So, it is a two-preview edge to introduce an area. Can you hear me now? So, it is a two-preview edge to introduce the speaker in the first Bristol Mayer-Squid lecture, distinguished lecture in immunology. Dr. Michael Cahill, and before I go on, I want to thank Stolm Lane, who made this possible using his connections at Bristol Mayer-Squid. So, Dr. Cahill is a distinguished professor at the Chamber of the Department of Biology of Physiology and Biophysics, and might have been a UCI for 33 years, the last three years, and has made a tremendous contribution to this institution. Mike received his Bachelor's in Science from Albany College and his PhD in 1974 from University of Washington, and after two short stints as a post-doc at University of Rochester and U Penn, he then joined UCI in 1977 as an assistant professor. So, at UCI, Mike has been extremely productive and successful, and has made a major contribution to our understanding of how the new system works. He chose to pursue a single-cell approach to investigate immune response, and he identified a pivotal role of ion channels in the activation, motility, and cell volume, motility for sites. He then went on and outlined the molecular structure and the functions of calcium channels. He identified the protein Ori as a calcium channel-forming molecule, and recently showed how steam can dinerize Ori diamonds to give form to the craft channel tetra. And then finally, pioneer together with young Parker, the two-photo microscopy to image the interaction of lymphocytes and the green cells in the immune system. Now, Mike has not always been an immunologist in his career. He claimed that he became an immunologist out of necessity and serendipity, and I let him tell you the story of the menome 81 and those squeaks in Newport. I'm sure he will manage to tell the story, so I let him do that. I have a different opinion of how Mike became an immunologist, and that is as a... as a... yeah, yes. As a... as an enthusiastic, vigorous and creative fellow, he could not resist the federal attraction of the grand and biosciences in an orchid. There. And, in fact, as I say, Mike received a number of awards for his work with the Anthony Crack Awards for Research, and has been incredibly productive and creative. In fact, he has held two NIH child awards, one for the last 32 years, the other one for the last 26 years, if I'm not mistaken. But he claims that he got his first grant almost 60 years ago. Yeah, yeah. It was a silver dollar from his grandparents, and I'm sure his grandfather gave it to him out of love. NIH doesn't give us anything out of love. So this is very good as a first step in his career. Now, the best acknowledgement of Mike's ability is his success in just a month, which is an action to the natural kind of sciences. And that is not the only achievement. Together with our own channels and everything else, Mike managed to produce a one family, like you said, here, with children and grandchildren, and how he managed to do this is only one guess, because when I talk to him, he's just about leaving for Paris, or he's just back from Paris. Anyway, he has done it, and without further ado, I leave the podium to Mike and his presentation. So yeah, I still have that silver dollar, so I'll take it. I'm really glad you didn't show any photos of me in the hot tub. Okay, so I've got my talk divided into three parts. The first is sort of a review of some of the immuno-imaging work that we've been doing over the last 10 years. And this is a collaboration with Ian Parker to visualize lymphocytes in vivo as they're doing things. The second part of the talk, we'll talk about electrophysiology, the ion channels that live in p-lymphocytes from all of us. And in the third part, I'll talk about the molecular aspects. So we've got imaging, electrophysiology, and molecular studies. And so this is the wild part, this is the zot part, and this is the crack part. Okay? And I'll try to focus attention in each one of these parts on the need for immunosuppressants. I'll talk about sphenocene 1-phosphate receptors and how agonist compounds can alter lymphocyte trafficking. And these compounds have been shown to be effective in vivo, and I'll talk about the strategy for why that works. And I'll talk about KV1.3. KV1.3 is probably my favorite, maybe my second favorite molecule. And how blockers of that channel, potassium channel lymphocytes, inhibited T-cell effector function and motility. And finally, maybe my first favorite pair of molecules is still one or a rye. I'll talk about molecular choreography and how these will form effective targets for T-cell activation, for suppressing T-cell activation. Okay, so this is a very nice audience. And I'm so glad that Powell invited me to do this. It's the first distinguished lecturer from Crystal Murray Squids. I'm really happy to do it, and I'm happy that you're all here on such a beautiful day. And since it is quite a diverse audience, I thought I would just give my own layman's version of the immune system as a practicing biophysicist, neurobiologist. The immunologists will please excuse me if I get some of the details wrong. But the immune system consists of a variety of cell types that mediate antigen recognition and effective functions and so forth. And these cells hang out in lymphoid organs and they're pumped around in the circulatory system by the heart through the vessels and they cruise through lymph nodes where they can locate antigen and then they move on if