Good morning or afternoon or evening depending on where you are. My name is Miriam and I'm a senior research scientist at the research team at the Wikimedia Foundation. What I'm going to talk about in the next 25 to 30 minutes is a result of a set of efforts which lasted around one year that the research team did to build what we call the knowledge gaps taxonomy. Before getting into the details of the taxonomy itself, since I speak on behalf of the whole research team, let me introduce herself. This is the research team at the Wikimedia Foundation. There are six of us, Leila who's the head of research and five research scientists. We come from different backgrounds from physics to human-computer interactions, natural language processing, and computer vision. And we use large-scale data to support and understand communities of readers and editors of Wikipedia and sister projects. To start our journey towards the knowledge gaps taxonomy, let me start with the principle which most of us in Wikimedia spaces are familiar with. Knowledge equity is one of the two goals of the 2030 movement strategy from the Wikimedia movement. Knowledge equity is encouraging us to focus our efforts in including in Wikimedia projects the knowledge and communities that have been left out by structure of power and privilege. It's pushing us towards removing those knowledge inequalities and break down the social, political and technical barriers preventing people from accessing and contributing to free knowledge. Now, while we're all working towards reaching knowledge equity, what would be also nice to have is a way to measure how well we are doing in terms of reaching knowledge equity, a way to track our progress towards this end goal. One way to do this is to do what we call operationalized knowledge equity. This means to isolate and identify the individual components and gaps which prevent us from reaching knowledge equity and find a way to measure those gaps. By doing so, we can track our progress towards the end goal of knowledge equity. To understand better this concept, let me give you an example from one of the most well-studied gaps in Wikimedia projects. The gender gap has been investigated by researchers and practitioners for years. Researchers have found that a very small percentage of biographies in Wikipedia is about women, for example. And for the Wikidata human gender indicators initiative is tracking the percentage of female biographies across different Wikipedia editions. Similarly, a recent effort from the research team and collaborators have tried to quantify the gender gap in Wikipedia readership. Through a survey, they found that 75% of the page views we receive in Wikipedia are from men. And this gender gap is even wider when we consider gender distribution of editors. So as you can see from these initiatives, by quantifying gender gap, we can understand how well we are doing in terms of knowledge equity on the gender front. But how can we do this more systematically? How can we measure and understand all gaps at this level? Different initiatives have focused on specific gaps or have tried to address specific gaps. However, what we missed when we started this project was systematic understanding of the types of gaps that we have in Wikimedia. And that is why we build this taxonomy of knowledge gaps that can map the different types of gaps that we can find in Wikimedia spaces. We define a knowledge gap as a disparity in terms of coverage of a specific group of readers' contributors on content across Wikimedia projects. So for example, the gender gap fits this definition. The gender gap in content is a disparity in terms of coverage of content about different gender groups. The gender gap for readers also fits this definition. It reflects a disparity in terms of participation of readers from different gender groups. So our task to build the taxonomy was to essentially find in Wikimedia spaces those gaps that fit this definition. How did we build this taxonomy? If you're familiar with the concept of taxonomy, you know that the taxonomy is generally represented as a tree, where the root are a few coarser grain categories and leaves represent finer grain categories or particular properties of the coarser grain categories. So to start our construction of the taxonomy of knowledge gap, we first have to find the root of the taxonomy. And to do this, we read different sources from literature, and we were inspired by this diagram of engagement of the Wikimedia movement, which was built by the product teams in the context of the medium term planning last year in 2019. Consistently, across the different sources that we went through, three main actors were identified as the main pillars of the Wikimedia ecosystem. Readers, contributors, and content. And so given this consistency in having these three pillars as main actors of the Wikimedia ecosystem, we choose content readers and contributors as their root dimensions of our taxonomy. Next, we had to populate the taxonomy. And how we did this in practice is that for each of the dimensions, the root dimensions, readers, content, and contributors, we did a thorough literature review to find elements of the Wikimedia ecosystem having evidence of knowledge inequity. And we identified those as knowledge gaps. And then we organized those gaps in a structured taxonomy that can be easily consumable and intuitively understood by technical and non-technical people. So the next step after having identified the root dimension was to identify the gaps for each of the root dimensions. In practice, how we did this is that we went through tons and tons of literature to identify those gaps. There were three main different types of sources that we analyzed to identify those gaps. The first one is academic literature. We read hundreds of papers from communication science, social science, and computer science that talk about, try to understand or quantify different types of knowledge gaps in Wikipedia and assist their projects. The second type of source that we analyzed is a set of strategic recommendations that the Wikimedia movement has released earlier this year. And the set of initiatives that the Wikimedia community has worked on throughout the years to bridge knowledge gaps. Both these community sources give us an idea of the important gaps for the community. The third type of source is what we call community surveys. These are surveys that have been carried out by the Wikimedia Foundation affiliates and user group to understand the composition of their reference communities. These surveys are especially about the composition of the readers and contributors community. And by looking at the dimensions they analyzed, we could also expose some specific knowledge gaps. So the final structure of the taxonomy is as follows. We have three root dimensions, contributors, readers, and content. For each dimension, we have identified through literature review a number of gaps. Now, because the root dimensions are few and the gaps are many, we added a middle layer of the taxonomy, a layer of what we call face sets. Face sets are a group of gaps which refer to similar properties or objective. They share similar characteristics and they're very useful to summarize the taxonomy and make it shareable in a smooth way. So for example, in the next few slides, I'm going to give you an overview of the three main dimensions of our taxonomy based on the underlying face sets. So let me start with the readers dimension. Gaps in the readers dimension reflect different participation in readership depending on different readers group. There are three main face sets in the readers dimension. And the first one is socio-demographic face set. Gaps in the socio-demographic face set, as the name says, reflects disparities in readers representation based on social or demographics characteristics. It was pretty easy to compile the set of gaps in these specific face sets because there has been a lot of work around characterizing readers in terms of their socio-demographics based on community surveys. There were a lot of academic papers that try to understand readers' socio-demographics and there are a lot of initiatives across the movement that try to bridge socio-demographics gaps. And so at the end of this research through these three sources we identified seven main socio-demographics gaps. The gender gap, which as we said reflects difference in readership depending on one's gender identity. The education gap reflects different volumes of readership depending on readers' education level. Similarly we have the background gap which reflects the background of readers including the religious belief, the age gap, the local gap which reflects the distribution of readers across different geographic area, the language gap which is about the readers' fluency in a given language and the income gap which reflects different volumes of readership depending on one's income. The next face set in the readership dimension is accessibility. Accessibility gap reflects all those technical barriers which prevent people from accessing free knowledge. Accessibility face sets are inspired by the improved user experience recommendation of the 2030 movement strategy which essentially encourages everyone in the movement to build more inclusive platforms, interfaces and knowledge services that can be basically so that Wikipedia and sister project can be accessed by people with different accessibility issues including internet connection or physical disabilities. And so based on the recommendations and the ongoing initiatives we identified four different accessibility gaps. The physical disability gap which reflects different level of readership depending on one's physical ability. The device gap which is related to the type of device that people use to connect to Wikimedia sites. The technical skill gap which we have found in literature technical skills to be a blocker to accessing Wikipedia and sister projects. And finally the internet connectivity gap which reflects different levels of readership depending on one's internet speed. And we've seen a lot of initiatives in the past few years to bring Wikipedia and sister projects into areas of the world without internet connection. The last face set of the readers dimension is the information need face set. Gaps in this face set reflect different volumes of readership depending on readers information need. The construction of this part of the taxonomy was inspired by a set of work around understanding why readers come to our site. The latest work is called Why the World Reads Wikipedia and was published in 2018 by members of the research team and collaborators. And so this work actually has a specific inner taxonomy to identify different information need and we port this taxonomy directly into the information need face set. As a result there are three knowledge gaps within the information need face set. The first one is a information depth which reflects different levels of readership depending on one's need for deep information in Wikipedia and sister project. The second one is the familiarity. People come to the sites with different levels of familiarity to a topic and these gaps reflect that. The third one is motivation and the motivation gap reflects different volumes of readership depending on one's motivation to come to the site. Now this concludes the readership gap. Next we're going to talk about the contributors gap. This part of the presentation is going to be fairly short because the contributors taxonomy is pretty similar to the readers taxonomy. There is a sociodemographic face set where gaps reflect different levels of readership different levels of contributorship depending on one's sociodemographic characteristics. There is an accessibility face set and then instead of the information need a face set we replace it with what we call a motivation face set. Gaps in the motivation face set reflect different volumes of contributorship depending on the type of work editors come to do on the site. So depending on the role an editor has they might do different types of work and depending on the