've heard there's a 50% chance of its failing. Now, this low margin of success wasn't true on the other shots, was it? No, it wasn't. Up until this operation, that is from about 1945 through 1951, the chance of failure has never been more than about 10%. In Crossroads, Sandstone, and Greenhouse, we had great confidence in the operation succeeding. We recognized the change in philosophy, however, when Dr. Ratbury spoke to a group of us at Los Alamos in 1951. Gentlemen, up to now, the laboratory has had sufficient time to compile information and revise weapon design before a field test of a weapon. As of now, the situation has changed. We must take risks. Calculated risk, it is true. But risk, nevertheless. According to the presidential directive, we must ascertain if a hydrogen bomb is feasible, and do this in the highest possible speed. We must ascertain if such a bomb is feasible, if we can, in 1952. Here is what I think we must do. We must set up a special staff under Dr. Marshall Holloway reporting directly to my office. He will receive from the theoretical division, the theoretical designs of such a system, have them fabricated, and shipped to NWETOC. There, they'll be taken by Dr. Graves and J Division and tested. It must be recognized that we're taking great chances. A great gamble. But a gamble, while there's a possibility of failure, nevertheless, there's a possibility of great gains. NWETOC will become our theoretical laboratory rather than a proven ground. And that's the way it is today. We're taking a gamble. I see that now. But then the uneasy state of the world puts everything on a gambling basis, I guess. Yes, but not as much of a gamble as you might think. Take that man over there. He and his company have put a great deal of thought into the engineering and design of Mike. Well, see you later. It's along, Doctor, and thanks a lot. I couldn't help coming back to the control room for another look. They tell me everything's going along without a hitch. I've been taking a closer look at the cryogenic screens there. You know, there's a lot too of this glow temperature work. In a good many respects, it's the most outstanding problem of the entire operation. And as soon as Bob Gibney can break away, we'll talk about some of these problems. But this I know. A lot of heads have been scratched, trying to figure out some way to work with various gases which have to be brought down to a liquid state. Oh, Bob, I've been browsing through this subject of low temperature physics. That's quite a subject. It sure is. The general problems connected with very low temperature operation had been met on the George shot of Greenhouse. The amount of thermonuclear fuel required for Mike's shot is considerable. Do you remember the buildings in the south end of Perry near the airstrip? Yes, I do, Bob. Well, that's the liquid-action plant. The original plant was established during Greenhouse. Because of the difficulties in transporting, the decision was made to produce liquid hydrogen at Inuita, rather than in the States. But for Ivy, it had to be expanded considerably. The capacity of the original hydrogen plant was doubled for the production of liquid deuterium. And a new plant was built for the production of hydrogen with the capacity four times the old Greenhouse model. The small nitrogen plant was expanded eight times its original size. For Operation Ivy, the Perry Island plant operated continuously for three months to produce the necessary low-temperature liquids needed in the experiment. But of course you don't put a material so difficult to make and so difficult to keep in milk bottles. You use special containers called doers, which are simply outside thermos bottles. Months ago, these doers were made in Massachusetts by the Cambridge Corporation, and driven out to Boulder, Colorado for testing. Each doer is mounted on its own truck, with its own refrigeration plant and generator. Against the picturesque background of the Flatiron range of the Colorado Rockies, these doers were filled with liquid hydrogen at the cryogenics lab of the National Bureau of Standards. This plant, set in an unpopulated area, is the largest source of liquid hydrogen today. And hydrogen is essential for a practical shakedown test. The climax for these doers came out here at Inuita when they were actually used for the job intended to transport. Since production of thermonuclear fuels might be needed on future tests at Inuita, it was thought best to put the liquefaction plant on Perry Island instead of on the Shaw Island, where it probably would be destroyed. But this meant a satisfactory means of transporting liquid hydrogen from Perry to the Shaw Island. This type of exaggerated thermos bottle mounted on wheels was the answer. A doer is constructed to isolate the contents of the doer from the outside temperature, so no heat can leak in. Contained within the boilerplate outer shell is an inner shell made of stainless steel with a vacuum in between. This is the container which holds liquid hydrogen. A radiation shield made of copper surrounds the bottom half of the inner shell. There are two