they don't find antigen and so forth. And so if you have influenza, if you contract a flu infection, what happens is the little viruses go into you and they're recognized by the immune system in lymph nodes first. And then there is a primary immune response that involves finding, locating that particular antigen and responding to that particular antigen. So T cells that are flu specific have to recognize the flu specific antigen and then respond to it. And then there's a phase of effective responses that lasts longer and involves clearance of the virus. And this happens not in lymph nodes but out of the periphery in the lung and everywhere else that flu virus might be. And this generates, this process generates memory cells that be a secondary immune response upon re-exposure to the same flu virus so that you can have a stronger, more rapid clearance of the virus and it's also the basis of vaccination. So these are thymocytes. Thymocytes give rise to mature T lymphocytes. Thymocytes are developing T cells in the thymus. There are two main flavors of T cells that are helper T cells that are CD4 positive. And T-cells are cytotoxic T cells that are CD8 positive. And we looked at these early on quite some time ago in their ion channel phenotype and so forth. So these green guys are the CD8 positive cells. These orange guys are the CD4 positive cells. And in the infection of AIDS, the AIDS virus manages to subvert the immune response by killing off the CD4 positive T cells, rendering people unable to fight infection. So the primary immune response to the color code red for red takes place out here in the lymph nodes where an antigen is recognized by the T cell responding to antigen presented by a dendritic cell, for example. And this could be CD4 cells or CD8 cells. And then the effective response takes place out in the periphery. Either CD4 cells or CD8 cells are involved in either the humoral immune response or the cellular immune response. So what's going on with those circles is actually depicted here in an in vitro imaging experiment done some while ago in the mid-90s by Paul Nagy-Lescu in my lab. And this is a T cell. It's specific for hen egg lysosyma for an antigen. This cell is the antigen presenting cell. It's a B cell that's got a look at the antigen previously and is now going to present it to this T cell. And the color represents the calcium concentration inside the cell. It's measured by a fluorescent thigh called Furotu, the Roger-Chatton invented. So let's take a look at what happens. And this is sped up as most of the videos that I'll show about 300 times. So it's time lapse. So you can see the T cell is crawling around and touches with the leading edge. And then the calcium signal is initiated as it's sustained. Then the cell kind of rounds up. And this is the period of time when the immunological synapse is being formed. And there are a variety of different kinds of T cells in B cells, T cells in dendritic cells, T cells in foreign cells. Here's a T cell B cell synapse that's forming between this T cell and this B cell. But right there you can see within 10 seconds of contact and it requires direct physical contact between these two cells to initiate a response. Calcium is pouring into the cells through crack channels. Calcium release activated calcium channels. And this is in my way of thinking about it the key to lymphocyte activation. This calcium signal not only stops the cell and you can see the cell stopping its motility here which helps to remain to keep it remaining anchored to the end of the presenting cell so it doesn't wander off. But this calcium signal also gives rise to a program of gene expression responses through the NFAT nuclear factor of activated T cells gene transcriptional pathway. I have more to say about that. I just wanted to show you the dynamics as we get started. So this would be for example a normal immune response to a foreign antigen. However, the immune response is powerful and protects us but there are also 80 different types or more of autoimmune diseases. These are usually classified by the tissue type or the organ that's mostly affected by an inappropriate recognition of T cells for a cell phantogen not a foreign antigen but a cell phantogen. So for example, rheumatoid arthritis is a crippling disease of the joints. It is one type of autoimmune disease. Multiple sclerosis. Here's an MRI imaging in a brain of multiple sclerosis showing plaques that represent the demyelination of central neurons which results in various kinds of symptoms in MS patients. Then we have type 1 diabetes T cells directed against pancreatic beta cells that are responsible for glucose regulation and secrete insulin. These pancreatic beta cells are inappropriately recognized by T cells and eliminated and you don't really notice the disease until 90% or more of the beta cells are eliminated and then it's kind of too late to do a whole lot about it and then the beta cells are wiped out and you have to be dependent on the insulin the rest of your life. Systemic lupus erythematosis is another example of an autoimmune disease here causing a rash but it also causes nephrotoxicity heart problems, all sorts of problems throughout the body. Inflammatory bowel disease in one kind is Crohn's disease another is ulcerative colitis in which lymphocytes are invading the gut and causing inflammation and psoriasis disfiguring disease of the skin in which you have these lesions. So corresponding to each one of these autoimmune