motivation editors have they might do different types of work and these two gaps reflect the different types of contributorship based on role and motivation. The final branch of the taxonomy is the content dimension. Gaps in this taxonomy reflect different volumes of content depending on different characteristics. There are three main face sets in the content dimension. The first one is the policy face set. To explain the policy face set I pasted I pasted a screenshot of the Wikipedia page about the core content policies. Most of us might be familiar with the Wikipedia core content policies. There are three neutral point of view verifiability and non-regionary research. For this presentation and the taxonomy of knowledge gaps we focus on the first two. Neutral point of view which requires that every article has a neutral perspective on the topic and verifiability which mandates that every material that is challenged or likely to be challenged has to be backed by reliable source. And we know that not all articles have the same level of neutrality and not all articles fulfill the verifiability policies in the same way. And so the policy face set contains these two gaps which reflect these two properties. Verifiability and neutrality. The second face set of the taxonomy is diversity. Gaps in this face set reflect different levels of content coverage depending on the topic represented. This face set is inspired by the identified topics for impact recommendation from the Wikimedia movement strategy which essentially encourages to develop and increase access to content that has historically been left out by structures of power and privilege. Similar aim at supporting diverse content creation we can find it in the strategic direction of the Wikimedia Foundation from the medium term plan which was developed in 2019. And we also find different organization beyond Wikimedia Foundation affiliates like Who's Knowledge, whose actual mission is to improve diversity of content in Wikipedia and its sister projects. So we identified, so based on the literature review and by looking at all these initiatives we identified four types of diversity gaps. The gender gap is the content counterpart of the readership and contributors gender gap and it reflects different volume of content depending on gender groups. Similarly the cultural context gap reflects different volume of content depending on the cultural background of a given article. And similarly the geography gap looks at the geographic distribution of articles or items in Wikimedia projects. And finally the impactful topics gap is looking at the differences in terms of coverage of topics that are considered interesting or impactful for different specific communities. The last face set of the content dimension is the accessibility face set. This is similar to its readers and contributors counterpart is inspired by the improve user experience recommendation of the movement strategy. And for this specific face set we focus on the accessibility of three types of content that we find very often in Wikipedia and sister projects. Three types of content are text, multimedia and structured data. The accessibility gap for text is the readability gap and it reflects the fact that not all articles have the same levels of readability. The multimedia gap refers to the fact that having visual and non-textual components in articles and items of Wikimedia project is important in terms of accessibility for younger generations or for non-native speakers. And so the multimedia gap reflects the proportion of multimedia content across different Wikimedia projects. The structured data gap reflects the different types and quantity of usage of structured data across Wikimedia project. We give importance to structured data in order to organize and manage large quantity of content in Wikimedia projects. Now this concludes the overview of the taxonomy of knowledge gaps. We have seen a very coarse-grain overview of what's in this taxonomy. At the end of the presentation I will provide you with a link where you can read a lot more about each individual gap and the sources that we use to mine this information. Now while this taxonomy has been built where do we go from here? How do we use this taxonomy? We identify three different groups of audiences that could be interested in using this taxonomy. The first one is researchers. We hope that researchers in Wikimedia projects or researchers who are using Wikidata from Wikipedia and sister projects will look at this taxonomy to either direct the future of the research and for example look at quantifying specific types of gaps or to use this taxonomy as a tool to understand the types of biases that they have in the data that they use to train their algorithm that comes from Wikipedia and sister projects. Organizations like Wikimedia Foundation and affiliates might want to use this taxonomy to address investments in different initiatives and events that address specific gaps or that address gaps that have not been explored before by the organization itself. And finally Wikimedia community might want to use this taxonomy to prioritize and or generate new initiatives about bridging knowledge gaps. For years already the community have had many initiatives to bridge knowledge gaps and this taxonomy can help support this trend even further. Finally let me talk about the future. I said at the beginning that the final goal of our work would be to have a tool to measure how well we are doing in terms of reaching knowledge equity. Now the taxonomy is essentially a first step to build a composite index which we would call the knowledge gap index that can help understanding and quantifying each individual gap so that we can monitor over time how well initiatives from different angles of the community are impacting our progress towards knowledge equity. To give you an example of how the knowledge gap index looks like take the gender equality index from the Europe Institute for Gender Equality. This index for each European actually for each country contains a series of indicators that monitor the progress towards gender equality. So the knowledge gap index would look something like this based on knowledge gaps to measure knowledge equity. And so with the knowledge gap index I'm going towards the end of the presentation before going to the end I want to thank everyone who made this presentation possible by making all the beautiful visuals available, the all the beautiful visuals that I use in this presentation available on the web. And I have two credit slides and a third slide containing the link where I took all the screenshots in the presentation from and with that I thank you very much for listening until now and I encourage you to give us feedback. This knowledge gap taxonomy is just a first step towards bridging knowledge equity and it's a first rough itself. So we would really like to hear from you any sort of feedback that you might have on individual gaps or individual faces, the root dimensions, everything that you want to tell us about the taxonomy you will find a way to give us feedback by following this link. And with that thank you very much for listening and looking forward to hearing your thoughts on the knowledge gap taxonomy.


Okay, so I'd like to begin by thanking Nawa and other organizers for inviting me You know, it's my great pleasure to come here and To speak in this really fascinating meeting and also take a break from my teaching in Colorado so Now this diagram shows you a membrane protein on the cell surface Now these protein are the windows and the doors of the cell Now this protein include receptors Channels and the transporters and they allow the cell to communicate with the outside environment Now as you can imagine the surface levels of these proteins must be precisely controlled now too much of a protein or too little of a protein Can often disrupt the physiological pathway associated with the protein and in humans this may often cause diseases So here I can give you one example many of you already know Now if you have two high levels activate EGR receptors on the cell surface This is going to cause uncontrolled proliferation of the cells and this may cause cancer Now on the other hand if you have a two low level or what we'll call transporter as you're going to see a moment This is going to disrupt the blue coat blood glucose homeostasis and in humans. This may cause type 2 diabetes Now how is the surface level protein is a determined? It's a determined by the balance of exocytosis and the endocytosis Now exocytosis is a Vesco fusion event which involves the fusion or exocytic Vesco with a plasma membrane and this delivers the cargo to the plaza membrane Indocytosis on the other hand removes the cargo from the plaza membrane and the returns it to the endocytic compartments Now remarkably both exocytosis and the endocytosis can be regulated by stimulus and And in this way the surface level of a membrane protein Can be adjusted acutely according to a physiological demands and here stimulus could be a second messenger. It could be a false relations Now a major model system we have been using to study a surface of protein homeostasis Is the insulin controlled for translocation? Now glu4 is quite an Euroglucal transporter Because under the basal condition glu4 is sequestered in intracellular compartments So it's not on the plaza membrane Now this is because your blood sugar level is normal and you don't need to uptake a glucose and At this basal stage the exocytosis and the endocytosis is at equilibrium So now imagine you have food So your blood sugar goes up and this triggers insulin secretion from the pancreas Which then travel in the bloodstream and reach the target tissues mostly adipocytes and the skeletal muscles Now when you insulin bind to the receptor on the plaza membrane this activates insulin signaling cascade Which eventually leads to the activation of exocytosis at the same time the endocytosis Is moderately reduced and the result is a translocation of glu4 protein from vesicle to the cell surface and This is a translocation allows the transporter to uptake a glucose into the cell either burning into ATP or storage Now this is your blood glucose level after you have food that's at time zero is the time you have meal Now you can see your blood glucose level quickly goes up But also it rapidly returns to the normal range and this Returning to the normal range is the driven by insulin stimulate glu4 translocation and the one your blood glucose returns to the normal range insulin signaling terminates and this also shuts down exocytosis at the same time Endocytosis resumes and this leads to the retrieval of glu4 from cell surface and the returning them to the vesicles Now you can see insulin control the surface level of glu4 Transporters by regulating exocytosis and the endocytosis now as much as we Love glu4 exocytosis the major goal we studied is to use as a paradigm to Establish the general principles of surface protein homeostasis For example, we expect our findings can be extended to understand the trafficking of neurotransmitter receptors in the neurons The transport of water channels in the kidney epithelial cells and the trafficking of immune receptors in the immune system Now today, I'm going to tell you two stories on the regulation of surface protein homeostasis I'm going to begin by a short story on the regulation of rap gtps by a protein called ribaf in the exocytosis Then I'm going to focus on a very recent finding on the regulation ap2 adapter formation by a gap in the endocytosis So I'll begin with exocytosis Exocytosis exocytosis as I mentioned is a vesicle fusion event Now this is after a rab and a tether bring the vesicle and the target of membrane together And the vesicle fusion is driven by two classes of molecules snares and the second one monkey team as I'm protein Now snares are membrane anchored proteins and you have a vesicle anchored snare kind of we snare and The target anchored snare called the t snares When the we snare and the t snare see each other they spontaneously assemble into a so-called trans