From around the globe, it's theCUBE with digital coverage of Dell Technologies World, digital experience brought to you by Dell Technologies. Hey, welcome back, everybody. Jeff Frick here with theCUBE. Welcome back to our ongoing coverage of Dell Technology World 2020, the digital experience not in person, like nothing this year, 2020, but the digital experience allows to do a lot of things that you couldn't do in person. And we're excited to have our next guest. He is Deepak Prasad, the Director of Product Management for Dell Technologies Cloud. Deepak, great to see you. Hello, Jeff, nice to meet you as well. You too. So let's back up like 10,000 square feet, because cloud came in with a big giant rage, I guess it's been a while now, with AWS and public cloud and people were putting their DevTest on there. And we've seen this explosion of public cloud and then we have hybrid cloud and multi-cloud. And then basically people figured out that not everything can go to a public cloud. A lot of stuff shouldn't. Some stuff's got to stay in data centers for all different reasons, but basically it's horses for courses. So we're a little ways into this. How are you guys at Dell really thinking about cloud and helping your customers think about what cloud is beyond kind of the hype? Well, that's a great question, Jeff. At Dell, we think of cloud really as an operating model and as an operating experience rather than a destination. So it's interesting that you bring up public cloud and private cloud, but we take a step back and think of what does that experience really represent? So if you think of what defines that cloud operating model, it's a democratization of technology, access of resources through APIs, through self-service portals, ability to pay as you go in a very simplified commerce experience and the agility of cloud, the promise of instant availability and infinite scalability. Now, if you look at the landscape around this, until now, that has only been delivered in a consistent way by public cloud vendors, which leads people to believe that really cloud is a destination, not an operating model. But we think that we are capable of bringing those experiences, those tenets of the cloud operating model through the on-premises experience and really taking location out of the conversation. So this really allows our customers to focus more on their workloads, the innovations they want to drive and then they can fit their requirements, their application requirements to the location where those resources are, regardless of having to worry about if this is public or private, they will get the same operating experience. They'll get the same scalability, the same simplified commerce, the same access to resources. Right. Well, let's talk about some of those things. Cause as you said, there's a lot of behaviors that are involved in cloud and cloud operating. Well, you know, one of the behaviors that I think gave the public cloud an early leg up was just simply provisioning, right? Simply, if somebody needs some capacity, they need some horsepower. Again, traditionally it would be test dev in the early days. You know, they didn't have to provision, they didn't have to put in an order with IT and wait for so long to get a box assigned to them or purchased or whatever, right? They just swipe the credit card and went. How have you kind of helped people have that kind of ease of use? Ease of, I don't know, ease of spin up, ease of creation, I don't know what the right verb is. Because I think that's a really core piece of what enabled early cloud adoption. No, absolutely, you're spot on. And that was a big part of it that if somebody needed resources instead of waiting for weeks and months, they could go and sign up for those resources and get almost instantaneous access. And we believe that what we're doing in this area is really transforming the business. Today we can deliver resources to customers in their data center in 14 days and really are aggressively looking to cut that down further. So what this really means is not just shipping resources in 14 days, but actually delivering a cloud experience in a customer's data center or a colo location, whatever location of their choice in 14 days. And making that available to the customers, not just through the traditional procurement process, but we're actually very proud to announce the cloud console, the Delta technology cloud console through which customers can in a self-service way order those resources and have it show up and be operational in their environment in 14 days. So we're really bringing that speed of cloud to the on-premise experience. So how does it actually work? Do you pre-ship some amount of capacity beyond what you believe is currently needed just to kind of forward cue if you will capacity? How does it work from both the implementation strategy in terms of the actual compute and storing capacity as well as on kind of the purchasing piece? Cause those are two kind of very different workflows. No, that's a good question. So for us, our strengths are really in supply chain management that allows us to build capabilities across the world in areas from where we can ship to customers almost on an on-demand basis. So as soon as we get an order that the customer needs a private cloud deployment in a certain location, we're able to mobilize those resources from those locations and have it instantiated in customer's environment. So it's really built to strengths over the years of optimizing supply chain, if you will, and just taking that to the next level. Okay. So we don't speak to customer environment. Yeah, no problem. I was going to say another great characteristics of cloud, right, is spinning up, which we hear about all the time, versus spinning down, right? The easiest example is always use if you're running some promotion. If you're a pizza hut, you're running a promotion for the Super Bowl, obviously write your demand for that thing is going to be huge. You want to spin up to be able to take advantage of all the people cash on their coupon. And then when the Super Bowl's over, you want to spin those resources down because you're not going to necessarily need that capacity. How do you guys accomplish that type of flexibility in your solution? So in our subscription model, we have different ways to address customer environment. So we allow customers to start very small and then grow the subscription as their requirements grow. And the key thing of our subscription, which is really unique is the ability to co-terminate. So for example, if a customer started off on a three-year subscription with resources for say 100 virtual machines and somewhere along the way, they needed to add resources for 50 more virtual machines. So they will pay for the 150 virtual machines, but that extra 50 virtual machines does not create an orphan or a child subscription. At the end of three years, everything terminates together. So it really give them flexibility with ability to start small and not have to worry about vendor lock-in. And now we started off with a sort of a reserved instance type of subscription model, but we're definitely bringing usage-based models as well, which allows even more flexibility with respect to speeding up and speeding down. Right. And then what are some of the real specific reasons that people go for this type of solution versus the public cloud? What are some of the real inherent advantages of doing this within my own infrastructure, my own data center, my own kind of virtual four walls, if you will. Yeah. We strongly believe that the decision should really be guided by workload requirements. There's certain workloads that work really well in an on-premises environment. For example, you could take virtual desktop environments, BDI. That works really well from a performance standpoint in an on-premise environment versus a public cloud environment. Similarly, there are other workloads. We're not public cloud deniers that are best suited for public cloud, but it really should be something that comes from understanding your application, understanding the latency requirements, understanding the data requirements for those applications, what are your egress issues or the profile of the workloads that you're trying to implement. That should really be the driving force in where the workload is placed. And then tell us a little bit about the partnership with VMware, because that's a huge asset that you have now, basically side-by-side, and you can leverage the technology as well as a lot of the assets that are in VMware. How has that changed the way you guys have taken the Dell platform to market? It really is a differentiating factor for us from a technology standpoint. It allows us to bring the best of both worlds, best of the hardware infrastructure, as well as the best of the cloud stack, the cloud software infrastructure together in one cohesive and well-developed package. So the Dell Technologies Cloud Platform from a technology standpoint is implemented with our VxRail appliances, which is a hyper-converge infrastructure, as well as VMware Cloud Foundation from a software standpoint. Now the code-developed and jointly engineered capabilities allow for a unique feature of VMware Cloud Foundation where it can do lifecycle management of the entire stack, both the hardware and the software, from a single interface. So it understands VxRails, it understands the different firmware levels and VxRail manager software versions, et cetera, and then it would automatically select what is the best and well-tested and supported software bundle that could be deployed without causing typical issues with version mismatches and trying to chase down different hardware compatibility matrices, et cetera, all of those that are limited. So it's an integrated lifecycle management experience. That's great. I don't want to go too far, sorry. I'm sorry, I have a little bit of a lag here. So I apologize. I was just going to say, you've been at this for a while, you're in product management. So you're really thinking about speeds and feeds and you're thinking about roadmap and futures. I wonder if you can share your perspective on this evolution from kind of this race to pure public cloud to this big discussion I think we had, Pat Gelsinger talking about a hybrid cloud back in VMware 2013. So then kind of this hybrid cloud and multi-cloud and really kind of this maturation of this space as we've progressed for a while now, probably 10 years. And the majority of our customers live in a multi-cloud world. They have resources that they consume from one or more hyper, sorry, public cloud vendors. And they have one or more on-premise vendors as well for their resources. And managing that complex environment across multiple providers with different skillset, different tools, different SLAs. While it sounds really interesting to have workload drive your deployment and place the workloads where they're best suited, it does prevent, it does present a challenge of managing a complex and getting even more complex by the day, multi-cloud environment. And that's where we think we have an advantage of based on some of the work that we're doing with the Dell Technologies Cloud Console to bring a true multi-cloud experience to our customers. Not one of the benefits of not being a public cloud provider is that we are agnostic to all public cloud providers. We are fully accepting that certain workloads need to live in those environments. And through our cloud console, we will make it easy for customers to manage not only their on-premises assets and on-premises cloud resources, but also cloud resources that reside in multiple public cloud vendors. That's good, yeah, because it helps, right? Because they've got stuff everywhere. It's like, you know, there is no Dell technology, right? There's a lot of people that work there. There's a lot of projects. There's a lot of, you know, kind of pieces to that puzzle. I wonder too, if you could share your perspective on kind of application modernization, right? That's always another big, you know, kind of topic. Should you take those little legacy apps and should you try to rebuild them in a more cloud-native way using containers and all this flexibility and deploy them? Or, you know, which one should you just leave alone, right? They're running fine. They've been running fine for a while. They've got some basic core functionality that maybe do or don't need to kind of modernize, if you will. And maybe those resources should be spent on building, you know, new applications and new kind of areas of competitive differentiation. When you're working with your clients, how do you tell them to think about app modernization? You know, we look at it from a business requirements standpoint of how and what end goals are customers trying to achieve through that application. And in some cases, you know, and you covered the spectrum right there, some cases modernization just means swapping out the hardware and putting that application on a more modern, more powerful hardware.  And in between those two extremities, there's, you know, virtualization, there's refactoring, there's containerization and microservices-based implementation. But it comes down to understanding what that application is meant to deliver for who and what business requirements and business objectives it fulfills. And that's how we use as a guiding principle on how to position application modernization to customers. All right, that's super helpful because I'm sure that's a big topic.  You should probably just leave them alone, they're running just fine, let them do their thing. All right. Yeah. I'm sorry. No, it's just interesting, I was on the station with the customer just earlier today where they have a portion of their infrastructure, some applications that they actually wanted to leave alone and just change out the underlying hardware. But there are other applications where they really want to adopt containerization and refactor those, rewrite those applications so that they can have more scalability and more flexibility around that. So it really is determined by the needs. Yeah. So last question. Dell Tech World this year was a digital experience like all the other shows that we've seen here in 2020. Just, but it's a huge event, right? A big, big show. And we're excited to be back to cover it again. But I'm curious if there's some special announcements within such a big show. Sometimes things get lost a little bit here and there. But any special announcements do you want to make sure that get highlighted that people may have missed within this kind of sea of content over the last several days? Two major things that I'm very excited to share with you. One is Dell Technologies Cloud Platform. We are actually discussing and talking about Dell Technologies Cloud Platform in the concept of instant capacity blocks. So in the past, we talked about it with respect to nodes of Dell Technologies Cloud Platform. You can have so many nodes in it to power your on-premises cloud resources, but really have changed the conversation and look into how cloud customers are consuming those resources. And we really want to drive focus to that and introduce the concept of instance capacity blocks. Instances are, think of it as a workload profile, a CPU and memory put together. And then in different combinations in a predefined way to address different workload needs. So this really changes the conversation for our customers, but they don't have to worry about designing or specking out the hardware platforms, but really understand how many resources they need, how much processing power, how much memory, how much storage they need. And they define their requirements to us in those terms and we will deliver those instance capacity blocks to them in their data center. So behind the scenes is built by best in class hardware from VxRails and best in class software from VMware, but it's really delivered in terms of instance capacity blocks. The second interesting thing that I wanna share with you and I've prefaced it a few times is Dell Technologies Cloud Console. We're building this single pane of glass to manage our customer's entire journey from on-premises to multi-cloud hybrid cloud with consistency of how you can discover services, how you can order services and how you can grow and manage your footprint. So those are a couple things from a Dell Technologies endpoint that we're really excited to share with people. Well, congratulations. I know you've been busting your tail for quite a while on these types of projects and it's nice to be able to finally release them out to the world. Well, it's my pleasure. All right. Thank you very much. Well, thank you for stopping by. Again, congratulations and we'll continue the ongoing coverage of Dell Technology World 2020, the digital experience. I'm Jeff Frick. He's Dipak Prasad. You're watching theCUBE. See you next time. Thanks for watching.