diseases and I've only shown you six here out of the more than 80 there are inflammatory cell networks so for example in rheumatoid arthritis in the joints there are lymphocytes that recruit other cell types that recruit inflammatory cytokines secrete inflammatory cytokines and we have kind of a mess that causes bone destruction and crippling disease another cell network inside the nervous system we have T cells and other kinds of immune cells coming in and destroying the oligodendrocytes that form the myelin sheaths that enable neurons to conduct action potentials very rapidly in type 1 diabetes it's either CD8 cells or CD4 cells helpers or killers that cause the destruction of pancreatic beta cells SLA's and stomach lupus erythematosis same kind of networks are going on inflammatory bowel disease and in psoriasis and each one of these involves a variety of different kinds of effector lymphocytes secreting a variety of different kinds of cytokines that cause inflammation and tissue damage so these cell networks and here I'm going to highlight some of the lymphocyte involved cell networks are producing damage in your own body to suppress an immune response in autoimmunity so with that said I would like to launch into some data and I would like to say that all of this introduction has highlighted the need for imaging in vivo we would like to see directly into this tissue and see what's going on in order to produce a diagnostic test or evaluate the course of therapy the course of the disease to be able to image ultimately inside inflamed tissue in autoimmune patients and the way to do that so far the only way to image live cells in vivo is with multi-photon microscopy so how does this work so about 10 years ago I had this idea that two-photon microscopy would be the way to do this and I realized that Ian Parker my good friend in neurobiology had just built his own two-photon microscope he was using it to look at OSI which are pretty cool big cells but I said to Ian why don't we image lymph nodes they're about the same size in small lymph nodes a xenophilic site but there are a lot of cells in there that are pretty important so this is the way it works so with single-photon excitation shining high energy light a fluorophore is excited to an excited state gives off a non-radiated decay of energy and then fluoresces at a longer wavelength in a photon in a cloud of photons with two-photon excitation low-energy photons that are long wavelength like near-infrared light excite the fluorophore to the same excited state and it gives off fluorescence of the same wavelength so why is that cool the reason it's cool is you can adapt it to imaging and obtain very good tissue penetration resolution of images that are right at the plane of focus so the way this works is you shine in a laser beam that has all of its energy packaged into unimaginably brief pulses so these pulses of light are only 80 femtoseconds in duration very short duration and there's a relatively long space in between these pulses so when the flashlight is on it's really really bright the photon density is already high going into the objective lens of the microscope the objective lens further concentrates these photons so that the plane of focus and this is what's happening at the plane of focus the photon density here is really really high almost as high as at the surface of the sun and so it's one of the few places on earth where the photon density would be that high and it's achieved by a pulse laser focus through an objective lens and so it excites the fluorescence only at the plane of focus because it takes two or more photons to do this it's a non-linear process and you have fluorescence only at the plane of focus so then what you can do is you can sweep this laser beam across the field much like you would do with a TV raster scan in order to form an image and the advantages are multiple it's inherently confocal here you can see a little spot of light little spot of light there that is the fluorescence excited in a cubat or fluorescein by the two photon excitation compared to the single photon excitation where you have fluorescence above and below the plane of focus it also produces photobleaching only at the plane of focus and you have better tissue penetration because near-infrared light passes through tissue better than the blue light and so we can image multiple probes in real time also because two photon excitation happens at the same time so we began to do this in lymph nodes and now we've been doing it also in periphery in the skin and lung in several other places but I'll start with the lymph node but we need to make a mouse that has fluorescent lymphoid cells so T cells, B cells, benign cells, natural killer cells, etc one way to do that is with an adoptive transfer protocol so you have a donor mouse that gives T cells or B cells that you can purify in vitro to homogeneity then you can label those cells up with a dye, different dyes for different cell types inject them by the tail bit through the tail vein and those cells will hone pretty quickly into lymphoid organs like the lymph node or you can generate a transgenic mouse that has yellow, green or some other fluorescent protein expressed in the cell type of interest either way it works so we developed two methods first of all these are lymph nodes super glued to a cover slit that's one preparation the other preparation is the mouse on the microscope and here's the front end of the mouse there's the front