snare complex And the trans snare assembly proceeds toward the membrane so you can imagine this is like a a zepering process Which forces the two membrane into a closed proximity to fuse Now SM protein is a soluble protein it can bind to the transnare complex and accelerate the transnare assembly Now snares and SM protein they're the core engine for vesicle fusion In addition to this core engine good for exocytosis also requires specialized regular regulatory factors such as the Sinep, Thomason and the Dr. B Our previous work focused on biochemical dissection of these molecules since the most of the work has already been published Here I'm not going to our details And from from here I like to mention this protein I believe Only represent a minor fraction of the entire regulatory network for gluophore exocytosis Now the sequencing of a human genome predicted a large number of membrane proteins And for the majority of these membrane protein we know virtually nothing. So therefore I think the most important Direction in the field is to comprehensively identify new regulators for gluophore exocytosis Our request coincided with the one the CRISPR-Cas9 genome editing So we decided to perform unbiased genome-wide CRISPR screens So we took advantage of existing genome-wide CRISPR libraries, but also we make our own customer CRISPR libraries in this CRISPR libraries So these are all lentil virus based you have a single guide array and Also the Cas9 the single guide array recruits Cas9 protein To specific location in the genome to introduce lots of function mutations and the two perform screens we Use a HAJP gluophore reporter Now this reporter has a HA epitope inserted into actual cellular domain Now HA staining as you can see here Can stain the surface gluophore reporters On the other hand the GIP tells you the total protein in the cell now if we calculate HA staining and GIP staining This will give you the information about a relative amount of our gluophore reporter on the cell surface and we start a stable cell line expressing this gluophore reporter and we mutagenize using the CRISPR-Cas9 library and then we treat the cell with insulin And then we use the flow cytometry to sort for the cells with the loglophore on the cell surface And then we repeat the process for three times and then we took the sorted population and Recovered the single guide arrays and use the deep sequencing to analyze the abundance of the single guide arrays Then we use the bioinformatic tools to calculate the significant hits and finally we performed secondary screens and the individual validations So we perform a screen in multiple cell lines. We start proof-of-concept screen in the cancer cell lines, but eventually we focus on myoblast and pre-dipsense So very briefly our screen Identified a large number of single guide arrays that were significantly enriched compared to the control population So we then use this equation to calculate the abundance of the single guide arrays So we can derive significant hits from the screens While the screens identify a larger number of genes, the majority of the hits were not previously linked to gluophore exocytosis But we did recover known regulators such as Rep10, exocyst and almost the entire insulin signaling cascade So here I'm going to focus on one new factor identified in the screen called the Rabbi F Now Rabbi F, Rabbi interacting factor. It's a very small protein. It's a 14kd soluble protein Now when I did a sequence analysis The only prediction was it's a putative guanine nucleotide exchange factor for wraps or gap for wraps now now why you're probably gonna Well, I think I remember you told me you didn't believe Rabbi F was a gap from very beginning But I have to say it's kind of ahead of the field because if you search literature and Search the motifs This is the only information you got the entire protein is a ribo gap of course a putative gap Now what's a gap? RAP GTP is involved in the Weisgau tailoring and the RAP is a G protein and the RAP cycles between GTP and the GDP bond form and the GTP bond form is active and What the gap does is to promote GTP binding. So gap is a positive is a positive regulator for RAP Now the story of Rabbi F begin 25 years ago with the two papers published by Peter Novick and the Pietro decommedians groups So their papers were mostly based on in vitro biochemical acids and actually RAP F is also known as MS4 or DSS4 This is the first putative gap for RAP GTPs. Let's think about that. There are about 70 RAP GTPs in the human genome and this protein is the first putative gap But the surprisingly that case later is a biologic function was still unknown Next we use the CRISPR-Cas9 knockout ribo F in deep sets and In the wild type cell here, you can see insulin strongly promote gulfo reporter translocation to the cell surface So all these are flow cytometry based Analysis But in the RAP F knockout cells, you can see the translocation was largely abolished and this is confirmed the screen data Now these are exocytosis I say if you look at the slope of the curves This represent the speed of exocytosis and you can see here in the RAP F knockout cells Exocytosis was strongly reduced and we could fully rescue the exocytosis by introducing the wild type RAP F gene and these are confocal images images which also confirm the flow cytometry data in the wild type cells you can see an insulin Relocate gulfo reporter from intracellular compartment to the cell surface so I want to point out these are Lepid droplets or a deep sets not the nucleus and here is the the boundary of the cells In contrast in the RAP F knockout cells you can see a insulin stimulated gulfo translocation was a largely abolished and Here I want to make an important the point is Discovery of the first the physiologic function of RAP F finally makes it possible to explore its medical mechanism Now since the RAP F is known as a RAP binding protein the first question we asked what's a RAP target? now if we Look at the screen again RAP F was identified in the same screen as the RAP 10, which is a non-regulator over Gulfo exocytosis so we speculated maybe RAP F controls Gulfo exocytosis by binding to RAP 10 and First we are prepared the recombinant protein and the test if these two protein bind to each other and This is the in vitro liposome called flotation assay and Here we anchored RAP 10 protein in the proteal liposomes Then we add a soluble RAP F to the liposome And after centrifugation the liposome migrate to the top over the gradient Together with the bonded protein Then we collect this fraction and analyze by STS page Okay, so here just want to you to focus on this link you can see a RAP 10 protein could bind to RAP F in the co-flotation assay and the binding appeared to be a stoichiometric By contrast protein-free liposome could not bind any RAP F protein So RAP F bind to RAP 10 directly And since RAP F is known as a putative gap we ask can RAP F function as a RAP 10 gap The structure of RAP F with a RAP 10 is still not known But we do have the structure or the complex with a RAP 8 Which is a kind of related RAP now based on this structure we We found that there are several residues at the binding interface So therefore we introduce the mutations either single island mutation or triple mutations to disrupt the interaction And this is the in vitro GDP release assay, which is commonly used to analyze RAP gaps Now without the RAP F you can see almost no change But when we added a wild type RAP F, you can see a rapid GDP release And when we introduced the mutant either a single mutant or the Triple island mutant you can see the GDP release rate was strongly reduced Now if you look at the initial rate of the triple mutation We estimate about a 24 the job in the In the GDP release So here came out the surprise when we introduced these mutants into a RAP F knockout cells, we found that these Gaff mutants Can rescue the group for access to the same level as the wild type gene Okay, and we repeated experiments in the multiple cell types several different conditions We always got the same results And these these data strongly suggest RAP F is actually not a gap you include for access atosis Now you put if a RAP F is not a gap. What's the biologic function? And then here came another surprise when we look at a RAP F Rep 10 expression in ribbon right back for knockout cells We found that the protein disappeared Okay, and we could rescue RAP 10 expression using either the wild type or the two mutant RAP F so The rescue of RAP 10 expression Correlate with the ability of this protein to rescue group for access atosis and Has nothing to do with their in vitro gap activities So therefore we propose RAP F functions by stabilizing RAP 10 protein in the group for access atosis Now to test this possibility we Also tried Lentil viral expression of RAP 10 in the knockout cells Now this work is important because we had to rule out right by if it doesn't control the transcription or epigenetic regulation RAP 10 expression so here we We found we could write it express RAP 10 in the wild type cells but very little protein was observed in the RAP F knockout cells and this data are consistent with a The previous data just showed you and the supported idea right by F stabilized RAP 10 protein and the one we treated the right by F knockout cells with MG 132 which is a proteasome inhibitor We found we could fully rescue RAP 10 expression and When we treat the MG the cells of is a both MG 132 and the secular hexamide Which is a protein translation inhibitor the rescue was abolished now this data suggest without a right by F RAP 10 protein could be Efficiently produced but they're quickly degraded without the function of a right by F Now here we proposed two models It's possible right by F stabilized Intrinsically unstable RAP 10 intermediate. So therefore it's more like a chaperone On the other hand, it's also possible right by F can protect the folded RAP 10 protein from proteasome degradation For example by masking a so-called a diagrams To distinguish between these two models we are reconstituted RAP 10 the right by F into E. Coli cells E. Coli as you know doesn't have any reps or right by F so we found RAP 10 protein could be produced in the E. Coli without a right by F But all the protein end up in the insoluble pellets. We couldn't get any protein from the supernatant we could Extract soluble RAP 10 protein only when right by F was co-expressed so therefore RAP F stabilize unstable RAP 10 intermediate and without RAP F RAP 10 is going to aggregate in E. Coli and then miss fold and get a degraded by the proteasome in the mammalian cells and This supports the idea right by F is a medical chaperone for RAP 10 not a gap So here I call it a HODIS chaperone because it's a different from the classical chaperones like HSV 70, HSV 90 both require ATP dependent cycles But RAP F is a small protein. It's not ATPS Instead it recognize the substrate and stabilize the protein which promote its folding and And then we perform the myspac analysis that we identified two additional RAP protein RAP 13 RAP 8 Also regulated by RAP F and this is our model RAP F as a chaperone they interact the substrate and stabilize These proteins which promote the folding of these RAP GTPs into the native confirmation So therefore they can promote exocytosis and without RAP F The RAPs cannot adopt native confirmation. They get degraded by the proteasome Brief summary we identified the first biological function of RAP F which enables us to Discovered unexpected function of RAP F in the GTP RAP GTPs regulation Because all the RAP GTPs function in the similar way We expect maybe this whole this chaperone model can also be applied to other RAP GTPs Okay, let's return to this diagram The surface level of membrane proteins Determined by the balance of exocytosis and the indocytosis now having talked about exocytosis I'm going to focus on indocytosis Yeah, go ahead. I mean Is this a chaperone for RAPs on the steady state conditions or Do cells have to go through something for this chaperone to be Not in group for exocytosis, but I think that's an interesting point Let's