bottoms to the radiation shield, and in the space between them, there is room for liquid nitrogen. The purpose of the radiation shield is to provide a barrier to the flow of heat. The heat which does leak in boils away the nitrogen first, thus protecting the liquid hydrogen. But even with all these precautions, another source of heat still occurs in the inner shell. This is mainly due to a special property of hydrogen. The molecules of hydrogen, as a gas, exist mainly in a state called the ortho. At first, when the gas is liquefied, the molecules stay in their original state. However, within a period of some two weeks, the molecules rearrange themselves into a state called the para. This is a natural phenomenon which always occurs. In this transfer, heat is given off. Actually sufficient heat to boil off two-thirds of the liquid hydrogen. To take care of this excess heat, a special refrigerator has coils located in the inner shell. The refrigeration plant, which supplies these coils, does a remarkable job for its coolant approaches absolute zero. Absolute zero, but unimaginably cold zone where everything stops. All life, all molecular motion. Minus 459 degrees Fahrenheit. A little above this fantastic figure, hydrogen becomes a liquid at minus 423 degrees Fahrenheit. This is the temperature to be maintained in the doer. Only 36 degrees away from the bottom. Absolute zero. The helium refrigerant used can affect the temperature sufficiently low to cause the gaseous hydrogen boiling off to return to a liquid state. Well, I went into more detail from the cargenic's end of this project than I intended. Well, so far we've pieced together quite a bit of this operation. I don't believe you've ever had a good look at our key test islands. The test islands for Mike are located at the top or the northern sector of any we talk at all. Some 25 miles from Perry and any we talk, the two base islands of this at all proving grounds. There are three main islands making up the test site. These are Eluge Lab, Teter and Bogon. Early in the game, these islands were linked together by causeways. The old business of men and machines digging and piling up coral sand. These connecting roads were built to make it easy to get from island to island and also to act as land platforms for some of the instrumentation. A sizeable camp was set up on Teter. First it housed construction people and later served as an advanced base for scientists working on the shot island. In the early months, Eluge Lab was just another small naked island of the at all. But by mid-summer it began to look like the thing it was selected for. A shot island. Actually the cab, so-called because it houses the weapon, is not a cab at all, but a building set flush to the ground. It has all the earmarks of a common worksheet, but in reality it's a laboratory building set on a Pacific at all. How do we stand at Quageline? Then they're all in the way except for the third and fourth sample elements. Right, are you picking them up on the slopes as they come into the area? Yeah, well from here in let us have an immediate modification if exact shot positions aren't maintained. Yeah, and the helicopters we expect to order them out in the fall. Parology was mainly required to move some of your units. This, as you have probably gathered, is joint operations. All atomic test operations in the Pacific so far have been run under a joint task force kind of setup. Operation IV is using the same organizational structure as Greenhouse. Four task groups. Scientific, Army, Navy, and Air Force. Members of these groups are here now sifting and coordinating the many details of this joint operation and passing key information on top sides to the command level. The Army is... The Army is the executive agent on this operation just as the Air Force was on Greenhouse and the Navy on crossroads before that. Whether this is J3, let me have your latest recon plane VHF report over. I've been talking about task force organization. Wish you would add something to help clarify this business of executive agent. Yes, I'll be glad to. First of all, we are organized along very simple conventional lines. I believe I have a chart which explains it better. The Joint Chiefs of Staff and the Atomic Energy Commission on this level coordinate on the instructions that are to be issued to the task force commander. The Joint Chiefs of Staff control the operation through the executive agent, the Chief of Staff of the Army. The task force commander has operational control of the Air Force, Navy, Army, and scientific task groups. Now, when we were in the United States, the Atomic Energy Commission maintained a control over the scientific personnel at Los Alamos scientific laboratory. However, for the operational phase, the commission appointed General Clarkson as its special representative. And in this way, he controls the entire operation. I get the picture, General. Okay, attention, you take this one. What do you feel are the unusual aspects of your operation? Well, we like to believe that IV is a milestone in atomic development. I believe that in many ways IV is another