pattypaw here is a simple skin incision which reveals the inguinal lymph node in a living, breathing, anesthetized mouse and in either case this is three-dimensional imaging because we can also move the objective lens up and down to get the Z axis here a cross section is the top of the lymph node with a fibrous capsule a subcapsular space and then there are lymphocytes these green cells are T lymphocytes that are honed into the mouse from the bloodstream and taken up residence in lymph node so like a naturalist observing for the first time a species or the behavior of something out of nature we have the first chance to see what the immune response really looks like and we've added some insights so here just for orientation is a lymph node now the difference between this and histology is this is a living lymph node these are B lymphocytes labeled red with CMTMR that have taken up residence in the follicle these B cells have the capacity to go on and produce antibodies if they're stimulated appropriately and T cells here in the diffuse cortex labeled green have the capacity to recognize an adrenic cell our volume is 200 by 250 micron it's only 2 naniliters in volume technically we inject enough cells into the tailbone so that we have less than 1% labeled cells so as you see the videos realize that there's a vast access of unlabeled cells and other stuff there and we're only looking at the labeled cells and there are 20 to 100 or so labeled cells per imaging volume so that we can trust that to track the cells and not have them too many or too few so just a little live music because to our delight we found about 10 years ago that these cells are very lively inside the living lymph node so here is the follicle with B lymphocytes outside the follicle are T lymphocytes it's time lapse of course but these cells are moving in about 8 micro-computers in a minute T cells in the surrounding diffuse cortex are moving 15 to 20 microns per minute but they move in a stop-and-go manner so that they kind of pause and that they change directions and when they stretch out they're really moving fast up to 30 microns per minute and you can see they respect this invisible barrier pretty well but they're occasional incursions of T cells into the follicle and B cells outside but usually they just turn around and go back to where they came from so here for example T cells are approaching the follicle they come right up to the edge and then they turn around so that's pretty cool isn't it you think it's pretty cool okay so what do we do with this data? so we track we track these T cells so here's a three-dimensional tracking just to illustrate the method so these are three different kinds of T cells there are foreign cells and self-cells foreign allogeneic CD8 positive T cells allogeneic CD4 positive T cells and self-CD4 positive T cells in a model of graft versus host disease this video was taken by Ying Yu some years ago and it's just to illustrate that we can track three or more individual types of cells in three dimensions and you can see their meandering paths through the interstitial space of the lymph node so we describe this except for right at the edge of the follicle as a random walk so tracking individual cells you can see here's a velocity histogram the average velocity is about 10 to 15 microns per minute so you just compute the number of counts this is a histogram and the average velocity is right here about 10 to 15 microns per minute and if you look at the tracks themselves these displacement plots you can see that from an arbitrary starting point they just kind of meander around and they displace themselves from the origin we can describe that mathematically as the displacement as a square root of time is a linear function so that's like diffusion it's like a random walk except of course these cells are not diffusing they're crawling all over each other they're calling particular fibers they're calling on dendritic cells nevertheless we can describe it mathematically with a displacement coefficient and calculate how far on the average will a cell produce cortex in a given time as a test for tino-taxis we summed the average tracks and showed that these cells just dither around the origin on the average so it's not like this cloud of cells is gradually moving in any particular direction it's really well described by a random walk so the default trafficking pattern of t-cells in the lymph node except for right at the edge of the follicle is randomly oriented autonomous motion with no evidence for directed motility and we can define a motility coefficient so here's the Paris Metro map my wife is from Paris I like to do these comparisons so people in Paris get around on metro trains and they go all over the place and they kind of look like those tracks except for the tracks of the lymphocytes really fill in the available space it's like these tracks are everywhere they can just move wherever they want to and Melanie Matthew in my lab made these nice videos using a transgenic mouse that expresses YFP on dendritic cells those are the green guys and red labeled B cells and you can see them just moving around all over the place all this is in the absence of antigen they're making contacts with this resident population of dendritic cells but if you then take these red tracks and you summate them you just allow the cells to etch out their own tracks and then you summate them over 18 minutes okay it just fills up the available space so the T cells really