come back to yeast. This protein is conserved in yeast I was cloned first in the yeast, but when you knock out the protein, there is no phenotype So at a normal condition, it's not essential for yeast But what I imagine is a maybe a durian stress condition. This protein is recruited To a traffic cause a trafficking pathway and they become essential, but in group for exocytosis You always need it Does it bind to both GTP bounders? Yeah So we didn't address that but according to the non-structure with a RAP 8 It's actually bind to the nucleotide free form Which I think suggests it bind to the more upstream over the folding pathway And so far I don't think there is evidence showing it bind to the nucleotide bond form of RAP GTPs Yeah, is it a chaperone or a co-chaperone? So at least they doesn't seem to bind ATP No, no, it's a very small protein So what I can say is that this protein form direct complex and it's about a one-to-one ratio and If there is any additional protein bound to the complex, we don't know yet On this universe University of this I have a lot of questions about that First these are I presume these three are the only rams you've picked up Right, so that's right. At least in the cell type we studied these three rams are only are only proteins controlled by RABF So question is are there other isoforms of RABF in cells that could explain the other ones and just a comment rams are very nicely expressed in a coli in generally and And in my experience RABF is the only one that really is difficult I haven't tested right or or or or or are 13 But RABF of all the rams that I expressed in the coli was the only one that was very difficult to express so my my Expectation would be that this is a very specialized thing for these three rams that you're writing there Or and and the question then becomes what's special about the structure, right? So I I think I can I can tell you something So why I show this work to a colleague in the group for field and he said finally There is explanation why they couldn't produce RABF 10 in E. Coli They had to produce in the inside the cells and the comeback to your question is anything special about these three proteins and You know, I talked to people in the field and there is no reason to believe they're special And so you said that in other rams could be expressed readily in the E. Coli But that doesn't really rule out that there is a bacteria chaperones that perform similar functions promoted folding But again, it's all open question Can I just comment first of all I didn't say I don't believe because I never said I don't believe I don't think I don't believe Not in science I just I don't think What you only say that in writing I Don't think especially when when I have data Because it was only shown to that GDP can be released but not GDP can be Dequired and like other Jeff's and here I want to just make a suggestion that maybe what's Similar in these three is maybe just about simple biochemistry of Nucleotide affinity there affinity to nucleotides and Nucleotide free is known to be Not stable a state of all GTP as so maybe these three but that's also That's a true for all the RABGTPS, right? So I'm Nucleotide free RABGTPS unstable, right? I know but I'm saying maybe these three Have lower affinity than all the other RABGTPS to Look low types and maybe that's why they need this Agree, I think they're certainly many question need to be addressed. Yeah, yeah So related to that if you get rid of the gap AS 160 for So as 160 is a gap. Yeah, it's a gap You mean repeat experiments in the gap Okay, yeah, yeah, that's a good idea we haven't tested so I could be next step Yes Okay, so here we perform a similar screen and It's a largely the same but I want to point out the difference we so here we didn't treat of the cell with insulin we We sort for the cell the way the high group for reporter on the plasma membrane without a steam stimulation And the idea here is we're going to recover the mutant cells defective in the endocytosis So the screen recovered known regulators of endocytosis such as AP to Adapter subunits. I'm going to come back shortly and TPC 1d4 is this is actually a inhibitor for Exocytosis, so not only the screen recovered endocytic regulators. They also can recover inhibitors or exocytosis But again, most of the genes were not previously known to be involved in the group for endocytosis So here I'm going to focus on one gene called a gap So before that I want to mention the screen recovered subunits our AP to adapter complex Now AP to adapter is a tetramer two large subunits alpha and beta and The one medium subunit mu and the one small subunit of Sigma Now our screen identified MP AP to S1 which encode the Sigma subunit We also identify AP to M1 which encode the mule subunit So we didn't recover the alpha subunit, which is actually expected because there are two genes AP to A1 and AP AP to A2 genetic screens cannot recover redundant genes and The a the beta subunit the same story The AP to B1 encodes the beta subunit, but the gene can be fully rescued I would say compensated by AP one B1 So we couldn't recover alpha and the beta, but the idea is fairly clear. We recovered AP to adapter subunits Now what's a gap now if you ask someone in the field nobody have heard about this protein It's a 34 KD soluble protein. That's why some people you've called it P34 Alpha and the gamma adapting binding protein. I'm going to come back to the naming shortly Now this is known as the IRC 6p in East, but the no phenotype in East I like to mention a gap is frequently mutated in the human disease called a punctate pommel planter Kyrtodoma PPK now. This is the high-flow insufficiency mutations Which means just one allele mutation given the phenotype we see if both alleles are mutated Apparently the organism cannot survive Now this disease is characterized by the thickening of the palm and the soul skin and Of course the cause of the disease By a gap of mutation is still unclear and this is because we still don't know how a gap works Again, we knock out a gap in adipose using CRISPR-Cas9 and you can see if you focus on the orange bars It cause constitutive Translocation of the reporter to the self-surface. So therefore insulin regulation is disrupted The reporter go to the self-surface even without stimulation and We confirm the flow cytometry data by confocal imaging Again, these are well-tap cells well-tap cells before mostly in the intracellular compartment But if you look at a gap of knockout cells, it's already on the self-surface and the phenotype is very similar to AP2S1 knockout cells And AP2S1 as you remember encodes a subunit of the AP2 adapter And next we examined The indocytosis of a cargo. So here is a measurement of indocytosis or the fluorescent labeled antibody Recognizing group 4. Now, this is a very commonly used as say for indocytosis in the field Now when we knock out a gap, you can see a indocytosis of the cargo was abolished and the phenotype was at least as severe as AP2S1 knockouts and the transfer receptor it's a classic Indocytic cargo of a class or media the indocytosis when we knock out a gap There was a strong accumulation of the protein on the self-surface and the phenotype was a fairly similar to AP2S1 knockout Now this data clearly showed a gap is essential to class or media the indocytosis Now I said virtually nothing is known about this protein the only hint that came from East to hybrid screens I think probably two decades ago Now Scotty Robinson's group found a gap Combined to alpha subunit in East to hybrid However, we still don't know if this binding is a direct interaction or if this interaction is a biological significance and next we prepare the recombinant protein and the test the direct interaction and This protein from E. Coli we found a GST tag alpha subunit indeed Interact with a gap and the GST itself couldn't and the interaction here appeared to be a stoichiometric Now how does a gap regulate AP2 adapter formation? There's just some background light But hopefully you can see in the wild type cells on a self surface. There are abandoned AP to a thank you AP to a punk to on the surface, but the strikingly When we knock out a gap this punk to largely disappeared And so this is accompanied by a translocation of group for a reporter from interstellar the compartment to the south surface and we confirm this confocal data by a Turf microscopy Which monitor the events near the plaza membrane? But so here again in the wild type cells we observed a large number of AP to punk to But they all disappeared in a gap of knockout cells And here I can imagine two possibilities Maybe a gap is important for surface recruitment of AP to adapter or They could be essential for the stability of AP to adapter Well, it turned out the second possibility was correct When we knock out a gap, you can see the alpha subunit largely disappeared At the same time the beta subunit was strongly reduced and the male subunit Was also abolished our antibody didn't work for Sigma so far But you can get the idea without a gap AP to adapters were degraded And of course, this is a highly reminiscent of a web app story. I just told you so therefore We are speculated maybe a gap also stabilized The AP to adapters since the bind to our sub subunit. Maybe it's a shaper for alpha subunit and Again, we recast it a gap and alpha subunit in E. Coli Which doesn't have rap which doesn't have a gap which doesn't have class remediated in those hypothesis Again, so here if you look at the pallet, this is after extraction which represent the insoluble fraction We could express the alpha subunit without a gap But all the protein end up in the pallet. We couldn't extract any protein in the supernet However, when we co-express a gap now we could extract the protein in the supernet and this again is similar to web app in the web 10 story, which suggests the alpha subunit itself is unstable in bacteria it's a Informal aggregates and in mammalian cells. It's recognized as a means for the protein and the degraded On the other hand a gap combined to offer subunit and the stabilized Therefore by coincidence, we think a gap is also hold this shaper on for alpha subunit And next we look at a subset of subsettler localization of the protein in the So this is the alpha subunit on the plaza membrane. You can see the punkta but a gap by contrast shows a diffusive pattern and Which which is a typical of cytoplasm Which leads to our hypothesis a gap Actually regulates upstream event in AP to adaptor formation And it does not follow AP to adaptor to the plaza membrane Now one prediction from the shaper model is a a gap function should be independent of its location in the cell To test this possibility we tether a gap to the ER surface by fusing it to a ER membrane protein cut ATF-6 And This is going to target the protein to the ER surface So this is a western blot you can see the fusion protein expressed at the similar level as a gap and there was a no degradation and I Think it's quite an interesting way. We found this fusion protein Could To a large degree restored transfer receptor indocytosis even it's not as close as a gap rescue But it clearly showed this a ER anchored protein can function. So therefore ER well a gap function is not dependent on its localization Which is consistent with a shaper model Now, this is the crucial test a Crucial prediction from the shaper model is forced expression of the alpha subunit should rescue a gap knockout phenotype and here we took the advantage of very strong promoter wires to derive the promoter and when we all expressed alpha subunit I Could fully rescue the transferrin receptor In internalization and as good as a gap rescue and The base subunit rescue our expression didn't work and I expected expression order for AP to subunits could rescue So I think we can do two key conclusions from this data The first is restoring AP to expression artificially Can bypass requirement for a gap and All these data indeed suggest a gap is a whole days of shaper on for the upper subunit But I think a gap is more than just a shaper on so I'm going to show you some data and