affinity, the beginning of something, and we hope the beginning of the H-bomb era. You know, it's been exactly this thought that General Clarkson has had in being so insistent that the task force support elements be emphasized. The fact that all of us must support the scientific people who are doing such important work, I would say that this is the keynote of the operation. Excuse me. While we're here in joint operations, it's a good time to think for a moment of the work of the military task groups. Take the Army, for example. This support group commanded by Colonel Georgie Burrett provides a variety of services which make it possible to conduct an atomic test such as IV at a proving ground outside the continental limits of the United States. The experience. Highly specialized, sometimes costly instruments help scientists bring home this vital data. An outstanding example of such specialization is the use of a helium atmosphere box by the Naval Research Laboratory. The plywood tube looking like a train of box cars runs from the shot island across the causeways to a detection station on Bogart, a distance of nearly two miles. Inside the box, the helium is contained in 200-foot polyethylene balloons. Some 45 balloons being required to fill the plywood tube. The detailed progress of the mic reaction will be studied by utilizing this device. The measurement starts in the mic cap with a columnating shield, a large concrete block designed to stop all radiation which might confuse the issue. The setup is a lot like having peep holes in a fence. DT neutrons from the mic reaction will pass through holes in the concrete wall and react on iron converters to produce gamma rays. These gammas will then be collimated through the long helium tube or column to the detectors. Helium is being used for a good reason. Gammas traveling through normal air will have a difficult time of it because of the relatively large size of an air molecule. Now the helium molecule is much smaller and the gammas will have a relatively free path in which to travel, the fence will be a thousand times more intense at the detectors. This experiment is one example of the kind of unique instrumentation needed for learning the secrets of weapon behavior. In addition to the diagnostic kind of measurement, many studies are being run on the effects expected from a high order detonation. These projects are being conducted jointly by the AEC and the Department of Defense. Today's there are many questions to be answered. Classical routine questions and special pertinent questions pertaining to the hydrogen device mic. For example, what will be the blast pressure on and near the ground? Many instruments like the Wiaco gate will measure over pressures at ground level. And from 15-foot towers. What will be the blast effects at low altitudes, say up to 300 feet? Water shells will label the air with smoke puffs for photographic study of their action. And what about high altitudes? For this, anti-aircraft guns are being used. The bursting shells from these guns will label the air with smoke up to 26,000 feet for a free air pressure measurement. Not only will the ground and air be measured for pressures, but also the water surrounding zero point. For instance, what will be the motion of the water waves in the lagoon? To get such data, photography will study the movement of these floating targets. And then, what about the underwater shark wave in deep water? And it's effect on underwater ordinance and vessels. This interesting pressure versus time measurement will be obtained by reading pressure gauges which have been attached to the mooring cables of canned boys. Then there are the questions concerning the direction and magnitude of the wind accompanying the shark wave and the after wind which follows it. The story of heat or thermal radiation needs continued study. The ever interesting history of neutrons will be recorded. What amounts of external neutrons are present? And what is their energy distribution? Because of the expected size of the shark, the fallout problem is being extensively analyzed. There are questions on the size of the particles when they got there and how hot they are. RAVs placed out in the lagoon constitute one method of collecting this information. 36 and a B-47 will be in the air to find out the effect of blast and heat on delivery aircraft. Should bring back a record which will indicate whether delivery techniques need to be changed or very high-yield weapons. Well, you could go on and on about the experimental projects being run on this operation, but in so doing, you run up against the same old problem. Each project is so specialized that it becomes a study in itself. So we have given but an impression of the 60-odd experimental projects being run on I-V. The latest word from the control room is that everything is going cutting to oil. Is the hydrogen level holding? Right, just as calculated. She's holding. She's holding. Isn't that what you were thinking, Crone? It wasn't too long ago, 50 days to be exact when you stood on the beach and watched Mike arrive in crates. A thing then dismembered. A