Do you want me to turn it on now or wait till I go? Well, it's because they turn it on now and make sure it's okay. Here we go. Life is good. Okay, I think we'll get started. It's my pleasure to introduce today's seminar speaker, Professor Brian Freeman from the University of Illinois, Urbana-Champaign. Brian and I met over Zoom, actually, when I gave a seminar at University of Illinois during the COVID pandemic. And while we work in different areas, I found them really fun to talk to you. Because he seems so curious about so many topics, which actually makes sense because chaperones are involved in virtually every aspect of cellular function. So you have to be curious about a lot of things, I think, to do good work in that area. Brian got his bachelor's in microbiology from University of Michigan Ann Arbor and his PhD in biochemistry and biophysics at Northwestern. So he is a real midwesterner. But after a successful postdoctoral stint with Keith Yamamoto at UCSF, he started his own independent career at University of Illinois in 2002, where he's now a professor in the Department of Cell and Developmental Biology. He also has affiliate appointments in both the cancer and genome biology institutes, attesting to his breadth of curiosity and his institutional commitment. And he has some very interesting ideas about promoting DEI initiatives at his institutions. And so, again, the breadth of thinking about different things and incorporating service into his academic life is impressive. He's the recipient of several honors during his career, most recently being elected a fellow of the American Association for the Advancement of Science, although the honors I was most envious of were the single-digit scores on multiple NIH grants, which I have never seen. So Brian's long, for myself, I mean, I've seen him for other people because I've served on study section. Brian's longstanding interests are in the conserved mechanisms by which chaperones guide the dynamic protein environment in our cells with an emphasis on central nuclear processes. And today, though, he's going to discuss recent progress on defining the cellular substrates of HSP90s. Awesome. Thank you so much, Catherine. It's been an absolute pleasure today. It was a wonderful meeting, Catherine, by Zoom and really getting to know her in person afterwards. And I've had just a terrific day talking with people. I think, if anything, the meetings were all too short because, as she said, I do like to chat and maybe talk a wee bit too much. That being said, thank you for the lovely introduction, Catherine. You're looking at, I will talk briefly about it today. And I put it up there because, honestly, I love this movie. It's something that keeps me thinking about how stuff works. And what you're looking at, I'm sure most of you realize this is budding yeast. This is mom down here. This is a daughter cell that forms. The nucleus upon budding is a closed nuclear environment. We have a cherry marking the nuclear envelope and the outer envelope of mom and daughter. And the green dot is actually one site of the DNA that's labeled with a GFP-LAC fusion. And what really fascinates me about it and one of the areas that my group studies, and I will talk a little bit about today, is watch when it gets over to the daughter cell and then suddenly it sort of zips all over the nucleus. And that's when the genome's being reorganized. And we know so little about how that process works. And my work on chaperones got us into that area. But I would have to admit, if someone says the word molecular chaperone to me, I don't think anything about the nucleus whatsoever. That's just not where my mind drifts. My mind really goes over. That got bright in a hurry. And the classic sort of chaperone biology, and that is really, I see Betty back there just did beautiful work along with Bill Welch showing that HSP-70 binds the nascent chains as they come out of the ribosome. And it's not that they guide folding, but they provide an environment, a friendly environment for nascent chains to fold so that those nascent chains as they grow won't aggregate with other nascent chains. And that's a beautiful function for certainly HSP-70. And of course, if a protein can't fold to it, and we'll talk more about this, the seeming native state, HSP-70 helps deliver that to degradation machinery such as the proteasome where Aaron Chekinover has spent a lot of time showing that that's another nice chaperone role. And of course, even if a protein does fold to a seeming native state, there's still chaperone usage. Proteins typically don't work as a single subunit. They work in protein complexes. Many of the chaperones contribute here, including GroYale or TREC homologs. And then there's the area that my group likes to spend a lot of time on. And we started with supporting metastable proteins where HSP-90 has a large role to help create an activatable state with sort of beginning hypothesis. 90 doesn't do this by itself, but it does it with a whole slew of co-chaperones. But here we already have four wildly different roles for chaperones in the cell. And that sort of starts to beg the question of like, all right, well how are chaperones doing folding, degradation, metastable proteins, complex assembly? And I think to get an idea of how is the chaperone functioning in the cell, I always go back to sort of an old school principle of in protein folding. As the nascent chain comes down its energy landscape, it often will encounter unproductive folding intermediates. And that is where the majority of us, including myself, think about chaperones. That chaperone binding or activity gets us over these energy barriers and leads us to my favorite misnomer, the native protein. And I say misnomer because proteins don't have a singular native state. They always have multiple native states. And those multiple native states are usually indicative of the work a protein does inside a cell. And so we think of there being energy barriers again between those different native states when they're trying to accomplish work. And you might be thinking like, what exactly are you talking about? And my group, when I started my own independent group, I started with what I thought was the easiest one to sort of get a handle on. And that is if we think about transcription factors or DNA binding proteins, we don't think of them as being non-native whether they're free or bound to DNA. These are both native proteins. And yet there's a clear energy barrier between the free and bound state. If you take a purified transcription factor, bind it to DNA, this complex has a half-life of typically on the order of two hours. There's a clear energy barrier, and I say that because inside a live cell, that complex has a half-life of about a half a second. So there's clearly a difference between in vitro and in vivo. Workthrough in my group demonstrated that a small molecular chaperone called P23, it also happens to be a co-chaperone for 90, could accomplish the change in those states. And what you're looking at is DNA anisotropy experiment where transcription factor called MCM1 is bound to the DNA. If we challenge this fluorescent label complex with a non-fluorescent DNA oligo, this thing happens in the anisotropy value, and that's because it's a very happily stable complex, not dissociating at all over an hour period. And yet if we challenge this P23, we quickly drop down to the anisotropy value of the naked DNA. And that drops down sort of on the kinetic values of what we see in live cells. And it turns out that P23 itself regulates about 70% of all yeast transcription factors, which is a very sort of chaperone-esque role inside a cell. And I say that chaperones are generally doing very broad activities inside a cell. All right, but what about HSP90, which is the topic of today? So 90 was originally discovered because it forms stable complexes of metastable proteins, typically signaling proteins, be it kinases or aposteroid receptors. A lot of beautiful work from Dave Toffbill, Pratt, early work, led to a model where HSP90 will bind two clients in conjunction with recent work from Johannes Buchner's group, showing that 70 will deliver those clients along with various co-chaperones to 90. With ATP and additional co-chaperones, we will eventually carry out late-folding steps to lead to an activatable state of those clients. And then when we have client activation, say, hormone addition, it will destabilize this complex and it will return to its cycle. For 90 to do this, it doesn't work alone. I've shown a few co-chaperones. In budding yeast, there are over a dozen co-chaperones. In human cells, there's over 20. It's a very complex system. And what was more intriguing to me is that HSP90 itself is a highly abundant protein. It's about 2% of your protein mass, way outnumbering the number of metastable proteins in your cell. So what are we doing with all that extra HSP90? And we didn't really have a good grip for that. We knew that 90 had a large interactome. So there are 1,400 out of 6,000 open reading frames in yeast that are linked to HSP90. But that linkage wasn't what you would think for a chaperone. It wasn't a physical linkage. We didn't know that these were physically associating. Most of these interactions were based on just genetic interactions between the various open reading frames and 90 inhibition. And they came out of three primary papers all really focusing on the genetic or chemical interactions between 90 and various open reading frames in budding yeast. Now, the genetic interactions have been useful for identifying what does 90 physically interact with? So my group has certainly taken a large advantage of trying to figure out what does 90 do in the cell? What does all that 90 do? What are its clients? One of the things that we focus on were telomere biology, where telomere's linear ends of chromosomes and eukaryotes have to be capped to protect them from degradation. 90 will actually attack this cap, much like P23 attacking transcription factors and dissociate it. But 90 does additional things at the telomere. It actually helps deliver telomerase, the enzyme that will extend the end, and facilitates elongation of that DNA. So it's doing a fair number of things at that DNA. And a lot of it being transient interactions. 