Look forward to your comments So what happens after a gap bind to upper subunit? To monitor a gap interaction with AP to subunits We divide the HA tag system In which HA tag is added to all the four subunits of the AP to subunits and You can see this is sigma HA this is mu HA and alpha and the beta They migrate at the same location in sts page Okay, so here is the co IP data We found a gap it could have put on alpha subunit as expected, but it could not put on the sigma subunit by itself However, when we put in all the four subunits a gap could have put on alpha subunit and the sigma subunit We couldn't we never see the mu subunit And also we perform additional analysis different the co IP Design using an untagged version of alpha subunit. We could only see Afra and Sigma subunits. We never could see a beta or mu subunits And if we confirm this co IP data by using a recombinant protein, so here I just want to see this lane We can find so we saw the sigma subunit bind to Alpha subunit and a gap. It's very small protein. So the staining is a very faint, but I think it's about a stoichiometric interaction So they're therefore the a gap alpha subunit dimer Subsequent they recruit the sigma subunit. So I like to put what I talked about so far into this model We think a AP to adapter assembly is not a spontaneous process as previously assumed instead It's a highly organized process the initiator of the process is a dimmer or a gap and offer subunit and The dimmer here recruits the sigma subunit Since beta and the mu subunit does not bind to a gap We propose they can replace a gap from the alpha and the sigma subunit Which allows the formation of the tetramer. So what happens after is well known So the story I just told you it's a prequel of what the story that has been told again again and What happens of without a gap the entire AP to assemble assembly process Just collapse all the subunits got degraded Well in summary We showed a gap is a master regulator of our AP to adapter assembly In addition as a shaperone for offer subunit we propose maybe a gap can prevent AP to binding to non-cognitive the cytosolic proteins in our many cytosolic protein contain the dilucine Signals, but they should not be recognized by AP to adapters Now if for the AP to adapter is a fully formed The beta subunit can block the binding site Unless until it's opened up on the plaza membrane But what happens before beta can mask the binding sites and we propose a Gap may be functioned by blocking the binding sites. So to prevent the binding to the non-cognitive proteins Now because other modern marriage a track trafficking factors like AP one AP three cop one Face the same challenges of assembly and the specificity issues We think it may be there are other a gap like molecules that control their assembly Now before I finish I like to return to the PPK disease Now here is a diagram of the skin cell contact the contact between skin cells are driven by a series of membrane proteins and the cell the solid proteins Which include a decimal glean decimal plaking dp here and the keratin interestingly Most of these proteins are also found to be mutated in the PPK disease Along with a gap now the mutations of these genes make a total sense because you can imagine these their mutations are going to Compromise in the integrity of the skin cell contact and therefore cause the skin disease But how does a gap fit into the picture? So here I just want to show you a preliminary observation we did a proteomic analysis of Surface of proteins in a gap knockout cells and we found a gap can Mutations downregulate a protein called a decimal plaking So without a gap this protein level on the surface was strongly reduced now This is a very preliminary data Additional work need to be done, but I think it has a potential to explain Why a gap of mutations can cause the skin disease? So before I rip up I like to convey the message There are still many mysteries in the mysteries in the Indocytosis and exocytosis None need to be solved Our screen identified many membrane proteins which show the very strong knockout phenotype But the virtually nothing is known about their biologic functions now if you do a sequence prediction you still got nothing In my opinion moving forward a very exciting direction is to find out how these a protein regulate cargo flow in the cell Okay, so with that I Like to thank the people Then Lauren great students hijab postdoc. So they did all the work. I show you today We return to this and thank you for your attention So this is very cool that you're regulating a P2 Assembly, but could you tell us why it's not legal? Yeah, so I Know class through a classroom is a list of because of the trafficking from the Golgi. It's actually Cause more lethality Actually, you can knock out the AP to and they're not a list of insert in cells It's lethal in you eliminate a P2 and it's lethal in the cell culture In animals I can understand Elimination is lethal in animals and if you eliminate Yeah animals for sure. Yeah, why this is so important for the Folding of the P2 The only phenotype you're getting as you said, well, that's a high-poly insufficiency, right? So just one a little mutation. So now remember I study a Westco fusion protein monkey team for many years high-poly insufficiency of mutation of monkey team one only cause Certain neuronal phenotype or all the patients are normal. Okay, so I think Why skin cells are more sensitive? Well, maybe some mathematic modeling I cannot tell so but overall I don't think other tissues are gonna be a sensitive and again, I Agree knock out to a AP to in animals gonna kill the animals, but in the cell culture levels I don't think there are essential genes Well, if you don't have a P2 and so I mean cells divide with no AP to they're very sick Anise's problems. I mean lots of issues. So I think you could be right. I think you are right For these clonal CRISPR knockout cells Eventually we select for the mutants that can grow right so it's it could be Compensation it could well, it's not gonna be phenotypic a compensation, right? Or could be a hypo morphic mutations Reduce the most of the activity, but there's some residual activity that keep the cells alive Yeah, well Well, when we knock out essential genes in the proteasome the cells can grow better sometimes No, but if you eliminated to So this actually can be This can be precessively check. There is a essential gene this We check a couple years ago with I don't think that these are essential genes Maybe surprisingly at least not in the cancer cell lines. Yeah Yeah, yeah, there are several screens Well, I cannot say for normal cells, but at least in cancer cells. They are not essential genes Yeah, it's a comment about the rabbeff story So if I understand you thought it's a sharp wrong if you remove it Rob tennis degree that rabbeff But I'm not so sure that this Excludes the fact that the real function could be something else No, it because it's common to find situations in which you have a diamond which both Proteins have Can I just check this question? Do you check it on indulgences or express proteins because the shopper all it would be different the reptile, right? Yeah, the reptile is all endogenous. Okay, and Also, we could For six press the reptile using a viral promoter. We restore reptile protein We totally rescue rabbeff knock out phenotype. So I think your question has a two parts first I think it's a fairly clear in our case. It's a chaperone, but there's a function In another way, you know, could be a gap In a different possible. I don't think I guess but in that case, I think it's a possible Yeah, it's good. Is it completely clear that it doesn't help Some I would call it we have function majority function on the activity around 10 But then of course it also stabilizes Because in diamond as this we did so in general this is the case for other diners I Think that's a totally possible for example a gap Even to chaperone, but I think as I showed it's more than that It's organized the whole assembly possibly maybe a rabbeff is doing that also Stabilize right, but also control downstream function. So right now we just don't have the evidence So quick follow-up on this and then I have another question Does it work on second form? Second second. Yeah, okay the east. Yeah, well the east I Remember if you knock out a rabbeff or homologue, you don't see anything, right? you see a little bit of effect only when you have a Rabbeff mutation that you overexpress is to rescue I think that only demonstrate those protein interacts, right? So we haven't tested in the east yet But I think you yeah, I don't see how you can test a function without a knock out phenotype It's kind of hard. So the other question was on the very last part of what you're saying about the phenotype I was confused how down regulation that there's no blacking would have anything to do with it right, so I Think that this is going to be an indirect effect We did a proteomics and knock out a gap cause a massive Dispersion Inbalances on the surface of proteins. So a large number of protein either you get too much or you get too less I think are going to be for example, maybe a cargo adapter on the plus membrane which is Reduced but in turn you get a moral another protein because of a lack of inhibition So I think it's going to be really hard to interpret at this moment. We don't know why It causes the disease. I just one possibility and I think I can likely can be a indirect effect For it all completely independent Yeah, so in that case, I would think we probably want to rescue a p2 Undercheck it, right? So Your phenotype which which has to do with critical site dysfunction Precise a patient Yeah, so that's going to be a crucial experiment that to study the disease we're thinking To introduce the precisely the same mutation into a PS out and the difference into into a keratinocytes I think That's going to tell us a lot about this protein function It could very well be that your protein Whatever the name is a gap He's doing what it's doing and it is involved in cell cycle specific cell junction disassembly And that is being affected and therefore what you end up with is a cell that does not Disassemble desmosomes when they have to divide and as a result what you end up with is creating a Pethenotype that is like that cell right to work, but you're still thinking the The more directed causes the endocytosis defect, right? Yeah, I agree So what happens after a p2 week? We cannot say for sure. It's just a speculation Okay, so we can choose to small comment question, but yeah, have you checked the mutant that caused the phenotype That's right, so we So many of the mutations actually are premature terminations Right around the middle and I see terminus. We didn't have with a precess mutation, but we have done a truncation which may make The disease in mutation. So that one was a totally inactive. So we saw in the cytosis of the fact So in our assay you need the entire protein How extensive it is this notion of how many chaperones to help So folding right because I mean the same is true with HSC Yeah, and so well Yeah, yes, so what we can we can talk more, but I think we're actually touching even Much a bigger problem for multiple subunits like AP2 or snares, you know, they are expressed at a different Chrome some location different promoters. How do you coordinate their assembly, right? Because they're expressed at different levels. I think maybe this is a one solution for that you have a initiator from one subunit and Just go from there and everything else If they're expressed more or less, it doesn't matter. They if too much they're gonna get degraded If we're too less, they're gonna wait for initiator to get ready. So I think this may be a general way to assemble Multimeric proteins in the mammalian cells How sensitive? But at least in a gap case is very sensitive because without a gap you don't get any AP2 adapters What's your second question is other substrates? Yeah, so I we have some preliminary data actually AP1 the gamma subunit also disappears with a gap knockout But the Delta Adapting was normal. So AP3 was fine, but AP2 and AP1 They were affected So All right, thank you