sleeping giant. A robot taken apart for the long trip across 4,500 miles of lonely ocean. And then it was home. Here in a building more like a laboratory than a workshop, it would be assembled for the last time. As you looked at the mass of crates and boxes, you couldn't help but wonder, could you, whether there would be time to make order out of this chaos? When you remember back, items moved into place like notations in a diary. Yes, for you, Crone, the days of preparation fly in kaleidoscopic fashion across your mind. The day is now in the past. Just as Mike itself will soon be in the past. That's General Clarkson, the Task Force commanding. And General Walk, Chief of Staff. The scientific deputy, Dr. Graves, you've already met. Captain Paul is deputy for naval operations. And General Wise, deputy for the Air Force. As you've gathered, a weather briefing is taking place. I should say another weather briefing. You've probably heard many times before how important the weather picture is in an atomic test operation. Weather can make or break a test shot. That's why you want to know up to the last moment just how you stand with the elements. The problem this time is especially acute because this entire area of the Pacific is subject to radiological fallout. And this area is inhabited by some 20,000 people. Plus, of course, the ships of this Task Force. That's why the RADSAFE officer works hand in glove with the weather officer. Oh, by the way, to help you understand this problem, these weathermen are covering an area larger than the United States, with 10 weather aircraft and 11 fixed weather stations. To put it mildly, that's quite a territory to cover. Let's listen in, shall we? This is a plot of the latest winds we've seen from in We Talk, showing that in the lower levels to 20,000 feet, we have a typical trade situation followed by southerlies aloft, by westerlies above that, which extend clear up to 85,000 feet. This is just as about as good as we can expect. This picture, in addition to the total weather consideration, indicates that all of the weather factors are favorable for our scheduled event. That's all I have, sir. Any chance of showers? Not within the next 48 hours or with the entire marshals. How about cloud cover? A few cumulus move in, but if we go off on schedule, there's nothing to bother the operation. Are you satisfied from the radiological standpoint come out a minute? Yes, sir. The situation is ideal since the entire fallout pattern is to the north of the inhabited islands. Thank you, John. Okay, I'll check with Sam. One quick question, Dr. Graves. Well, that'd be quick. We haven't much time. I see. How soon after the shot will you know how big Mike went at? We will get rough estimates, like from the bang meter a few minutes after the shot. See you later. Right. You have a grandstand seat here to one of the most momentous events in the history of science.  The blast will come out of the horizon just about there, and this is the significance of the moment. This is the first full-scale test of a hydrogen device. If the reaction goes, wherein the thermonuclear error, for the sake of all of us and for the sake of our country, I know that you joined me in wishing this expedition well. It is now 30 seconds to zero time. Hold on, goggles, or turn away. Do not wait until 10 seconds after the first light. 15 seconds. Fighters are being used exclusively on this operation. Experience is proven, if they're just as efficient, and much less costly to put in the air than our drones for sample collecting. Jets were selected because they can operate at high altitude. Since the force of mic shot has pushed the cloud up to around 100,000 feet, it is necessary to get aircraft up high enough to get reliable samples. Even at that, they're scraping the bottom of the barrel, so to speak. Some 14 F-84Gs are being used at staggered intervals to get a good cross-section of the cloud at different times. The jets which are based on Quarjolin also have the speed necessary to make them good sample collecting machines. For the relatively short length of time the pilot is in the area, he gets a good sample per rate of contamination. One of the disadvantages of using jet fighters is their range. To handle this problem, KB-29 tankers, flying gas trucks, are in the air to refuel the jets on the way back from their sampling mission. The jets are also short on over-water navigation aids. Charlie 1, this is Red Leader. Request air to dog 2. Peter, this is Charlie 1. Steer 310. A B-29 control aircraft gives the samplers headings and rendezvous points and generally orientates them to the remote region of the Pacific. The control aircraft is fully equipped with radar and communications, and in essence performs all the functions normally thought of as being controlled from a ground air operation center. A B-36 is also in the air as another kind of control ship, like a football coach running the game from the sidelines. A scientific controller watches the formation of the cloud and sends the samplers in for their runs. When a sampler aircraft has obtained its allotment of contamination, it