90 will work with other things. It will work with chromatory molars. But here, the chaperones are doing different things. Before I invented P23, it kicks things off DNA. Here P23 doesn't kick it off, but actually greases this machine to slide the nucleosome along. And then 90 will then attack risk, and this is from the risk chromatory molar, at the DNA binding subunit to kick it off the DNA. So we were able to use the genetic interactions to establish what does 90 physically interact with, but those two quick little examples was five to ten years of work. We have 1400 things to truck through. Did we really want to keep taking that approach? The answer was obviously no. That was just, it was non-tenable. How can we get at what 90 physically interacts with? Because if we looked of 90 interacting with the capping complex or the chromatory molaring complexes, we wouldn't find it as stably associated. When it does what it does, it does it in a very transient way. So we needed a way to stabilize what 90 physically interacts with. And I was once again inspired by Betty, Craig. So Betty had used a non-natural amino acid called BPA to look at HSP70's interaction with its partners in live cells. And I thought, well, that, I like that cross-linker. Typically I'm not a big fan of cross-linkers, especially with chaperones. I always worry about, like, once I break them open, I'll capture artificial interactions. But here, BPA, we're going to integrate it into the HSP90 structure. We're going to activate it with UV light, and that will create our cross-linker. And we only have the cross-linker when we are activating it with the UV light. And so we're controlling when and where we're going to have a cross-linker. And we took advantage of the crystal structures, our crystal atomic information from various groups, including Dave Agar and Lawrence Pearls, to pick where we're going to place the different moieties of BPA within the HSP90 structure. 90, this is a dimer of 90. Here's one protomer. Here's the other one. N-terminal domain, middle domain and C-domain. And we did try a lot of other spots, but they seemed to interfere with function of 90. These seemed benign, and they seemed like really great spots for us to utilize. And one of the things that drove where we picked, what locations we picked, was we knew they were close to sites of protein-protein interactions, be it co-shaperones or the really limited amount of client interactions of where 90 would bind to a client. The CDK4 kinase was one of the few that we had information on. So we thought we could then take advantage of this BPA screen, incorporate mass spec to answer the following four questions. We could identify, first off, what are all the proteins HSP90 physically interacts and build out a really good solid physical interactome? We could answer a really seemingly simple question that had not been answered. How does HSP90 interact with so many different clients? And that's why we actually were covering the three different surfaces. We didn't know where all, what HSP90 used. There was some good clues that the middle domain was going to be used, but we needed to study it in further depth. And then also I will say I naively thought we'll be able to use this BPA as a tag to identify where 90 interacts with a client. Because BPA is a non-natural amino acid, I thought that would just fall right out of the mass spec data and you'll see later we have some success with it, but it certainly wasn't to the level I was hoping. And then finally, of course, we wanted to discover new pathways that were governed by HSP90 by knowing what it interacts with. We could then figure out what it regulates. And so we started all this with a proof of principle screen with Matthias Meyer at Heidelberg University to figure out exactly what were the samples that we wanted to test, how did we want to go about it. And then eventually we did a large scale screen with Alex Campos at Sanford Burnham Presbyterian. And what we ended up deciding to do is take a wild type HSP90. We do grow it in the presence of BPA, but this doesn't have any integration of BPA. And this would allow us to isolate those stable, the meta-stable proteins that stable interact with HSP90. And then once it contained BPA, we would capture both the stable interactions and the transient interactions. And then, of course, importantly, all these are histagged. We also use a non-histag to screen out what were sort of our background hits on our beads and were interactors. Through this approach, we were able to identify about 1,100 hits roughly half of those are dependent upon BPA. When I say dependent, we either have to have BPA there or we at least enrich over wild type levels. And if you read around the circle, you'll see HSP90 is really interacting. Anything that's going on inside a cell, we're hitting. And unfortunately with my level of curiosity, I like to float all over the cell and try to address where is 90 working within the cell. One of the things I was really happy to see is that it was about split half and half of clients in the cytoplasm and the nucleus. And I say that because it wasn't too long ago that chaperones weren't even believed to have a presence in the nucleus. All right. So first things first, how does 90 interact with 1,100 different proteins? Client interaction surfaces that were known are shown here when we started this work. Tau, glucocorticoid receptor ligand binding domain and CDK4 kinase. The contact residues with HSP90 on one of the protomers primarily in the middle domain, a little bit for Tau off in the end domain and a few client interactions down in the C domain. We thought, well, what we chose should hit most of those surfaces. But the prevalence really seemed to be on the middle domain. And when we look and ask where do we, what residues are capturing what clients, all the surfaces seem to be used. There is a small bias for the middle domains where they have about 350 hits, but the end domain is 225 and the C domains are around 200. And notice, you know, in this quasi-Venn diagram there's a lot of sort of co-usage of the different surfaces. And I think this is how 90 achieves such a broad binding capacity with so many different clients. It's basically a combinatorial use of binding surfaces. We have clients that are dependent only on the end domain, middle domain, or C domain. We have clients that will use both the end and middle, and C, or even the end and the C. We have some clients that use all three domains. And then, of course, this is all within a single protomer. We have ones that will cross the protomers. And that is how you have one protein that is able to reach out, or at least in part reach out to almost 20% of all the proteins inside a cell. All right. Now, let me move on to what I thought was going to be a simple straightforward question. We have this non-naturamino acid. It's a chemical cross-link to its client. This is going to get us, what is 90 recognizing on a client to make it a client?  They recognize short hydrophobic peptides. Yes, for HSB 70. And that's the only chaperone we actually know the rules for. We had no idea how 90 was recognizing a client, and we thought we could get there with that. And I totally convinced my student that this was going to be easy and doable. These were our non-naturamino tags coming out of 90 where we put the different BPAs. And when she started looking, nothing. And we had to search and go through so many different computational approaches until she hit upon MIROX. And John B. Coley is a grad student doing this, and she just dug in until she found a program that could at least recognize the cross-link peptides. And that got us basically the needle in the haystack because mass spec data, there are just hundreds of thousands of peptides. And finding the ones that represented cross-link peptides was really difficult, but it didn't get us... We had a lot of false positives in that, a lot of sort of nonsense information out of that. She figured out we had to add two more criteria. And then we could finally, with confidence, identify BPA-containing cross-link peptides. STI-1 is a classic co-shapron for HSP-90, and by adding in these two, we could read across the sequence in STI-1 across to an X that's BPA, and then more sequence density from HSP-90 itself. STI was a really nice one to hit because we knew it's one of the best well-behaved co-shaprons in terms of forming stable interactions with 90. I mentioned that 90 kicks risk chromatary myelin complex off the DNA. It does so functionally through risk-3 and we capture risk-3. So we're gaining sort of confidence like, okay, these hits are actually real. We also hit B&I-1. B&I-1 regulates actin dynamics. I'll get to that story in a little bit. It's one of the clues that help us how chromosomes are moved in interface cells. To really sort of drive it home for us, were we hitting or were we getting valid hits? If we look at where we placed in the cross-linkers in STI-1, I mentioned as being a really good co-shapron, when we dock it on with the known sort of atomic structural information, wherever the yellow is where we get a cross-link in the structure of the co-shapron, and this is the red is where we have a BPA moiety. And so these two moieties under here cross-link to those two, there wasn't any structural information for STI at the tail, but we did capture the tail with a BPA there. And so all three hits made sense where the co-shaprons should interact. And we hit a lot of different co-shaprons and chaperones, really convincing us and hitting some chaperones we didn't really expect. We actually, CCT is the trick, Gro-Yell homolog and Budding East. We didn't expect to hit them all. 90 really does look like it's a central hub for chaperone interactions. And when we dock any of the ones where we had that information, we could always make good sense of that's why we captured them, where we captured them. And so our confidence that this looks like this is working and capturing physiologically relevant things was making us happy. Using Javi's sort of combination of Mirox and her additional criteria, we have 1100 hits, we didn't get all the BPA peptides, but we did get out to 180, well over 180. And that I felt was enough for us to start figuring out at least some of the rules for how HSP90 might interact with either co-shaperone, chaperones, or even clients. 24 of those hits are coming from co-shaperones and chaperones. When we ran for sequence conservations or any commonality, there wasn't any. So it wasn't simple like HSP70, it was recognizing a short hydrophobic peptide.   So there's a preponderance of client interactions at intrinsically disordered regions. And some of that is shown here because I have to say the field in intrinsically disordered regions can be a little rough. It's still a developing field. If you run, there are many different software prediction programs for where IDRs are found. We were fairly dissatisfied by using one. So we typically run about eight. And this is where we got a mass spec hit for not nine. And all of them are predicting it intrinsically disordered region. The nice part about not nine is we actually have crystal structural information. And sure enough, it's a disordered region. And usually what we see is 90 is right next to not just an intrinsically disordered region, but it's usually right next to a structural motif like an alpha helix or a beta sheet. And we see that for wherever we had structural information, that is usually what we see except for PRI1 where that is truly just within an IDR itself. All right. So that was actually, to us, fairly huge because it's telling us how 90 is now recognizing this client. And for us, it was making a nice sort of contribution to the chaperone biology. 