My name is Jeremy Bradshaw. I'm originally from California. So when my mother had found out that she was pregnant with me, she had told my dad, and they weren't together at the time. It was like, hey, we need to raise this child together. And he was like, that's not my kid and stuff like that.  And so he chose her. So because of that, you know, he was never in my life. I was born without him. And it really like damaged me. Like I feel like from the moment that I was born, even in the womb, I was like born into rejection. And so I really grew up really kind of feeling alone. Even though I wasn't necessarily alone, I felt alone a lot of the time. And even as a child, you know, I'd be laying in my bed and it was like I could hear voices downstairs talking like almost like a crowd of people talking downstairs. And so like there was that that was going on too, because it was just very like demonic. And I realized where it's from now, my grandma, she practiced a lot of like tarot card, things like that. And so because of that, you know, that allowed a lot of that in there. And so we had like this line of psychics, I guess. So in that, you know, it just led to more darkness filling my heart eventually led me into doing heavy drugs like marijuana starting and then into shrooms after that. And then eventually leading to meth and heroin. And so they always say like, you know, oh, marijuana's not the gateway drug and stuff. And yeah, some people could smoke it and be fine and be done with it. But for me, it was like a thing where I was like, oh, this wasn't as bad as I thought it was. Well, this ain't bad. Maybe the shrooms don't be that bad. So then I did that. And I was like, well, if the shrooms and the marijuana weren't that bad, and then you could kind of see where I'm going from there, led into smoking meth and heroin and such. So in that, it just really kind of broke me and got me to this place of darkness that was so intense that like, I started allowing these things what I didn't know at the time, but now I do it was allowing these demons to come in and fill me. And I remember being like on shrooms and like, I would almost call upon these spirits to come fill me up in a way. I didn't know what I was doing at the time, but.  And my mom had to actually take me to a hospital because I was just not myself.  And they brought me to the hospital and they had a psychiatrist come in and talk with me and she deemed me unsafe to be out. And so I got put on what is called a 5150 hold, which is basically danger to self or danger to society. And they put me into a mental facility for, it was supposed to be three days, but it ended up being seven. And so I finally get led out of that. I ended up doing heavy drugs one last time. And then I for some reason, and I have no idea why I just decided to stop. Like I was still smoking weed, but I was like the meth, the heroin, the shrooms, all that I need to be done with, you know?  And a couple of months later, I was working at Chipotle actually and this girl got hired there. You know, she always spoke about Jesus. And so I was like, all right, I mean, she's cool, but she's weird and this whole Jesus thing, I'm not about it. Cause at the time I was atheist, you know, I didn't believe in God. So I'm at Chipotle and Ashley is the girl that worked at Chipotle. She was like, hey, I need a ride home. You know, my car broke down. Can you give me a ride home? And I was like, sure. So I like drove her home and all of a sudden I just knew in my heart, I wanted to give my heart and my life over to the Lord. And so like I told Ashley that I was like, hey, I think I'm ready to give my life over to the Lord. And she's like, what, really? That's amazing. You know, I was like, yeah, literally I went through the repentance prayer and the sinner's prayer and just like repented for my ways and, you know, asked the Lord to come and live in my heart. And it was in that moment I experienced him for the first time.  But this was like his presence came so heavily on me in that moment when I received him. And I actually got baptized in the Holy Spirit in that moment as well. So I was able to get not only saved but baptized in the Holy Spirit at the same time. And so I knew without a shadow of a doubt that the Lord was real, you know? I remember in 2019, I was sitting on my deck and I was reading my Bible and I was smoking a joint at the same time. And I remember the Lord's voice so clearly spoke out to me. He said, Jeremy, you will never be able to do what I've called you to do while smoking weed. And it was in that moment that I was like, yeah, I need to be done with this. I found a church home and it was amazing. But like I said, I was still struggling really bad with just depression and feeling lonely and disbrokenness. And so eventually I went to raised to deliver there in Seattle or it's federal way in April of 2021. And there I received some deliverance that was just amazing and like I was so changed. So I ended up leaving Arkansas and coming up to the discipleship program here at Hungry Jen and it changed my life. So I remember delivering Sunday and I get up there and I close my eyes and I'm just sitting there. And all of a sudden I hear Brittany's voice. I didn't know her at the time but she's just like started praying for me. And then she said, I see you in there hiding and you are no longer allowed to hide. And all of a sudden I just started manifesting. And I was like, I knew there was something hiding in there. And so I'm going through this and she's like asking me like, where are you from? Telling me that it was a spirit of witchcraft that was in generations that came from generations ago and it was using my eyes to cause me to be impure and all these things that had been torturing me. And so like finally that thing gets yanked out of me and I finally delivered and I remember sitting there. And I was like crying so heavily because I was like, I'm finally free. This is the moment I've been waiting for and praying for my entire life.  I finally got to that point. I'm like, I'm free. After getting set free, it was like the cage was open and I could walk out of it and just be free. And I have not been the same ever since. And because of that, man, the Lord has just been doing so much in my life and he's changed me completely. And I just, I'm so grateful to him for everything he's done and he continues to do even more so every single day. And so I just hope that this is an encouragement to somebody out there just saying like, no matter how much darkness you've walked in, you know, there's still hope for you. My name is Jeremy and this is my testimony.


Hey everyone, we got some exciting Zelda news for you, some first details on Hyrule Warriors Age of Calamity DLC, also some stuff surrounding the Zelda 35th Anniversary, including some juicy rumor, great stuff, you know we've been covering this all on top of it, and I know what you're thinking right now, you're looking at me and you're going damn Nate, that is a fly officially licensed Legend of Zelda hoodie, look at this, look at this, you see that shield, yeah here's the deal, I would love to link to you guys where you can buy this, here's the problem, they don't make it anymore, that's right, this officially licensed product it doesn't exist anymore, however you know what does exist, this PVC first four figure Revali statue that's 10.7 inches, two different versions, one of them that lights up at the base, one of them that doesn't, the one that doesn't is about 108 bucks, the one that does have a light at the bottom is about $127, I will put links down in the description, if you guys would like to buy it, they are officially licensed, they are made by first four figures, they are supposed to be shipping by this December, might be the perfect time to get them with Breath of the Wild 2 potentially on the horizon also, I know, you really want this hoodie, well, you know, the thing is it's a good thing that, I've got two of them, oh wait wait wait, what's that, oh this one too, this one's brand new as well, the tag is still on it and everything, I don't know if I can get it for you, the tag is still here and everything, it's the first time I've ever worn this thing, these are just two items that we will be giving away during our massive E3 shenanigans this year starting on, as far as we're aware, June 12th, unless there's other E3 things going on earlier, then our four-day show becomes a five-day or six-day show, we are giving away thousands of dollars worth of stuff, we're giving away Nintendo Switches, we're giving away these hoodies, we're giving away games, we're giving away accessories, we're giving away things like this collectible Legend of Zelda Breath of the Wild coin from E3 2016, that's right, I only have two left out of the 18 that I originally had and this is one that is fully sealed, has never been opened and this one is one I'm giving to you, I'm keeping my other one for myself as a memory, but that is something that's being given away during our event, yes lots of Zelda stuff, but not just Zelda, we have so many giveaways going on, just what are you doing, why are you not subscribed, why are you not liking this video, why don't you head down in the description, click the link to our first stream set up for E3 2021 and hey, set notifications for it so you know when we go live, so you don't miss an opportunity to win a bunch of cool free stuff, also we're going to have gaming competitions where you can beat me in one-on-one matches in Super Smash Bros to win prizes and you will also see Eric from the Nintendo Prime podcast and myself go at it together, 1v1 in various video games throughout E3 with the loser getting various forms of punishment, hot chip challenge, egg on the head, I don't know, we're still gathering up what all those punishments are going to be, in fact, you guys let me know what you would like those punishments to be down below. Alright folks, let's dive deep into this, you've waited long enough, we're getting information here from SamusHunter2 who again has been right on a whole bunch of stuff and yes, Nate the hate of all places, because Nate the hate is great, known as Nate Drake over on the recent era in NeoGap forums, he does appear on the Spawncast podcast that happens every Saturday night over on Spawnwave's channel and then yes, he has his own podcast as well, which is very very informative, he does have industry insiders and sources on things, he's very coy about it, he doesn't talk about his sources that much, but every now and then he'll kind of put two and two together over things he's hearing and throw it out to the masses, so we got some juicy stuff to talk about, first I was getting to SamusHunter2 who's been teasing for weeks now that we were going to get something Zelda related this month, was it 35th anniversary, is it Breath of the Wild 2, is it just Hyrule Warriors, Age of Calamity DLC, it turns out it might be a combination of multiple things. First off, new details from SamusHunter2 today for Hyrule Warriors, Age of Calamity DLC that will be unveiled today, in fact this image you're seeing up here or at least a partial image on the screen, that's actually part of the leak, now it's just got various characters on there, but the point is some of these characters that weren't playable in the past will be playable in the future and we have some more details, if you don't want to know what the Hyrule Warriors DLC is going to be, maybe skip this part of the video, if you don't care, here we go, don't worry, there's not really any major spoilers I would say except for whatever's behind that question mark which I have no idea what is, it says spoiler, I have no idea what it is, it says, back in December I tried to create a picture with some elements from the promotional material used in the Direct and here are things that are going to be announced for this DLC, you're going to help Robbie and Pura learn more about Shiko weapons, you're going to take on missions to unlock new weapons, what else is new there, and you're going to