has done its job and returns to quag. That's part of the air story going on right now. Other aircraft are getting ready to track the cloud and also to collect samples for long-range detection studies. Dr. Graves! Do you know the yield on Mike yet? I've just been to find out now. Come along. The bang-meter records are in. The dots indicate the time in milliseconds. Well, what's the verdict term? About 12 megatons, Al. Nice going. What this tremendous blast did to the atoll nobody knows. Re-entry parties are leaving the Rendova now by helicopter. The Navy Task Group commanded by Rear Admiral Wilkins has the problem of providing the means to re-enter shortly after the blast to get exposed film, samples, and other scientific data. Since no land mass is available, the problem is complicated. Re-entry must be from a ship. Further, fallout will be very high starting at about M plus one hour. Helicopters must get in quickly and get out again before that hour is up. I can't go along, but you can and see for yourselves through the eyes of the camera what has happened back on the atoll. His king shot will be an airdrop of a large-yield fishing weapon. And for this, I'll leave you to the air. Medally, monotonously, it wings its way from Quasulane to a point in the sky over any we talk atoll. For the first time since Operation Crossroads back in 46, an atomic weapon will be airdropped on our Pacific proving ground. Will you take over for a while? And you, Colonel, what are you thinking as you relax for a moment from the controls of this strategic bomber? This bomber whose cockpit now resembles a closed room curtained off from possible dangerous effects of heat and light. Are you thinking of the mission? Or of Quasulane? Or perhaps of home? Home, you certainly can't call Quasulane home even though it now looks like an extension of the Carswell Air Force base. Talk about crowded. In addition to the normal complement of the island, Quasulane has been the major base for our Air Force ever since this operation began. This small atoll island is the jumping-off spot for the air group, commanded by Brigadier General F.E. Glansberg. Operation Ivy isn't like greenhouse when any we talk island was used in addition to Quasulane. In a sense, we talk is still used but just for L-13s and copters. Aircraft easy to evacuate. Look at this lineup on the Quasulane. Sankers, tankers, weather ships, photo-aircraft, trackers, control planes, drop aircraft. Looks like it's harder to find a parking space here than in downtown Fort Worth on Dollar Day. But now Quasulane is behind you, Colonel, and you're on your way to doing something big.  So much for the operational plan. Perhaps some of you are wondering about the idea behind the dropping of such a high-yield weapon. General Glansberg has a few words he'd like to say to you on this subject. The Department of Defense has indicated that certain targets may require a bomb of such yield as the one you will be carrying tomorrow. And it's your job to put it down a pickle fowl so that they can do a decent job of poop testing. One other thing. There's been a lot of talk about why we selected the B-36 to do this job. The reason the B-36 was selected is because it has metal-controlled surfaces. There is still some argument as to what will happen from thermal radiation on fabric surfaces. So now you know you will have a heat problem. AC to weapon air. How's the IFI coming along? Weapon air to aircraft commander. IFI proceeding satisfactorily. As you carefully check off each item of the in-flight insertion, Captain, you can tell that the preparation of the King weapon is moving along on schedule. But not too long ago, this weapon did not exist. To get King ready for IV, the weapon was being worked on in the States almost up to the last minute. So the weapon, minus the nuclear component, was ferried out to QAGE by a C-124. In the meantime, after the mic shot, the Curtis sailed to QAGE alone. Assembly people from this ship work in conjunction with Sandia in preparing King for the airdrop. Following normal safety measures, the nuclear components of King arrive from the States in a separate aircraft. And now this super-efficient weapon, completed, ready, is taken from its ship-borne assembly shop and moved under the cavernous belly of the waiting B-36. As the B-36 moves on its way to the target, the weather situation suddenly changes and becomes unsuitable for the test. And this means that the finally coordinated effort has to be canceled and the drop plane recalled to QAGE. For 72 hours, the King phase of Operation IV waits on weather. Men and machines who have been geared for the mission have to start all over again. Finally, shortly after dawn on November 16th, the go-ahead is given. This time, there will be no turning back. This time, King is on its way to the proof test for which it was created. Weapon air to aircraft commander, IFI, completed. AC to engineer, set climb power. And so, the King bomber climbs into the thin air of the stratosphere and levels off at 40,000 feet its bombing altitude. Here, the radar operator, or bombardier, if you will, guides