90 is typically thought of as a protein that interacts with seemingly native proteins. And the question always was, well, how's that happening? If it's recognizing IDRs, that's a really nice substrate for chaperones to bind to. And also may explain why so many proteins contain intrinsically disordered regions. Prediction of software usually runs about 30% of all proteins have sizeable IDRs. So are these sort of co-evolving so 90 can regulate those native proteins and sort of feedback on each other? Moving past that, we wanted to really drive home our confidence and our hits meaningful. And right away, I was pretty pleased that about two years ago, we published a paper using DNA seek to show that DNA footprints when you inhibit 90 are radically modified. But we didn't have a physical interaction with, no one had shown physical interactions with all these transcription factors. Our network of transcription factors is rather large. Almost all of the hits in the transcription area are simply DNA binding proteins. The work we had done before is within a DNA hypersensitivity site. If we look at the footprints in wild type cells, we see lots and lots of footprints. Some of them have consensus motifs. If we inactivate 90 for just 15 minutes, it's a radical change in those footprints. We use that information to look across a genome at all footprints. And at the 15 minute time point, we see typically a dramatic loss in the number of footprints. And that changes when we get out to six hours. It's a totally different sort of event inside the cell. And what we see is later on, we're basically just losing those transcription factors. And some of that came out in the biochemistry. If we look at steady state levels of transcription factors, late, there's a loss of falling inactivation of HSP90. In this case, we're using a temperature-sensitive allele of HSP90 called G170D, isolated by Sue Lindquist lab. Or if we use a small molecule inhibitor called radicicol at the six-hour time point, there's a loss of any transcription factors. But at 15 minutes, there isn't any loss. And yet we lost DNA footprints. And so it was a bit of a quagmire, like, well, they're not being lost, yet they're not binding DNA. And it turns out, 90s require for a late folding step for many transcription factors. Within the transcription field, people often just go with, well, I can't express my transcription factor in E. coli because the recombinant proteins are notoriously inactive. We went ahead and purified a few, RAP1, CBF, ABF, and MET31 in E. coli. And when it's just a transcription factor, there isn't any DNA binding activity. Because that transcription factor has never seen a eukaryotic version of 90. If we simply expose it to yeast HSP90, we gain DNA binding activity. And so we think that's the issue. 90 was required to physically fold these proteins. But if we look and ask, can we see 90 associated with any of these, we never could. And the BPA screen was really nice for that because it captured all these transient interactions and told us that 90 is actually folding all these proteins. Not all transcription factors require 90. RAP1 was a really nice negative control. All right. A couple of years ago, we published a really nice paper, I think, anyways, on chromosome motion. And we didn't mention in that work what really facilitated us to delineate the chromosome motion pathway was this BPA interactome. That was classically a really difficult and challenging area. People were trying to figure out how do chromosomes move within interface cells. We had a fairly large contingent of players thought to be involved in chromosome motion. And chromosomes, of course, I'm sure most of you already know, have a really high level organization inside of cells. They are organized not only by chromosome territories where each chromosome is placed in the same spot after every cell division. And they're also organized, in this case this is nomenclature related to high C experiments of compartment A and B. You can think of compartment A and B as heterochromatin and uchromatin. Always well positioned after every cell division going back to the same location. And the question became how does that happen? How is such unique precision achieved? And one of the things that's, you know, to bear in mind are chromosomes are huge and heavy polymers within the cell. So it's not a little task to position these long polymers. For us to address does 90 have a role in chromosome reorganization? We took advantage of a system developed by Jason Brinkner at Northwestern University. He started this when he was a postdoc with Peter Walter. He's using the eno-1 gene locus that he can activate just simply starving cells for anocetal. When you starve for anocetal, you go from the center where you're inactive to the nuclear periphery really associated with the nuclear pore where you are then active. And he's incorporated a lac-i response element array where lac-i fusigee-f-p will bind. This is where that movie was I came up with in the beginning. And we can starve and move the lac-i over to the nuclear periphery. And it always moves to the same nuclear pore, which is really fascinating. It's a really precise motion mechanism. The downside of this system is, you know, yeast is a relatively small nucleus. This is a rather large spot. So the relative enrichment never looks great, but it's a very consistent two-fold enrichment. It's enough to follow. And one of the nice parts of this system is if we impair different chaperones. Here we're impairing P23. It's that chaperone that kicks transcription factors off the DNA. We don't get any movement. We saw the same thing with HSP90, except now we're inhibiting with a small molecule inhibitor. We don't get any movement when the inhibitor is there. But if we take those live cells and simply wash it out, it moves. So 90 is actively contributing to the motion of that DNA locus. I'm not going to go too deep into this. This was a rather complicated story. And I'm going to just tell a couple of sort of points highlighting 90 and highlighting what we use the sort of interactome to dissect out how this pathway works. But the E1 locus has different transcription factors, you know, 2 and 4, that will recruit in chromatary molars and will recruit in RNA polymerase. And then putt3 is a transcription factor. Jason had shown binds a very unique DNA element called the DNA zip code. And that is the element that tells this locus to go from the middle of nucleus to that specific nuclear pore. We know, you know, 2-4 binds to these and putt3 binds to, well, you know, 2-4 recruits in chromatary molars. This was information that was known before. You know, 80 is going to have a really important role in motion. And then putt3, we found recruits in a myosin motor. And that myosin motor is one of the substrates for HSP90, and so is you know, 80. And both of those fell out of our interactome. HSP90 is going to work with both P23 and another Coshack around for she4 to recruit in our nucleate actin at this site. And the myosin's then going to join the actin. It's going to be held there in part by the chromatary molar. And what 90 does to facilitate this pathway, if we do what's called an actin spin-down assay, where we're just adding this eno80 interact with actin, and it does, it will spin down with actin into the pellet, because eno80 contains actin-related protein subunits that interact, that are known to interact with actin, although the complex itself wasn't known before this. 90 really directs it towards whether it's interacting or not interacting with actin. And it changes its behavior quite a bit, along with P23 will contribute to get a very different behavior pattern for eno80 interacting with actin. And so that was one of the bits of this story and how we were able to show it. What I found really intriguing is it's not just a stable interaction. And we found that out by going to a single molecule approach where we were trying to recapitulate the chromosome motion with purified components. And so what you're looking at here, we did single molecule work with Paul Selbin's group. Actin is in purple and in green are nucleosomes. In this movie, it's just the mixture of those two. And not surprisingly, you don't see nucleosomes interacting with actin. We didn't really expect to, it was really more of a negative control. If we add eno80 with labeled histone, there's a little bit more interaction, but not much. If we then add in 90 to that same one, now you see a lot of interaction of that labeled nucleosome bound to the remodeler, but it's really dynamic. It's constantly on and off, but it's always returning to the actin. This we think will become a very important characteristic to the chromosome motion. And I'll talk about that in just a sec. So that's where 90's role with eno80 is coming into play to favor interactions with actin. The other one is myO3. We would have never really hit upon myO3 if we hadn't already isolated it in our BPA interactome. MyO3 is a myosin motor. It's to move cargo along actin. In this assay, this is called an actin gliding assay.  If the motor's moving, the actin will slide across that glass surface, which is why it's called an actin gliding assay. And we isolated myosin from yeast. We put in the actin gliding assay and we were severely disappointed. Nothing happened. So it seemed like we just destroyed our myosin motor potentially, but if we put in 90 and she4, the motor's perfectly fine. It's just always actively dependent upon 90 being there. And we think that is why we hit myosin. And the way this system works is we have short actin polymers, and I'm not showing that data to you today, but it's a highly dynamic, polymerizing, depolymerizing actin network in the nucleus. It's short actin polymers. That transient interaction of 80 with the actin is important to move along that actin polymer as the myosin power stroke goes, pulling it over to that nuclear pore. What we're yet to identify is how that locus knows which pore to go to and that stuff that we're working on now. So those are things that either were published before we did the network, things that facilitated us publishing work, but that's already out. I wanted to tell you guys something new in terms of what 90 is doing. And the one I'm going to focus on today is in translation. 70 has a big role with ribosomes, nascent chains. It was a known nascent chain binding protein, and the data's not out there because it was all negative data, but people looked hard. Does 90 have a role with nascent chains? It doesn't. But it does have seemingly important role with the translating ribosome. And that's where I'd like to go. There was a few publications that said 90 had a role and with the elongating ribosome, I never like to chase what somebody else has done, although I know there's a lot of important things in there for validation. I wanted to chase something we didn't know anything about. And a lot of our hits were for initiation factors. We didn't know anything about 90's role with initiating ribosome, so I was excited. The student got excited and we asked, okay, what goes on when a ribosome initiates? Why might we get hits there? A lot of things go on. There's a lot of sort of reasons for protein. Protein protein dynamics, protein RNA dynamics, but we have the 40S that's going to form primarily with what's called the multi-factor complex. And this is going to then interact with the RNA capping proteins to form an initial complex. And then this structure is going to move along the RNA until we get to an AUG. The multi-factor complex is particularly important for finding that AUG. Once we get to the AUG, we'll have some more protein remodeling events. We'll have the 60S will join, we'll have the 80S, and off we go for elongation. All right, so there's a lot going on. What got us excited is that our BPA Interactum had a lot of hits within these complexes, and I thought, okay, cool. This looks good. Let's move forward sort of in a cautious way and ask, well, is 90 associated with the 40S or the monosomes? And so we just did your sort of standard sucrose gradient as Betty would have predicted. 