cover the territory within the diffuse cortex it suggests that the basal state of the T cell is constantly crawling around in a perpetual search for antigen so I'm going to now summarize a whole bunch of work over these past several years and beginning with the random walk that I've already told you about B cells in the follicle moving somewhat slower than the diffuse cortex you can see that by eye right these T cells moving faster than those guys now we tracked T cells and dendritic cells together this is the first ever view of a dendritic cell it's an vivo labeled dendritic cell we put dye under the skin along with an adjuvant the dendritic cell took up the dye and then moved into the lymph node and here it is and you can see that it has a very different kind of motion it's sending out long dendrites and retracting them dynamically so over here on the right we have a depth encoded view so this allows you to look at the third dimension the z-axis as a pseudo-colour so down at the bottom of the imaging column their pseudo-colour blue at the top of the imaging column they're red and this enabled us to rapidly tell whether a cell had actually made contact with one of those distant dendrites on the dendritic cell and the rest are moving above or below those dendrites and we came to the conclusion by counting these contacts which last only about two or three minutes that a single dendritic cell can contact 5 to 10,000 T-cells per hour so this whole process that we call stochastic scanning is an amazing touchy-feely exercise between T-cells and dendritic cells all the time touching each other this is a homing video so T-cells enter the lymph node and again it's depth encoded view on the right showing the T-cells move in all three dimensions here's a cell as it's tracking, it's moving in pseudo-colour meaning that it's moving up and down in the z-axis but the circles on the left in the same data set represent homing events where cells move a thousand times faster in the hindothelial venials and then they suddenly stop they detect something and then they extravasate the venial and enter the intrastitial space where they begin to crawl once they're in the lymph node these T-cells last in there 12 to 24 hours and then they move on and we're able to measure the process of moving on in this case the lymph nodes the lymphocytes move across a different barrier an endothelial barrier which we've stained red here these are the lymphatic endothelial cells that lead to the efferent lymphatic vessels where they return to the bloodstream and you can see T-cells moving across these sinus walls at particular places we call portals of entry and eventually they're going to move off into the sinus and out through the lymphatic vessels now there's another population Kim Garrett in my lab she's now past her institute tracked natural killer cells inside the lymph node in the presence of either foreign dendritic cells or self-dendritic cells so the foreign dendritic cells are green the self-dendritic cells are blue there's one up there and you're about to see a murder take place so if you're shy just close your eyes so these natural killer cells are first going to take on this guy and then take on this guy so here comes a little pinch pinch right there you see the dendritic cell kind of flinch and look it falls apart and then this guy is pinching and it kind of tries to get away and falls apart so this is a very efficient process by which natural killer cells can detect the presence of foreign cells in our body and it's a major barrier in transplantation so in the presence of antigen everything changes here are transgenic T-cells now from a mouse that has only T-cells that are specific for ovality so you can be sure that these guys will respond to the foreign chicken egg white protein ovality but in the absence of antigen they move just like wild type T-cells and we enter around they have the same velocities and so forth but in the presence of antigen we immunize this mouse a day before imaging there are big changes so the cells are bigger they're about to divide at 24 hours after recognizing antigen and there are clusters here's a cluster it's kind of a dynamic cluster so when we saw this we wanted to do this at the same time that we could image the dendritic cells presenting antigen so we tracked the process of an immune response and first we tracked T-cells interacting with T-cells and then we tracked T-cells interacting with T-cells so these are helper T-cells and let's look at the choreography the musical so at the beginning T-cells at the beginning of an immune response recognize foreign antigen but they still made transient interactions with a lot of different dendritic cells the interaction time begins to prolong however so that by the end of an hour with these transient interactions they're already responding, they're up-regulating an activation market called CD69 and after about an hour they start forming really long lasting contacts here's a T-cell that got snagged by a dendritic cell and taken for a ride so that's a long lasting contact it's forming a mature immunological synapse and after about 8 to 10 hours most of the T-cells are stably engaging dendritic cells in a polygamous manner too so here are a dozen T-cells moving around a particular dendritic cell that's presenting antigen at this time the cells are starting to discreet in a lukin II, a key cytokine for the rest of the activation program after about 16 hours the T-cells then begin