the aircraft to that four-dimensional, yet imaginary spot so necessary for pinpoint accuracy. Radar to AC approaching target area. The radar operator positions the aircraft in space with the movement of his hand, continuously running across check between the radar bomb site and the visual system. Back and forth, first peering into one scope and then into the other. During these critical moments, the vital facts split across his mind. Ground zero, 1,900 feet to the north of run-it on the coral shelf. Height of burst, 1,500 feet. Allowable margin of error from ground zero, less than 500 feet. Zero. In the 56 seconds, it takes the bomb to fall, the men of Operation Ivy wait on Perry and any We Talk Islands. Unlike the mic phase of the operation, the king phase will be observed from land. The bomb is now at 8,500 feet. At 6,500 feet. At 4,500 feet. At 2,500 feet. Approaching 1,500 feet. Watch the air overrun it and point up the statistical highlights of that device. Remember those final last seconds? Fireball ever produced. At its maximum, it measures about three and one-quarter miles in diameter. Compared to the skyline of New York, this means that with the Empire State building a zero point, the mic fireball would extend downtown to Washington Square and uptown to Central Park. In other words, the fireball alone would engulf about one-quarter of the island of Manhattan. The tremendous upsurge of air from the detonation rapidly pushes up the mic cloud. Again, nothing of this height and width has ever before been witnessed. If the picture is stopped at this point in the cloud's growth, the height of the cloud is approximately 40,000 feet. This means that 32 Empire State buildings at 1,250 feet per building could be piled one on top of the other before they would attain the cloud's height at this time, roughly two minutes after zero. Some 10 minutes later, the cloud approaches its maximum. At this time, the mushroom portion of the cloud has pushed up to around 10 miles and spreads out along the base of the stratosphere to a width of about 100 miles. While the stem itself has pushed upward deep into the stratosphere to a height of about 25 miles. Later figures put the mic yield at around 10 megatons or 10,000 kilotons. This means there was more energy released in this one shot roughly 10 times more than in all previous atomic blasts combined, including probably those of Russian origin. Or to put it another way, four times more power in this one shot than from all the high explosives dropped by the entire Anglo-American Air Force on Germany and the occupied countries during the last war. The results of this tremendous power can be shown at the Atoll. Here is an aerial photo of the test area of the Atoll before the blast. This is the same area after the blast, showing the crater caused by Mike. The outlined island in the center is former Illugilab, the Zero Island. Sections of the islands on either side have been chopped off. The crater is roughly a mile in diameter. When it is illustrated that some 14 pentagon buildings could be comfortably accommodated in this hole, the size of the Mike crater becomes more real. In profile, the crater gradually slopes down to the maximum depth of some 175 feet, or equivalent to the height of a 17-story building. The lateral destructive effects are the greatest yet observed from a single explosive device. Without getting into the areas of target evaluation or secondary effects, it can be safely assumed that there was complete annihilation within a radius of three miles, or out to and including all of NJV, that there was severe to moderate damage out to seven miles, or even to Rujoro, and that light damage extended as far as 10 miles or down to run it. Relating this area of damage to a city like Washington, D.C., would present a picture something like this. With the capital as zero point, there would be complete annihilation west to Arlington Cemetery, east to the Anacostia River, north to the soldiers' home, and south to Bowling Field. Complete annihilation, and that is mentioning merely the primary damage. King shocked our history, and we're back here at Perry Island winding things up. It's been a pleasure to help bring you the story of Operation Island, the work of Task Force 132. Now that it's all over, I have sort of an inadequate feeling. There's so much more that could have been said. But then in a presentation of this kind, one can only hope to give the broad brushstroke. And the man who told you the story, we're only able to pick out a few of the many. You get a feeling even now that nothing is really over, that this is a breathing spell, like a lull in battle before the next attack. The feeling is on this island, with the man here. Yes, the coral sands of any we talk at all have viewed events in the fall of 1952, which less than a year and a half before would have been colossal accomplishments. Today they are calmly accepted. This is characteristic of the progress being made in the weapons development program. What is new today is old hat tomorrow. And of the day after tomorrow, who knows what these specific sands may see.