70 is found both with all the 4060, the monosome, and the polysome. HSP90 is primarily, though, with the 40S, a little bit with 60 monosome, and then it teeters off pretty quickly. But it's there. And that gave us some, you know, okay, let's keep going. Let's see, is there a role and an impact of 90 on finding the AUG? We took sort of a classic approach where we put in plazins that are going to express runeloluciferase with a canonical AUG or non-canonical start codon with Fireflyuciferase. So this will get barely used in wild-type cells. But when we inactivate 90, we're going to use a G170T temperature-sensitive allele and shift to 37 degrees. And inactivate 90 and just ask, how does the usage of that non-canonical go up or down? It goes up. So we start that ribosome cares less and less about starting at a proper AUG. Great. All right, let's move forward into, you know, a more high-throughput ribosome profiling approach. And because there's a lot of other non-canonical start sites that could be used. So we joined forces with Natalia as a postdoc with Judith Freeman group. And we did the ribosome profiling. They did the bioinformatics. The first time in my career, usually we do the bioinformatics. Ribosome profiling was a whole new nut for us. That was not so easy to crack, especially at the first question we wanted to ask. What is the ribosome profiling over the AUG? So if we do a meta-analysis just in wild-type cells, here's the 5' UTR. All the peaks are where the ribosome is pausing. And of course you have the highest occupancy over the AUG. And very nicely, when we do G170D, after 15 minutes we lose that peak. And so when you inactivate 90, the ribosome really doesn't care so much anymore about finding an AUG. It's there on the RNA. It will lock on to the RNA and it will start making peptides. And we thought, well, who are the targets that are sort of contributing to this loss of finding the AUG? The multifactor complex is one that I had mentioned. This is associated with the 4DS and it's important for finding the AUG. Jami's going to use a sucrose cushion to look at monosomes and the 4D and 6DS. What components are there? And in G170D, both without temperature rising or with the temperature rising, peer T1, which is EIF3B, homolog in budding yeast, is always clipped. So it's not even dependent. And I have to say G170D is not the happiest mutant. That non-happy mutant already has peer T1 clipped. And if we inhibit it in a different way with a small molecule inhibitor, we actually just see degradation of peer T1. And so we think that's one of the contributors to why it's no longer caring so much about finding a start site. But of course, there begs the question of, well, is translation even happening when you inhibit 90? So we label nascent chains. And much to our surprise, 15 minutes at 37 degrees where G170D gets inactivated, yeah, we're making nascent chains. If anything, we're making more nascent chains. We looked in our ribosome profiling data and I'll take you a bit through this. This is the proper frame where you see in a heat map highest level of occupancy and wild type. These are read lengths. The ribosome does have different size footprints along that RNA. They can tell you a little bit about what's going on. But for in the coding region, in general, wild type is in frame as expected. And in G170D, after just 15 minutes, it's no longer in frame. That's across the entire coding region. It doesn't matter what footprint it is. And so that told us, oh, this is a significant problem that may actually correlate to some old clinical data. And it's not even terribly old clinical data. About five years ago, five to 10 years ago, there were 60 clinical trials targeting HSP90. Those clinical trials all failed with the same sort of phenotype. All the patients exhibited a heat shock response. You can't inhibit a heat shock protein if you're making more heat shock protein, which explains why it failed. But it wasn't quite clear why it failed. At the time of that, there was one paper that suggested HSP90 kept heat shock factor 1. It's the main transcription factor for delivering a heat shock response to cells in a quiescent state. And that heat stress would cause release and HSF would go to the nucleus and up-activate genes. And so by inhibiting 90, they thought, well, maybe that's why we get a heat shock response. But the funny part is, no one really believed that paper. And I thought, well, is our data telling us why we would get a heat shock response? If we're making nonsense peptides, that essentially would mock a heat shock response. If that's true, then we should be able to sort of tweeze that out. And we took a shot at tweezing that out with the following experiment. Where we used cyclohexamide in budding yeast as a way to inhibit translation. And we're doing heat shocks after 15, 30, or 60 minutes. These are our controls. Heat shock, we're producing a lot of the yeast HSB70, allele, SSA4. If we treat with cyclohexamide, the only thing we do is exacerbate that heat shock response. Not surprising. You want to produce HSB70 to turn off the heat shock response. We're just keeping it on because we can't translate anything. The stress response can be brought on by more than just heat stress. You can add a proline analog, azide. And azide will get a heat shock response, but since it's a proline analog, when you add cyclohexamide, you don't get a heat shock response because you never incorporate that proline analog, so there's no sort of mal-folded proteins to get you a heat shock response. If we do that same thing with radicicol, we see the same thing. It's very translation-dependent. If we inhibit translation, we don't get a heat shock response. And we now think we're in a good position to sort of go back and ask, oh, in those clinical trials that failed, is there a way we can increase the fidelity of the ribosome for its AUG, and can we return to those clinical trials? All right. And I'd like to thank John B. Coley, the grad student who did the majority of the work. She's carried out all the BPA. She's carried out the translation work. I didn't have a chance to talk about Audrey's work. She looks at the role of the nucleoskeleton and genome organization. Anna Mankiewicz is another student who looks at the role of 90 with tail anchor proteins, where we think 90 actually contributes to insertion of tail anchor proteins in the membrane. Nietzsche does aging studies. Ancien Zlada did the former work on chromosomal reorganization or the transcription network. Former students that contributed to various areas of all of his work. Alex has been just a great collaborator in the mass spec work. Bill Breyer was our collaborator with Acton. Judith is with the ribosome profile and Matias helped us set up the right conditions for carrying out the BPA screen. Craig is a long-term collaborator on chromatory modeling, and the Brinkner's helped us immensely with one relocalization. And I need to of course thank you guys for your patience during my talk. Thank you kindly. Thank you. Can you hear me? Yeah. So I'll open it up for questions. Does anyone have any questions? Okay. So you implied that the effect on translation was due to direct interactions between HSP90 and the initiation factors rather than like an indirect effect with like the other 1200 proteins about that also interact with HSP90 and I was wondering if you've is there a way to disentangle that issue or even have you tried looking at initiation from an iris to see if there are changes for CAP independent translation? So we're now moving towards a more sort of we're certainly moving towards an in vitro system to get at that because it's always a concern for any of the chaperone work that we do we typically try to take it all the way to a purified system I know I won't be able to do that here but at least take it to at least like a retic system and ask where we're not going to have so many sort of competing influences. One of the things that sort of brings a little bit of doubt at least in my mind that it's not some indirect effect is we're inactivating after just it's a 15 minute heat treatment takes 5 minutes and activates a 90 so whatever is happening is going to happen within 10 minutes that certainly won't rule out kinases it's still one of the possibilities but that's it's the best I think that we have right now and I will feel better about it and hopefully you will too once we move to an in vitro system and ask okay can we see the same things? Does the oxygen system work with the HSP90 or is it too concentrated to decorate it? I think the oxygen system probably could work but the small molecule inhibitors are good specific inhibitors and when we can use different 90 inhibitors the one that we are using does have at least two other weak targets but we can use C-terminal inhibitors and also sort of tweeze it out that way the temperature is sensitive to 90 and the small molecule inhibitors do attack it in two different ways and say are we still seeing that same impact? Thanks can I ask you to elaborate a little bit more on HSP90 interacting with IDRs IDRs are all the rage now forming these liquid phase separation things in vitro or any in vivo system that you will examine whether HSP90 modulates the type of liquid-liquid phase separation that you see? I don't know of anyone who's looked at 90 per se because there wasn't any real reason to look before but probably half a dozen of other chaperones have been looked at and most of them do modulate that phase separation do they promote more or do they promote more LLPSs usually it's less so that's I would say the general take on it okay can I follow up on the IDR question so some IDRs are enriched in certain types chemical flavors of residues and I'm wondering if when you do your analysis do you see any no selectivity of sequence that was the thing we focused on the most in the beginning nothing came out of it even if we sort of looked at it in different ways of just categorizing and in classes of amino acid and there's no sequence specificity to it the only thing that we found was the presence of the IDR for the clients okay question about the I-080 and the interaction of the nucleus