learn more background on the characters in the world, I know, very very limited information on the Hyrule Warriors DLC, they're beyond helping Robbie and Pura specifically with the Shiko weapons, the rest of it is kind of a general thing, but we're going to be hearing about it and this image does contain legit spoilers for Hyrule Warriors Age of Calamity DLC, so I'll put it full screen for you guys so you get a quick look at it, and then I was obviously curious, hey is this all, you know, is this what you were teasing and this is what Samus Hunter said, this is what she said, yeah all of the elements included in the image are related to some of the contents of the entire expansion pass, like playable characters, weapons and armor and theme, and then I said hey so are we going to get any other announcements this month or is this it for the Legend of Zelda, and this is what Samus Hunter 2 said, likely yes, but I would also expect Nintendo teasing other things for E3, but that is speculation on my part, right now I don't know about any game specific plans for E3, obviously beyond the prior tournament stuff, and we're a little bit further away, based on how I know Samus Hunter gets her information, she wouldn't really be privy to what's happening at E3 until much closer to E3, like a week or so away, so we'll see what happens, beyond all of that, Samus Hunter 2 threw this out there a couple days ago saying starting this month we are going to see more news in the Zelda series, that's part of the news, this is Hyrule Warriors, Age of Calamity DLC, and so also likely be an appetizer before the main course at E3, but we are entering the Zelda 35th anniversary, so Samus Hunter believes Zelda 35th anniversary stuff, it's happening, E3 big blowout, let's go, now what might happen at E3, what can we expect, what is going on, Nate the hate on his most recent episode of the podcast, linked below, gave the following statement and paraphrasing because he has long form discussions like we do on this channel, and he says he stated firm belief that for the 35th anniversary of Zelda, so he too believes Nintendo celebrating that 35th anniversary of Zelda, that we will be getting Twilight Princess HD and the Wind Waker HD with minor improvements packaged together, so when you're talking about that 3D All-Stars type game, this is what we're going to get, these are two games that Nintendo already HDed over on Wii U, so there you go, also that we are going to get Ocarina of Time and Majora's Mask by the end of this year as well, however this is where this goes against some of the other rumors and reports out there, he's saying that it's going to be a digital only release, now for Ocarina of Time and Majora's Mask, now if they were actually like remastered or whatever, Nintendo would obviously give us a physical package for them, digital only kind of suggests a bonus to the Nintendo Switch online, something like that, now we did get bonuses, we did get like Super Mario All-Stars from the SNES as a bonus during the 35th anniversary, so that kind of feels like that's what this could be, although there aren't any N64 games, so maybe this is like spinning off and do are they going to add N64 games to the Switch and it's just going to be headlined by Ocarina of Time and Majora's Mask this winter, I have no idea, but that is something he has thrown out there, again now this is Personable Leafs, obviously he's heard some stuff, he's not going to confirm or deny a certain way, but this is the kind of bee's knees of all of that information, so there you have it, that's all the new info on Zelda, I promised people I had a Zelda video today, I was busy gathering it all up, realized I had a sick merchandise I can give away because I don't even use this, I love this jacket but I don't know man, I feel like you guys, one of you guys out there can make use of it, though unfortunately because these jackets aren't available anymore, you have to get the sizes I have, which I think this is a 2XL and the other one is a size large, either way be given away during our E3 event, along with so much stuff, holy crap, I still have stuff arriving for the set I'm building, which I, guys, you're going to want to tune into our E3 show, we're going to have the greatest, the best, the most exciting E321 show, the side of the entire world, that's right, tune in here over Nintendo even, forget Nintendo, because all of the Nintendo stuff is going to be on our channel anyways, we're live reacting to it all, anyways thank you guys so much for tuning in and I'll catch you each in the next episode


Now, the Roma Wine Company of Fresno, California, presents Suspense. Tonight in response to requests from many thousands of listeners, Roma Wines bring you the Metro Golden Maier star, Miss Agnes Moorhead, in Sorry Wrong Number, a suspense play produced, edited and directed for Roma Wines by William Spear. Suspense, Radio's Outstanding Theatre of Thrills, is presented for your enjoyment by Roma Wines. That's R-O-M-A, Roma Wine, those excellent California wines that can add so much pleasantness to the way you live, to your happiness and entertaining guests, to your enjoyment of everyday meals. Before we bring you Agnes Moorhead and our suspense play, here's a message from Roma Wines. Today, big transports were warped into piers on both sides of the continent. Thousands of Americans came home, home for good.  One of the good things to enjoy is Roma California Sherry, as first called a dinner or later in the evening. In the words of famed hostess Elsa Maxwell, there is nothing friendlier, nothing more heartwarming than delicious Roma Sherry. A good and happy wine, gold and amber with a rich nut-like taste. Serve cool. Roma Sherry, like all famous Roma Wines, is made from carefully selected grapes from California's choicest vineyards. Grapes gathered at the peak of their flavored goodness when every grape is hanging firm and full on the vine. Quickly but gently pressed, then by a process as slow as time, brought to delicious liquid perfection and bottled in Roma's famed wineries. Always unvaryingly good, yet Roma Wines cost only pennies a glass. Remember, because of uniformly fine quality at reasonable cost, more Americans enjoy Roma than any other wine. R-O-M-A, Roma Wines. Yes, right now a glassful would be very pleasant as Roma Wines bring you a remarkable tale of suspense. And with sorry wrong number, Lucille Fletcher's incomparable study in terror, and with the performance of Miss Agnes Moorhead, Roma Wines hope indeed to keep you in suspense. Operator, I've been dialing Murray Hill 4098 now for the last few quarters of an hour, and the line is always busy. I don't see how it could be busy that long. Will you try it for me, please? I will be glad to try that number for you one moment, please. I don't see how it could be busy all this time. It's my husband's office. He's working late tonight, and I'm all alone here in the house. My health is very poor, and I've been feeling so nervous all day. Ringing Murray Hill 4098. Is Mr. Stevens in there? What number am I calling, please? I'm here with our client. Oh, good. Is everything okay? The post clear for tonight? Yes, George. He says the coast is clear for tonight. Okay, okay. Where are you now? The phone booth. Don't worry. Everything's okay. Very well. You know the address. I know, I know. At 11 o'clock, the private patrolman goes around to the bar on 2nd Avenue for a beer. That's right. 11 o'clock. Sure that all the lights downstairs are out. There should be only one light visible from the street. Okay. Just a minute, George. Oh, our client tells me that an 1115 train crosses the bridge. It makes a noise in case our window is open, and she should scream. Oh, hello. What number is this, please? Oh, do you remember everything else, George? Yes. That's right. You'll use a knife. Yeah, those are rings and bracelets. Oh, how unspeakably hot. Operator, I've just been cut off. I'm sorry. What number were you calling? Why, it was supposed to be Murray Hill 40098, but it wasn't. Some wires must have got crossed. I was caught in the wrong number, and I've just heard the most dreadful thing, something about a murder. Operator, you'll simply have to retrace that call at once. I beg your pardon. I don't quite... May I help you? Well, I know it was a wrong number, and I had no business listening, but these two men, they were cold-blooded beings, and they were going to murder somebody, some poor innocent woman who was all alone in a house near a bridge, and we've got to stop them. We just got to. What number are you calling, please? Well, that doesn't matter. This was a wrong number, and you dialed it for me, and we've got to find out what it was immediately. What number did you call?  What time is it? Do you mean to tell me you can't find out what that number was just now? I'll connect you with the chief operator. Oh, I think it's perfectly shameful. Now, look. Look, it was obviously a case of some little slip of the finger. I told you to try Murray Hill 40098 for me, and you dialed it, but your finger must have slipped, and I was connected with some other number, and I could hear them, but they couldn't hear me. Now, I simply failed to see why you couldn't make that same mistake again on purpose, why you couldn't try to dial Murray Hill 40098 in the same sort of careless way. Murray Hill 40098, I will try to get it for you. Oh, thank you. Operator. Operator. Operator. Your call, please. You didn't try to get that wrong number at all. I ask you explicitly, and all you did was dial correctly. I am sorry. What number are you calling? Well, can't you for once forget what number I'm calling and do something for me? Now, I want to trace that call. It's my civic duty, and it's your civic duty to trace that call, and apprehend those dangerous killers, and if you won't, I will connect you with the chief operator. This is the chief operator. Oh, a chief operator. I want you to trace a call, a telephone call, immediately. I don't know where it came from, or who was making it, but it's absolutely necessary that it be tracked down because it was about a murder that someone's planning, a terrible, cold-blooded murder of a poor, innocent woman tonight. At 11.15. I see. Well, can you trace it for me? Can you track down those men? I'm not certain. It depends. Depends on what? It depends on whether the call is still going on. Well, a live call, we can trace it on the equipment. If it's been disconnected, we can. If the parties have stopped talking to each other. Oh, well, well, of course they must have stopped talking to each other by now. That was at least five minutes ago, and they didn't sound like the type that would make a long call. Well, I can try tracing it. May I have your name, please? Mrs. Stevenson, Mrs. Albert Stevenson. But listen up. And your telephone number, please. Plaza 32098. But if you go on... Why do you want this call traced, please? Why? Oh, well, no reason. I mean, I merely felt very strongly that something ought to be done about it. These men sounded like killers. They're dangerous. They're going to murder this woman at 11.15 tonight, and I thought the police ought to know. Have you reported this to the police? Well, no, no, not yet. You want this call checked purely as a private individual? Yes, yes, but meanwhile... Well, I'm sorry, Mrs. Stevenson, but I'm afraid we couldn't make this check for you and trace the call just on your say-so as a private individual. But I... He had to have something more official. Oh, for heaven's sake. You mean to tell me I can't report that there's going to be a murder without getting tied up in all this red tape? Why, it's perfectly idiotic. Well, all right, all right, I'll call the police. Thank you. I'm sure that'll be the best way to... I never heard of this, no. Police, you have to go. Police department, get me the police department, please. Thank you. Oh, dear, do you have to dial? Why can't you ring them direct? Oh, it's... Please, Sergeant Martin speaking. Police department, this is Mrs. Stevenson, Mrs. Elbert Smy Stevenson of 53 North Sutton Place. I'm calling up to report a murder. I mean, the murder hasn't been committed yet, but I just overheard plans for it over the telephone. Over a wrong number that the operator gave me, and I've been trying to trace down the call myself, but everybody is so stupid, and I guess in the end you're the only people who could do anything. Yes, ma'am. Well, it was a perfectly definite murder. I heard their plans distinctly. Two men were talking, and they were going to murder some woman at 11.15 tonight. She lived in a house here, a bridge. Are you listening to me? Oh, yes, ma'am. And there was a private patrolman on the street. He was going to go around for a beer on 2nd Avenue, and there was some third man, a client, who was paying to have this poor woman murdered. They were going to take her rings and bracelets, and he was a knife. Well, it's unnerved me dreadfully, and I'm not well. I'm very nervous. I see, I see. And when was all this, ma'am? Well, about eight minutes ago. Then you can do something. You do understand. What is your name, ma'am? Mrs. Stevenson. Mrs. Albert Stevenson. And your address? 