Hey Psych2Goers, welcome back to another video. To start off, we'd like to thank you all for the support that you've given us. Psych2Go's mission is to make psychology and mental health more accessible to everyone. Now, let's begin. Are you trying to find the one? You have trouble figuring out whether your partner is right for you. While there are many factors that lead to a healthy relationship, there are also some qualities to look out for in your partner that are beneficial in fostering a long-term, healthy relationship. And even though the ones we will mention aren't the only qualities to look out for, they are some of the major ones that would be most beneficial to a healthy relationship. So if your partner has these nine qualities, you should not let them go. Number one, they're supportive.  Having a supportive partner who can provide you with the emotional and mental support you need is essential for a lasting relationship. Researchers from Carnegie Mellon University had even found that supportive spouses played a role in increasing a person's chances of success. Their reassurance and support had encouraged their partner to take on more challenging and rewarding tasks. In the long run, these couples showed an increase in personal growth and happiness. Number two, they engage in your personal growth. Do you have a partner who encourages new experiences that allow you both to grow as individuals and develop new perspectives? Another important determinant for a successful relationship is self-expansion, which is the desire to strengthen or increase your potential efficiency. Experiencing new things together makes you feel happier with your partner and more committed to them. Even if you don't have time to engage in new hobbies or activities with them, simply witnessing their self-expansion can boost your level of happiness too. Three, they're emotionally intelligent. Is your partner an effective communicator who can express their emotions and understand yours? Emotional intelligence is the ability to use, understand, and manage your emotions. People who are emotionally intelligent often have great social skills, acknowledgement and regulation of their own emotions, and an understanding of how others feel. Licensed mental health counselor, Carrie Ann Brown even states that emotional intelligence in a relationship can help couples express and manage difficult emotions that they may confront, which helps forge a strong and lasting relationship. Four, they are understanding. Is your partner able to relate and empathize with what you're going through? As mentioned by Dr. Bernstein in his self-help book, understanding between couples is vital to a relationship. A partner who truly understands what you're saying and expressing can connect with you at a deeper level and strengthen the bond in your relationship. Number five, they're kind. Is your partner kind and generous towards you? Psychologist John Godham studied newlyweds and found that the key to a long-term healthy relationship is rooted in kindness and generosity. Couples who were kind to each other were able to foster an environment of love, trust, and intimacy. It allows couples to emotionally connect and become comfortable with each other, which helps them overcome conflicts and disputes and maintain a healthy and loving relationship. Number six, they're respectful. Does your partner respect your opinions, experiences, and boundaries? It's crucial to a relationship that you and your partner see each other as equals. You each have your own interests, opinions, and feelings that are valid and should be respected. An unhealthy relationship, however, will involve one partner dominating the other. They disrespect any ideas or opinions that go against theirs by telling them that their opinions are crazy, dumb, or worthless. Therefore, the ability for both of you to speak on equal terms and be listened to is vital for a long-term healthy relationship. Number seven, they know how to compromise. Are you and your partner able to meet each other halfway when you're in a disagreement? Compromising does not mean that one person wins and the other loses. Both partners need to be heard and understood to reach a happy medium, especially when confronting a particularly difficult problem. You and your partner both deserve to be heard and a healthy compromise helps you and your partner become the most authentic versions of yourself. It promotes each other's personal growth and leads to overall happiness in the relationship. Number eight, they accept your flaws. Does your partner accept your flaws and differences without judgment or annoyance? Someone who accepts you and your flaws is open and emotionally honest and is able to see the perfect in your imperfections. There is less resentment and more peace in relationships where partners accept each other. This acceptance can also encourage more personal growth and high levels of self-esteem in yourself and your partner. And number nine, they share your core values. Do you and your partner share the same set of values? While it's normal to disagree on the minor things such as your favorite show or takeout order, it's much more important you share the same core values. Where they stand on topics like marriage, family and religion can be important in determining the compatibility and durability of the relationship. If you and your partner have opposing core values and are unwilling to compromise on most of these topics, future conflicts may arise. Does your partner show any of the qualities we mentioned? Tell us about it in the comments below. If you enjoyed this video, please like and share it with others who may find it helpful too. Don't forget to subscribe to Psych2Go and hit the notification bell icon for more content. See you soon.