cells with the actin that was it molecule stuff so the I-080 I think it has some actin related protein subunits in it and I'm just wondering like you know which subunits of I-080 complex are responsible for that all the ARPs contribute sort of mildly to the overall affinity that was a bit of a disappointing answer you can't you can't get rid of all the ARPs in the I-080 but you can get one at a time and the overall affinity went down just a little bit depending on which ARP we got rid of but it's not particularly satisfying but I suppose biologically it might make sense especially given the way it interacts with actin in such a dynamic fashion having multiple sites that all contribute would make that sort of multi-valent behavior or readily occur Thanks Peter I think there was one question before you Dave Hi, great talk my question was also about IDRs so you said you have looked at the amino acid composition and I am really curious whether there is any hydrophobicity versus hydrophilicity pattern that you could identify especially because you are saying that many of the clients are binding DNA and there may be burial of non-polar surface from the IDRs perhaps so I thought that there could be a way perhaps for HSD the evidence of the whole interactome are not a large chunk of that interactome so the transcription pie of the pie chart that was almost all transcription factors that bind DNA but that is maybe I don't know it's not even 10% of the entire interactome and so there but there those IDRs aren't even going to be near the DVD either a few of them are but again we were left with no sequence sort of common signatures in terms of amino acid sequences where we found 90 binding not even a broad character like hydrophobic versus hydrophilic yeah that's what I was I guess poorly conveying we broke it down to that not even specific amino acids but let's just go with negative you know nope we got nowhere that was very disappointing in the beginning I don't remember I'm trying to remember how we even tripped upon to look at IDRs actually I believe John V was looking at okay let me take a look at crystal structures what do I see there and that's when she was finding like oh wait it's where 90 cracks there isn't actually structural information to go with and then we went back and asked what about IDR sort of structural prediction software I see and so within that context can you say whether the function of HSP90 among all of those 1100 substrates is mostly to help final stages of folding or maybe hiding binding surfaces or something else oh I think that's a mix of lots of different things just in the limited time that you know we've spent asking functionally what does 90 contribute to a client we see all sorts of different things you know for today I just happened to focus on DNA binding events and they're depending on the client 90 might kick it off the DNA or 90 might put it on the DNA for most of transcription factors it's putting it on the DNA and that's because it's partner p23 is kicking it off the DNA but then for chromatory modeling 90 off the DNA so it we don't have a rule for what will 90 do to a DNA binding protein let alone you know in a lot of ways what 90 does at Eno80 in actin is very comparable to what it does with some transcription factors in DNA actin is just a polymer and it's helping to modulate whether it is or isn't interacting with actin it helps more to facilitate or disrupt interactions rather than help folding would that be accurate? I think there are clients where it is certainly helping we found that with the transcription factors that's a very late folding step require for DNA binding I didn't show you today but by limited proteolysis we see conformational changes in those factors so that we believe is a folding one but for a lot of things it's helping to promote protein protein dynamics and protein nucleic acid dynamics so is HSP90 ATP dependent or not? HSP90 has an ATPase it's a very sad ATPase it hydrolyzes ATP about once every 15 minutes and most of that ATP in the hydrolysis of it is really to control the conformational sort of what Conformation 90 is so I showed you it formed the dimer comes together and I have an open surface this is nucleotide free this is ATP this is ADP it doesn't look like the chemical energy of the hydrolysis goes to the client but rather to the conformational change within 90 and how about P23? P23 has no ATPase activity so when it what it does to all of its clients it's just through binding energy and so how do you see that being dynamic I guess how does it change states and do you see any cold sensitive mutants in these proteins that get stuck? so P23 the deletion of P23 is non-essential in budding yeast is essential in mouse but the yeast are cold sensitive P23 does not directly when it interacts when it kicks something off the DNA it doesn't interact with the DNA binding domain it always interacts with an adjacent domain that is often known to allosterically alter the DNA binding domain a good example that was with steroid hormone receptors the ligand binding domain and steroid receptors is known to be able to shut down the DNA binding domain P23 requires the ligand binding domain to kick it off the DNA in an odd sense it does that not just with DNA binding proteins but GCN5 which is a sonocidal transferase has an odd two interaction domain next to the hat domain that is used to control hat activity that's where P23 regulates GCN5 through that domain it's always some sort of allosteric interaction never with the activity that we're directly following it's always using a sort of regulatory domain that's used elsewhere in the biology of that client but then to recycle it it's just presumably using thermal energy when it recycles once P23 kicks it off we can see P23 kicking off a transcription factor off the DNA we can't see it bound to the transcription factor once it's free in the DNA we have one nice example MCM1 and I showed the data for P23 kicking MCM1 off the DNA if we remove P23 MCM1 will not rebind to the DNA but if we give it 90 90 will convert that conformation into a DNA binding form and now it can happily bind to the DNA but 90 won't join it to the DNA bound complex it's just changing its conformation and allowing it to now bind to the DNA so that's the only one where we have this sort of a stable state of it's free but not DNA binding component 90 gets it to the competent state P23 will kick it back off the DNA and leave it in a non-DNA binding it's a competent way does HSP90's ATPase change when it's associated with clients its rate change it can go up or down as frustrating as that is so some clients will get the ATPase the usual ATPase will go up with a client but there are certain clients that will make the ATPase go down Presumably the ATP binding and ATPase activity of HSP90 are essential despite it being a function given it there was an initial publication that said ATPase was essential and Johannes Buchner's group a couple of years ago published another a follow-up paper that said the ATPase is not essential ATP binding is essential but not its ability to hydrolyze it those yeast are very unhappy and slow growers but life still goes on even if it can't hydrolyze ATPase so something as fundamental as whether or not this thing hydrolyzes ATPase is still somewhat controversial as to what it does I think the people that published that it was required agreed with Buchner and said yes its ATP binding is more important than hydrolysis can live if they can't hydrolyze ATP but it is an extreme slow grower so life isn't happy without that did you have another question I'm just wondering instead of thinking about HSP90 may have evolved to interact with a certain set of proteins maybe it evolved to just not interact with another set of proteins and so like if you look at the things that don't cross-link does that give you any insight we haven't done that to any extensive degree it's like the negative of the picture I think that's a great angle to sort of go at it and I think we might we sort of are playing with that idea with telanchor proteins which I didn't talk about today because we think for telanchor biology get345 is this complex that delivers telanchor proteins to the translocase get12 90 delivers has a bias set that it delivers but if you get rid of get3 it'll deliver those if you get rid of 90 get3 will deliver night so they can cross over but they certainly have preferences so I guess in some ways we're pursuing that but I don't have for you yet because that's relatively we're like halfway through that project I guess okay well let's thank Brian for a stimulating talk lots of questions so that was great yay hmm that's good so I sent you that oh thank you I have my name with the check okay I got bond or so bond well thank you


We have already discussed the stability of nuclei and the decay modes that allow an unstable nucleus to eventually evolve through the emission of alpha, beta and gamma radiation to end up as a stable nucleus. However, you might have some questions, such as how long do these processes take? What is the time scale for the nuclear decay of unstable nuclei? The answers to these questions lead us to the concept of a nuclear half-life, and that is the subject of this video. Let's do a thought experiment. I mentioned in the last lecture that the mass of a calcium-forte nucleus was less than that of potassium-forte, and I described how potassium-forte can be to decay into calcium-forte. So we said that this process can happen. But here is the interesting question. When will it happen? To be more precise, if we have a single atom of potassium-forte, can we say exactly when it will decay to calcium-forte? The surprising answer turns out to be that no, we can't say when it will decay. In fact, it turns out that the only thing we can know is the probability that the nucleus will decay in a given time interval. What does this mean? It means that if we watch a potassium-forte nucleus for some time interval, let's say 100 seconds, we can know the exact probability that it will decay during this time. Let's call this probability x%. But let's say that this particular potassium-forte nucleus doesn't decay in that first 100 seconds. Perhaps we walk away and we come back an hour later, and it still hasn't decayed. Here we have to deal with something really interesting and somewhat counter-intuitive. The probability of that particular potassium-forte nucleus decaying in the next 100 seconds is exactly the same as it was in that first 100 seconds over an hour ago. This might seem really weird the first time you think about it, but the fact is that the past history of the atom is irrelevant. In any new observations of a potassium-forte atom, the probability of decay per unit time is constant and unchanging. This constant probability leads us to the following observation. For a large number of nuclei, we can predict with some accuracy how many decays will happen in a given time. However, for any one nucleus, the exact time of its decay is inherently probabilistic and cannot be predicted with any certainty.