5353 North Sutton Place. That's near a bridge. The Queensborough Bridge, you know, and we have a private patrolman on our street, and 2nd Avenue is the next bridge. And what was the number you were calling, ma'am? Murray Hill 40098. But that wasn't the number I overheard. I mean, Murray Hill 40098 is my husband's office. Well, he's working late tonight, and I was trying to reach him to ask him to come home.  Well, we'll look into it, Mrs. Stevenson, and see if we can check it with the telephone. But the telephone company said they couldn't check the call of the party that stopped talking. I've already taken care of that. Oh, you have. Yes, I have. Personally, I feel you ought to do something far more immediate and drastic than just check the call. What good is checking the call if they stop talking by the time you drag it down, they'll already have committed the murder. Well, we'll take care of it now, don't you worry. I'd say the whole thing calls for a search, a complete and thorough search of the whole city. Yes, ma'am. I'm very near the bridge, and I'm not far from 2nd Avenue, and I know I'd feel a whole lot better if you sent around a radio car to this neighborhood at once. And what makes you think the murder's going to be committed in your neighborhood, ma'am? Well, I... I don't know. Only the coincidence is so horrible. 2nd Avenue is the patrolman, the bridge. Well, look, you see, 2nd Avenue is a very long street, ma'am. Well, I know that. You know how many bridges there are in the city of New York alone? Yeah. Not to mention Brooklyn, Staten Island, Queens, and the Bronx. Well... And how do you know that there isn't some little house on Staten Island on some little 2nd Avenue? You never heard about it? Well... How do you know they were even talking about New York at all? I heard the call on the New York Dialing System. Well, maybe it was a long-distance call. Oh, I... Well, telephones are a funny thing. But I did... Now, look, lady, look. Why don't you look at it this way? Now, supposing you hadn't broken in on that telephone call.  So upset with you. Well, I... No, I suppose not. Only it sounded so inhuman, so cold-hearted. A lot of murders are plotted in this city every day, ma'am, and we manage to prevent almost all of them. But a clue of this kind is so vague, ma'am. Well, it ain't much more used to us than no clue at all. Like, surely you can undo... Unless, of course, you have some reason for thinking this call was a phony and that someone may be planning to murder you. Me? Oh. No, no. I hardly think so.  Well, here... I'm alone all day and night. I see nobody except my maid, Eloise. She's a big girl. She weighs 200 pounds. Uh-huh.  Uh-huh. And the only other person is my husband, Elbert. He's crazy about me. He just adores me. Yes, ma'am. He was on me hand and foot. He seriously left my side since I took sick 12 years ago. Uh-huh. Well, then... then there's nothing for you. Now, if you'll just leave the rest to us, we'll take care of it. But what will you do? It's so late. It's nearly 11 now. We'll take care of it.  Lady, lady, I said we would take care of it. Now, just now, I've got a couple of other matters here on my desk that require immediate attention. Good night, ma'am. And thanks.  Now, why did I hang up the phone like that?  Oh, why doesn't Elbert come home? Operator. Operator for heaven's sake. Will you ring that Murray Hill 40098 number again? I can't think what's keeping him so long. I will try it for you. Well, try, try. I don't see why it doesn't... I didn't hear it. You don't have to tell me. I know it's busy. This telephone tonight, but it's positively driving me crazy. I've never seen such inefficient miserable service. Now, look. Look, I'm an invalid, and I'm very nervous, and I'm not supposed to be annoyed. But if this keeps on much longer... What seems to be the trouble, please? Well, everything's wrong. I haven't had one bit of satisfaction out of one call I've made this evening. The whole world could be murdered for all you people care, and now my phone keeps ringing and ringing and ringing and ringing every five seconds or so, and when I pick it up, there's no one there. I am sorry if you will hang up. I will test it for you. I don't want you to test it for me. I want you to put that call through whatever it is at once. I'm afraid I cannot do that. You can't ask. The dial system is automatic. If someone is trying to dial your number, there's no way to check whether the call is coming through the system or not, unless the person who is trying to reach you complains to his particular operator. Oh, well, I've got to sit here in my bed suffering every time that phone rings, imagining everything. I will try to check the trouble for you. Check it, check it. That's all anybody can do. Oh, what's the use of talking to you?  Oh, I'm only impudent. Young woman, I don't know your name, but there are ways of finding you out, and I'm going to report you to your superiors for the most unpardonable rudeness and insolence. It's ever been my privilege. Give me the business office at once. You may dial that number direct. Dial it direct now. Do no such thing. I don't even know the number. The number is in the directory, or you may secure it by dialing information. Listen here. Oh, what's the use? Where is she? Where is she? Why doesn't she answer? Didn't you answer at once? Give me the police department. This department can't be busy. There must be other lines available. The line is busy. I will try to get them for you later. Oh, no. I've got to speak to them now. It may be too late. I've got to talk to someone. What number do you wish to speak to, please? I don't know, but there must be someone to protect people inside the police department. A detective agency. You will find agencies listed in the classified directory. But I don't have a classified. I mean, I'm too nervous to look it up, and I don't know how to use the directory. I will connect you with information. Perhaps they will be able to help you. No, not at all. You don't care, do you? Whatever. I can die in you when you can. This is Plaza 32099. This is Western Union. I have a telegram here for Mrs. Albert Stevenson. Is there anyone there to receive the message? Mrs. Stevenson. The telegram is as follows. Mrs. Albert Stevenson, 53 North Sutton Place, New York, New York. Darling, terribly sorry. I tried to get you for the last hour, but lying busy. Leaving for Boston 11 p.m. tonight on urgent business. Back tomorrow afternoon. Keep happy. Love signed. Albert, would you like to deliver a copy of the message? No. No, thank you. Thank you, madam. Good night. Good night. No. But he couldn't do it. Oh, Diane, number for me just once more, please. Telephone number of Hensley Hospital. Hensley Hospital? Do you have the street address? No. No, it's somewhere in the 70s. It's a very small, private and exclusive hospital where I had my appendix out two years ago. Hensley, H-E-N-T. One moment, please. Well, please hurry and please. What is the time? You may find out the time by dialing Meridian 71212. Oh, for God's sake. I've no time to be dialing. The number of Hensley Hospital is Butterfield 8. Nurse's registry. Who was it you wish to speak to, please? I want the nurse's registry at once. I want a trained nurse. I want to hire her immediately for the night. I see. And what is the nature of the case, madam? Nerves. I'm very nervous. I need soothing and companionship. You see, my husband is away. And I've been recommended to us by any doctor in particular, madam. No, but I really don't see why all this cataclysm is necessary. I want a trained nurse. I was a patient in your hospital two years ago. And after all, I do expect to pay this person for attending me. We quite understand that, madam. But we're not completely back on a peacetime basis, you know. I know that. Registered nurses are very scarce just now. And our superintendent has asked to send people out, only on cases where the physician in charge feels it is absolutely necessary. Well, it is absolutely necessary.  I'm very upset with this house and I'm an invalid. And tonight I overheard a telephone conversation that upset me dreadfully.  I couldn't say. She went out to suffer at 11 o'clock. Just now on my own telephone. Although someone has lifted the receiver off the hook off the extension telephone downstairs. Downstairs in the kitchen and they're listening to me now. Call someone now while they're still down here. There'll be no time. Operator, I'm in desperate trouble. I don't dare. There's someone listening. Can you hear me now? Got to hear me. Oh, please. Please, you've got to help me. There's someone in this house. Someone who's going to murder me at the extension phone. I'm in the police department. The police department. One moment, please. I will connect you. Oh, I can hear him. Police department? Yeah. The wrong number. Don't worry. Everything's okay. And so closes, sorry wrong number in which Roma Wines have brought you Miss Agnes Moorhead, a star of tonight's study in... Suspense. Suspense is produced, edited and directed by William Spear. Before Miss Moorhead returns to the microphone, this is Truman Bradley with a word for Roma Wines, the sponsor of Suspense. This is the time of year when the air is clear and the hills look as if they had been burnished in gold. At dusk, the color glory of Indian summer paints the countryside. A perfect setting for a simple supper with friends. For a meal brightened by distinguished Roma California Burgundy. In the words of famed hostess Elsa Maxwell, next time you have friends for dinner, add to their enjoyment of the food and give the occasion a gracious festive touch by serving delightful Roma Burgundy. My guests especially enjoy the fruity, robust tart pecancy of Roma Burgundy. A simple way to lend enchantment to dining. Yes, like all Roma wines, you'll enjoy Roma Burgundy. Delightful, delicious, distinguished wine at its best in uniform, fine quality. And here's a hint on how to make better cocktails. Make them with zestful, full-flavored Roma vermouth. The vermouth of almost a hundred rare herbs, made and bottled in the heart of California's famous vineyards. Yet surprisingly low priced. Try Roma vermouth soon, won't you? This is Agnes Morehead. It goes without saying that it has been a major thrill to appear again on suspense with sorry-wrong number. If you enjoyed it, I'm sure you won't want to miss next Thursday's Suspense Show, which Mr. Spear tells me will present the premiere of Lucille Fletcher's very newest suspense play. It is called The Furnished Floor, and in it you will hear Mildred Natwick and Don DeFore. Agnes Morehead appeared through the courtesy of Metro-Goldenmayer Studios and is one of the stars in their forthcoming production. Our vines have tender grapes. Next Thursday, you will hear Mildred Natwick and Don DeFore as stars of Suspense, radio's outstanding theater thrill. Resented by Roma Wines, R-O-M-A, made in California for enjoyment throughout the world. This is CBS, the Columbia Broadcasting System.


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