All right, it's the middle of the night right now and I just woke up with an idea and I'm just gonna barf it out on camera, so I don't forget On the solar snarler Replace one of the flexible solar panels with one glass solar panel that has a higher voltage than the flexible ones And that'll solve a bunch of problems. That means I can keep the batteries at the full voltage and And I can charge them through the glass one Which will be a higher voltage so it'll be able to charge them and then I Can run the batteries in parallel with the solar panels the flexible solar panels without risk of damaging the batteries right So I barfed out some gibberish about solar panels and batteries at 3 a.m Here's what I was talking about Well, first I'm running a 48 volt system on the boat because I'm getting a free motor That's 48 volts all my other boats are 24, but since I'm getting this free motor. I'm making this one 48 So here let me just I'm gonna draw this so it doesn't get confusing Okay 40 excuse my upside-down writing 48 volt system is generally 48 volts up to like about 56 Yeah, about 56 volts now the solar panels I have The flexible solar panels are 28 volts each like maximum voltage So two of them together is 56 volts. So I have my solar panels here They are also 56 volts a 56 volt thing can't charge something very well up to 56 volts Sure, you can leave them connected to each other for like four months and it would eventually get there. However When the voltages get close together the electricity flow slows down a lot and then eventually stops So you need a higher voltage to charge the batteries so my first Thought was well, I could reduce this because I'm making the battery banks, right? so if I leave out a couple cells and I'm gonna have to confirm these numbers, but it's somewhere around this I'm using lithium iron phosphate batteries, but I could leave out a couple cells and get down to like 50 volts or something like that and this would be like 40 42 to 50 volts But then the maximum voltage is what we're concerned about here so then the 56 volts because there's a six volt differential here the electricity is still gonna flow and I can charge the the batteries to 50 volts Now the problem is I'm not gonna connect 12 solar panels I'm not gonna connect all 12 of these solar panels to those batteries because it's it's gonna be a waste They're not gonna be able to force the electricity in there fast enough and even if they could they'd like melt everything. I Just want to so I'm gonna do what I usually do I'm gonna take two solar panels. Let's say these two here Those ones will charge the batteries and All the rest of these will just go directly to the motor This is a marvelous diagram here Now the thing is When okay the the batteries also go directly to the motor, right? so there's going to be times when I'm I'm powering with the batteries and the solar panels and that means these two will also be connected and If the motors using all the electricity from the solar panels, then it'll also get electricity from the the batteries however, if it's not using all the electricity from the solar panels some of that electricity is gonna go into the batteries and Could overcharge them This is a problem. So my first thought was okay. I put a voltmeter on here and I make sure it stays at 50 volts or less That means I have to pay attention What if I'm not paying attention or what if some some idiot is come comes over and starts messing with switches on the boat when I'm Not there I need to plan for non optimal situations, right? So I don't want to have this situation where I could blow up the batteries so what I thought of last night was Okay, we keep this 56 volts jup These two solar panels can't charge it all the way point over there now Because they only go up to 56 volts. So I take one of these solar panels out say this one and Make it one of the glass ones which is slightly longer. This is totally not to scale So this left to be a little bit longer Now this will go up to say this is a 36 volt instead of a 68 So that's a six volt difference. No an eight volt difference. So I get eight extra volts So it's not 56 anymore and now it's 64 volts going into the 56 that will charge it all the way with these two solar panels and Then if these are connected to the motor and this 56 volt is connected to the motor and the motor is not using all the Electricity this 56 volts could flow into the battery But only up until this reaches the maximum of 56 it won't overcharge it boom And yes, there are also complicated Computerized solutions to this which I'm not using because it is not useful in What if I'm out in the water and the computer dies like what I'm dead stranded because this tiny little chip no This is this is a good way to do it. I Think I want to steal one of these solar panels Let's see open circuit voltages 40 The rated maximum weight rated voltage 31 yeah, so it would be 31 to 39. That's that's enough It's gonna be a meter or less. Oh, it's lately over. No Oh There's one I can get a good look at right here. Oh, no, it's right on Yeah, it's dead on a meter so it will fit with no space My flexible solar panels are one meter actually 99 centimeters by 138 centimeters and the glass one is one meter by 168 so 30 centimeters extra in this middle row Totally fine. I think I Better confirm that I actually have The solar panel frame stop some way of just attaching a glass solar panel in there and then another chunk of this Flexible Well, I guess I could just do that Just a recess a chunk in this is only like a Two or three centimeters the thickness of the glass one Yeah, it should be fine. I can't just make it all in one shot I was gonna be so nice just all flat super simple thing, but yeah, that's not that Yeah, I mean other than being a tiny bit more irritating to build I don't see any problems with this at the moment I'll let it dwell in my head for today. I think it's a good plan. I don't see


Some people are naturally concerned about VR because you're strapping a supercomputer to your face and it's sending all kinds of things up into your brain. Shouldn't we be afraid? This is a mixed bag. We know that plants near Wi-Fi routers are prone to dying a lot faster. So yes, we should be careful about all of our technology. But we're also on this evolutionary pathway where the technology is very important for us. Humanity is not in a place right now where we can just shut down the whole internet and be okay. I mean, yes we could, but it might not be the ideal reality that some people think it would be. We have to pass through this stage and we, through our intelligence, need to recognize how certain technologies may be harming us and develop better ones so that they don't. As far as VR goes, I think a lot of what it can offer us can be helpful if it's done with a lot of love and care. Perhaps we just need to remember to be moderate and not go overboard until we can reach some kind of point of transcendence or we develop technologies that can help us integrate into more natural ways of living.