Hey everyone she bear 1,000 here today. We're going to be doing an unboxing video and a review I Had to order something that I really didn't want to order but I really had no choice so Stick around and I'll show you what I had to get guys. So here's what we got one unbox it like I said We didn't really want to have to buy this thing, but we did So there we go as you can see it's Dell The new computer It's a video editing computer size gaming computer So what we want to do is we're going to get this thing out of here and we'll get inside It must be the keyboard here's the mouse Hope it's better than mother mouse We're gonna get inside it and make sure it has all the components That I ordered Okay, so this is why it's supposed to be brand new. It's not we didn't get a rebuilt one or anything like that so Hopefully this is the one I ordered monkey's helping Let's get a Stood up here What is this the 8940? Dell 8940 what we're gonna do is take it out of here Got the drive It should be it should be fast enough for what I'm gonna be needing to do with it, which is live stream and edit 4k videos My other one just did not have the power It had trouble it actually had trouble with 1080p videos here recently so and it's an all-in-one so I can't expand it. I can't upgrade it So what we got here a screw Shiny solar screw Open it up here See what we've got. Okay Looks nice Okay, we've got the two cards in here Let's get you okay for the You see that this is the ram. It's got two sticks around so It should be eight gigs on each one Yeah, eight gigs there. Thank you mum found it hell and The thing about this like I said guys is I can expand this Later instead of having to buy a whole new PC my fan It's supposed to be the i7 have the i7 processor It should have and I'm not seeing it Somewhere in here You should have maybe that's it We'll have to check It should have the What is it 525 SSD solid-state drive? so Won't have to check that out. So I'm going to check it all out and make sure it's got everything That I ordered and then we'll hook it up plug it in boot it up and See how everything works stick around guys. Okay guys So I got my new PC up and running seems to be doing pretty good Everything seems to be installed. That's supposed to be installed And the good thing about this is because see that thing right there There's there's just no way you can't You know you can't go any further than that. That's it but this Does have everything Ordered it's a core. It's an i7. I know it's not the grays For you guys that you know are big gamers and stuff. I'm not a big gamer This should do what I needed to do but later on I'll put a new Processor in it, you know a bigger processor a hundred sixty or sixteen gigabytes Installgram so Which you guys saw it's got two eight gig sticks in it, so It is running windows home so Everything seems to be working good the way it should be I'm not having any issues with it, so it seems to be all right Everything's loading fast.  I'm gone for now And hope you all have a great Friday and an even better weekend monkey's looking at me like I'm crazy or something I'm sorry, and I'm tired. I've been messing around this trying to get stuff off that old computer onto this drive So I can put what I need to put onto this PC. I'm still not done yet There are a couple more things I got to do and then I got to tidy up everything so Once that's done, I'll be done. So anyway guys Like I said, we didn't really I didn't I didn't want to have to get a new PC, but that other one just I mean it was just constantly creation and It's alright to surf the web and stuff like that But it started having trouble just doing 1080p videos There's you you just it was near impossible. It took hours didn't hun for that thing to process a 4k video hours upon hours and took hours just to edit it because it was so jerky and It just it just didn't want to do it. So anyway, hopefully this will be better. So anyway guys again.  I'm burning up. So anyway guys.  Yeah So tomorrow won't try a live stream and we'll see how that turns out. So I don't know what time or anything like that probably be afternoon sometime But anyway guys, we'll see you guys later. Y'all be safe and take care. Bye. Bye


Hey, I'm at this resale shop near downtown St. Pete called Art Pool. It's a big place like a resale mall Here at 2030 Central Avenue right across the street from Haslund Books. They've got this section with games here all sorts of playing cards, but I noticed down at the bottom They've got this small section of board games, and it's a small selection, but interesting stuff here. Look at this There's this thing called magnetic steel playing cards For use on boats, cars, and trains Steel playing cards. That's awesome. I wish I could open that up This is the Mad Magazine game, which I had as a kid and Which I think I'm gonna get from them before I leave They've got two versions of careers a part cheesy Starbury Shortcake, but this is what really caught my eye here Patty Duke game Take a look at this That's a beautiful piece of work there Patty Duke, how can you resist? Yeah, I'll be picking this up Remember, this is at the Art Pool near downtown St. Pete 2030 Central Avenue


In Second John, as in our lives, finding the good news isn't as hard to do as it is to figure out what to do with it. The elder rejoices that the people know the truth, that our hope and salvation come through Christ, the Word made flesh, and that they celebrate that by loving and caring for one another. That is good news. But how do we act towards those who deny that truth and reject that love?


The kids came over and helped us take Christmas ornaments and decorations down so we got that done. I had an interval of sanity broadcast today so we got that done. We are going to be changing up the time of the weekly live broadcasts to I think 11am central. I will put the channel link in the description below. I'm sorry I ran up the stairs. Anyway, so it's just been not a super busy day just quietly steadily busy and I completely forgot about vlogging and so that happens, right? I did some cleaning in the art room and I rearranged my daily drawing case which is downstairs and added some gel pens into it some things that will show up on black paper because I want to work more in the little black sketchbook doing some abstract doodles. Vicki, Peg, Michelle, it's all your fault. Peg Robinson, Vicki Brown and Michelle Mitchell. Vicki Brown of Messy Table Studios, Michelle Mitchell of Lady Blue Studios. I will link all their channels in the description below all three are fabulous artists in their own right and yeah they just started something anyway. I'm enjoying doing daily drawing just for me sharing with you all on Instagram and or Twitter and Facebook posts if you haven't followed me on any of my other social media you have no idea what I'm talking about. There's a tree there's a tree there's a link in the description below called link tree and then slash and my name click on that you're going to find every single place you can find me support me follow me stalk me whatever on the internet so go look at it. I also was watching a video just now on from Kent from Oz. He hasn't been actually had any videos on it I think a year. He is the one that told me about the Filofax Maldon and I saw it first on his channel and that is a planner organizer that I actually carry as a wallet. I actually had a spare one I have two of them and I was watching his video and reminded about that he took the rings out of his a while ago. Good morning everybody it is Monday morning January 7th. I only know that sort of because I have an appointment this morning with a new doctor new general practitioner. Hopefully things work out and I like her and everything so that's a little stressful so we're going to go do that this morning and I need to run some errands and I have of course always projects to work on laundry to do oh that reminds me I gotta put some laundry in the dryer before I leave. I'm thinking about how much I want to say and how much I don't want to say. Christmas time is always really stressful for me for those that don't know why I had my first miscarriage years and years ago at the beginning of December plus I worked in retail for a lot of years plus there's always family drama over the years those things just build up to where you're just not super thrilled about the holidays you just kind of dread them. That being said I enjoy spending time with my family doing things we really enjoy leave the drama at home if you don't have anything nice to say keep it to yourself yeah and I just going forward I would just rather do things with people who I really enjoy being around than having to deal with drama I just yeah I'm done anyway it's all I'm gonna say about that don't ask because I won't say that's probably too much and I may cut it out anyway I'm sitting here in the library for a few minutes because I'm ready a little early because I'm anxious to get the doctor visit over with probably I do need to show up a few minutes early but I don't think I need to show up an hour early so that's a thing I may just pop over to Safeway I need some tinfoil and some hand soap and some bottled water and I may just go do that and then go to the doctor that'll kill some time and I won't drive myself crazy just sitting around the house anyway that's it for now I will be back hey guys how are you I'm downstairs after working upstairs I tried to shoot a little film with my camera upstairs with my camera with my phone upstairs I tried a new mic which I'm going to return to Amazon it didn't work and and honestly the footage is crap I'm going to show off what I did with my Filofax and inspired by Kent from Oz and I am loving it I've barely used it and I'm loving it already I am gonna though shoot proper footage because the footage I showed was crap and I need to do a better job at speaking at filming turn the lights on like I just need to do a better job I also have a project I'm working on for a friend for a surprise she may not want it she doesn't know about it she didn't ask me to do it but I'm gonna do it anyway I'll clue you all in once it's been mailed but yeah so I'm gonna work on that and I did get all my paperwork done all the 2018 bookkeeping is finished California State sales tax are paid I'm done and finished with California it's been nice for so long I had my appointment with my new GP yesterday and had some blood work done and I went on earlier to just see if the results were in yet not really expecting them to be but to my surprise they were and everything was good which is great the numbers looked better than they actually have in a long time so yay I think the move was really good for me I am back to tracking what I'm eating and drinking using lose it it's an app for just watching what you're eating I don't make a point of working out but I do walk up and down the stairs a lot and I'd like to do I like to walk so I do a lot of walking she also has me weeding off of the daily inhaled steroid for my asthma which I am over the moon about and as long as I'm able to control the asthma without it and all the coughing doesn't come back and stuff like that I'll cross your fingers people because I'm so excited um but I had to come down and eat some lunch because it's I think it's after three o'clock um I was starting to feel lightheaded it was probably because I hadn't eaten um and I need to do a load of laundry and stuff like that so anyway just a quiet nice day at home in my oversized uh red cross t-shirt and baggy pants and you know nothing special I'm sure I look like a just a dream on the way to the mailbox anyway um that's it for right now uh tomorrow I have to hang out around the house waiting for ups to come pick up the mic that I'm returning to Amazon and I want to get some more stuff done and then Thursday I'm going to go run errands and I might hit a couple thrift shops or or Tuesday morning or something our footstool that we had out here in the living room that we brought with us from California broke um not because we moved just because it was past it and so I'm kind of on the hunt for a new one anyway that's it for the moment I'll be back good morning it is it is Wednesday morning January 9th yes you know I should know the iPad is right in front of me um January 9th I've been up for a while I got all my daily postings done and I started working on a dot mandala project which I will put a link to um a link I'll insert a picture here somewhere but it's turning out really well and I'm having a lot of fun most importantly I'm trying out some new dotting tools that I'm absolutely loving not sponsored I purchased a set on Amazon I'll put the link in the description below super easy to use I have other dotting tools and I am filming the project um so I'll give you my thoughts on the tools uh but hint I love them uh in the in the video so there'll be a video coming out on the process soon I do need to go out and run a few errands but I am waiting for UPS to come by and pick a box up so I may not get any of those errands done until tomorrow because it's like a giant waiting game but I do have dishes to do and laundry to do and all that stuff so it's not like I don't have things to keep me busy all right guys if I don't catch it on camera you're gonna think I'm insane there's a baby deer in our front yard hang on he's eating our bushes it's the same baby deer who's been coming I just wonder where his mom is at so cool cool is that I'm gonna go try to catch him he's in the driveway now he I'm probably gonna scare the crap out of him but he is so pretty I wonder where his parents are though that's what I'm worried about there's two of them oh my gosh at least two maybe three I'm feeling right there it's just watching me so cool just watching me like come on let me go back in the house so we can have some lunch now I know why they're in my front yard they're eating the bushes and here there's lots of wildlife out today there's a whole family of baby deers I don't I'm pretty sure they're not grown-ups I don't know anything about deer they maybe does I don't know the one I saw that was in the front yard wasn't very big accidental photographs when you're not completely awake or a bad thing good morning five months after moving I'm still trying to figure out where things got put when we moved in anyway fun fact years ago when I was pregnant with our daughter who is now 24 I was in a head-on collision with somebody who ran a red light and that's how I found out I was pregnant fun fact anyway I've had problems with my um head and neck and back since then and so I was looking this morning for my little hard rubber peanut shaped thing which is this to do uh there's an exercise I can do to relieve this the pressure in my neck so I must have slept on it wrong or something periodically over the years I have an issue especially um if I sleep on it funny so I need to do that this morning because my neck is killing me and it'll lead to a migraine if I don't do the exercise which the exercise hurts but it's better than dealing with this so good morning everybody Thursday morning January 10th I think I think it's the 10th because yesterday was my baby sister's birthday happy birthday Elizabeth everybody tell her happy birthday um it's 746 am I'm gonna have some more coffee and some breakfast watch some youtube do my daily postings and then we're gonna get dressed and go run some errands I can leave the house today because I'm not expecting people I have work to do but nothing that requires me to just stay home all day and get it done so yeah we're gonna get to it maybe find the local Tuesday morning so uh I love this Tuesday morning and it's next to a different dollar tree than the one I was going to I like this shopping center a lot better than the other one so anyway but the other one has Joanne's fabric so um either or but this is a nicer shopping center when I just want to go to dollar tree I did just realize as I was putting in um to my GPS app for admire to go there next then I forgot the shopping list off the refrigerator so I'm gonna just wing it and yeah pretty much guarantee you I'll forget something but you know and I'm gonna try to reduce our amount of waste I there's no way I can go zero waste I don't think but um I did well in dollar tree but then I forgot to tell the young man on Tuesday morning I didn't want a plastic bag so um I think Fred Meyer has a thing where I can stick the plastic bags hopefully so I'm gonna put this in there and I don't know what they do with it but I think they repurpose it or something anyway oops all right I'll be back okay so I got most of what I needed I think I don't again I forgot the list so I don't know what's on the list I am shooting for healthier um um low waste alternatives to things including food less plastic packaging I am at the moment unable to avoid it completely but I'm doing my best to buy alternatives when they're available including I'm gonna try switching body wash to I back to a bar soap which I haven't used in like a million years um we'll see how that goes I like my liquid body wash um there were a couple things I wanted that Fred Meyer didn't have and when I wasn't looking the very nice clerk at Fred Meyer put my greeting card that I got for my dad's birthday in a plastic bag so now I got two plastic bags and they don't have anywhere to recycle them so um I don't know I'm gonna head across the street to new seasons market which is sort of a combination of an organic grocery store and a regular market I was in the wrong lane dang it um I think I'm still in the wrong lane that's okay um anyway so I'm going to um go head over there and see if I can find what I need over there we'll see it's gonna be a journey I yeah so I'm hoping between the two stores that when I do need to go shopping that I can get the bulk of stuff at um you know one store and when I can't find there I can go to the other store I like Fred Meyer and it's right across the street from new seasons so hopefully that works I'm becoming an Oregonian the sun is blinding me what is that not used to the sun it's very bright out I love new seasons market so I um was successful so they had frozen Marion berries which I've been looking for yay they were back in stock and they did tell me in all fairness when it was here last week that they were getting new stock in any day and so they had some so I got a bag for the freezer because I want to try baking with them I also found half and half in a glass bottle I keep forgetting to look every time I come over here and I finally look today and um the bottles aren't very big so I got two of them and they're also you know they don't have a super long shelf life but we drink a lot of coffee um but they're in a glass bottle which can be recycled uh returned um the plastic bottles and the waxy coated cartons are none of that is recyclable so um in these glass bottles it does have a plastic cap but that's the only part that's not recyclable so um I love that and so I might have to have an extra cup of coffee today so I can try the new creamer that might be a thing hand here then we have that um good morning it is Friday morning January 10th maybe 11th maybe I don't even know I just know it's Friday it's 9 13 a.m the only reason I know that for sure is because I'm sitting in the car and the clock is like right there um I started off yesterday I think I said already um in yesterday's vlog with my old neck and back injury bothering me and you know I got it worked out I didn't found my physical therapy tools did my exercises as you saw um and I worked out most of the pain and and stiffness but because I started my day off on a misstep note um I hesitate to call it a bad note because it wasn't a bad day but uh because that happened I you know led to a series of things like me forgetting the shopping and errand list which meant I forget to forgot to do a couple of things forgot to pick up a couple things so yeah so I'm headed back out to the store so anyway we're gonna go to I'm just gonna run to Safeway I forgot a couple things at the store and I need to mail some stuff over at McCann's um so I'm just gonna go do that really quick and then come back and work on some art and journaling and look at my to-do list and figure out what needs doing and stuff like that so I'll be back good afternoon everybody it is after 12 o'clock I don't think it's not one o'clock yet it's like 12 30 ish anyway it's Saturday I almost said December January 12th um had an impromptu intervals of broadcast intervals of sanity live broadcast this morning and now we're out just running a few fun errands we probably will go antiquing a little bit but I had to stop at McCann's you see behind me um they have a little post office in the pharmacy because our former PO box sent us a piece of somebody else's mail they forwarded our mail for us like once a month which is great but when I got the envelope today I took everything out there was a piece of someone else's mail in there so I called them and let them know and I'm sending it back my husband dropped me off he went to go to the bank across the street real quick and then we're gonna drive into Newberg and Sherwood and just go drive around for a little bit maybe grab a sandwich or something on the way home and yeah have some fun we'll take you with us we'll be back and yeah that thing out there that bright blue thing and the bright light yeah the sun's out I don't know what to do with that anyway I'll be back all right so that's been our week it's been an interesting one pretty good I think overall this next week we celebrate my dad's 80th birthday and I just ran up the stairs sorry um I'm taking him he's gonna stay overnight I'm gonna take him to the airport he's going down to Southern California to work with my brother for a little bit and what else have another doctor appointment with a new doctor um colonoscopy doctor yeah it's that time I think so oh joy I'm gonna see what she says she's gonna review my records and oh boy anyway um I'm also gonna continue my journey trying to reduce our plastic packaging consumption and the amount of trash we produce can I go zero waste probably not but I got some more stainless steel straws because I'm gonna switch the family over including my husband who's very reluctant from disposable straws I also bought some silicone uh ziploc bags they're reusable you can put them in the dishwasher to sanitize them between uses so I got a few of those to try I'm on the hunt for products and food that are in less packaging so that's my current obsession I mean it does mean I'm probably going to have to cook more but you know I probably should be doing that anyway it's healthier so yeah that's a thing anyway it's been a great week I hope you guys have enjoyed it if you have any questions comments or concerns please leave them in the um comments below you can check out all my links and places to follow me on social media support me on social media in the description there's a few gonna be a few links in there but also there's a link to a place called link tree and then slash my name click on that you're going to find every single place I am on the internet plenty of places to follow me and support me including all the different places you can find the products I've designed or made including etsy spoonflower red bubble society six I knew there was one more in there um my tip jar patreon I do have one um and all of that sort of thing so check it out let me know what you think and if you guys have any specific requests for content please let me know leave something in the comments and we'll see what we can do about that I'm finally getting into the swing of things it's only taken almost five months but you know it happens anyway that's it for right now I hope you all have a great one don't forget to like share and subscribe hit the little bell icon if you want notifications of new videos when they come out there's some great content coming up and go out and have a great week do something nice for yourself because you deserve it I'll see you later bye guys


Automation, artificial intelligence, blockchain, virtual reality, the Internet of Things. Innovative technologies are developing at an exponential rate that have the capacity to help address the world's challenges in the long term. WISIS forums is innovation as a powerful tool to accelerate digital transformation. This year's innovation track provides a unique opportunity for high-level cooperation and partnerships among all the WISIS stakeholders. Regulators, government, UN agencies, civil society and the private sector are all working together to create the innovation ecosystem that fosters the sustainable growth of the digital economy. AI is not only a technology, it's a system that you need to put in place. Imagine that you want even to do the efficiency right on energy. That means that you need the stakeholders to make sure the AI is diffused and at the same time you want the public to push for this resource to be better utilized. At WISIS Forum 2018, the ICT for development community aims to foster innovation across all sectors, from health to education, agriculture and of course urban development. The world's urban population is growing fast, with 60% of us living in a city by 2030. Innovation is therefore crucial to the successful and sustainable development of smart cities. From a smart government perspective, we work together to redesign the process to look into how can we change the service to make the customer a happier customer. We even took an extra mile on which we sat with the customer themselves. We heard them, we co-created the service along with them for something that will actually make them happier. The United Nations 2030 Agenda for Sustainable Development has embraced the spread of ICTs to accelerate human progress, bridge a digital divide and develop knowledge societies. So that's it from us today, but do join us tomorrow for more highlights of WISIS Forum 2018.