to let go of the dendritic cells before they divide so then they make repetitive contacts with other dendritic cells in the area now during the beginning we were able to measure the calcium signal I showed you a calcium signal earlier well this is what it looks like in vivo these T-cells have a die into a 1 in this case here's the calcium signal in the absence of antigen this is just absolutely flat but in the presence of antigen there is a calcium signal that leads to activation of these cells and this calcium signal leads to rounds of cell division here's a cell division event in a stereo video cells will suddenly just round up and then undergo cytokinesis in about 15 minutes and then the daughter cells begin to migrate away and when they divide they dilute the die equally into the two daughter cells and you can keep track of how many divisions have taken place by measuring the fluorescence intensity of these daughter cells so that by day 5 in the lymph node the bright cells shown here are the ones that didn't divide much or not at all and the dim cells you can see everywhere else are the cells that have divided multiple times up to 8 rounds of division within the first 5 days of activation okay meanwhile there's a drama up in the follicle because these are helper T-cells and the reason they're called helper T-cells is they gotta help these B-cells to differentiate plasma cells so they can secrete antibody so how do the B-cells ever find the T-cells they're stuck in the follicle and it's achieved by chemotaxis let me show you that so here is an experiment which we've got two populations of B-cells one antigen specific the green guys one antigen non-specific the red guys and we gave the mouse the antigen one hour before starting this video and you can see what's happening is the green guys are moving to the edge of the follicle which is over here leaving behind a space which is relatively depleted of the green antigen specific B-cells and this is by chemotaxis the red cells do not do so the green cells upregulate a chemokine receptor, CCR7 and they then detect a pre-existing chemokine gradient, CCL19 and CCL21 and then they begin to migrate in a directed manner rather than a random walk toward the edge of the follicle and when they get there they form conjugate pairs and they turn off waltz okay never mind that music but they waltz okay so here is the T-cell B-cell forming conjugate pairs and you can see the red B-cells the B-cells are red here and the green T-cells and if you look at them carefully the conjugate pairs are always led by the red B-cell and they are quite actively motile meanwhile there is some solitary bachelor T-cells looking for somebody to hook up with and these green bachelor T-cells are moving rather faster than the conjugate pairs which are led by the B-cells now I don't know which is male and which is female but it's interesting to speculate on this now the B-cell T-cell conjugate pairs migrate around so robustly that they usually just leave the imaging volume that we are looking at but they can sometimes exchange partners so here is a partner exchange video a B-cell led a T-cell in and if you were quick here it comes you can notice right there another B-cell came and took away that guy so there is a menage à trois that lasts about 2 or 3 minutes but it's really serial monogamy unlike the polygamous relationship of this dendritic cell with the T-cell and this then leads to a block of egress involving sphingocene and phosphate receptors and here we are studying that looking at agonists and antagonists so the S1P receptors in the lymph node and you can see the cell block, block, block wash the agonist off and then it moves in so this is the first illustration and this leads me into pharmacology using this imaging preparation Melanie Matthew and Cindy Wei did the imaging in my lab in collaboration with Hugh Rosen who provided these nice agonists and antagonists specifically for sphingocene and phosphate one type one receptors and what these agonists do is they prevent the lymphocytes from leaving the lymph node they keep it trapped inside the lymph node so what you see when you apply an agonist in vivo is empty sinuses and chocolate block with lymphocytes crowded around the sinus lining this is sphingocene and phosphate this is the N-log which is phosphorylated in vivo called FTY720 and they both do the same thing SEW, S1P or FTY720 but FTY720 is made in the clinical trials about which I'll have a little bit more to say later this is Fingolimum it reduces the rate of relapses in multiple sclerosis by over half compared with placebo but there's a catch that's a little bit there have been some adverse events involving acute infections so we use 2871 SEW and we use the competitive antagonist W146 and we could show that when we wash off this agonist compound the lymphocyte motility increases and they start crossing into the sinuses you can see motility as we wash is increasing and then moving into these formerly empty sinuses on the other hand vehicle control wash, they're just moving all over the place and we can even do kinetics here's an SEW wash-in fitted with an exponential curve here's an SEW wash-out fitted with an exponential curve so it's nice to see exponential relationships actually applying in biology to something as complicated as this so we developed a model in which the agonist compounds are acting on the barrier cells the lymphatic endothelial cells trap up the gates preventing lymphocytes from crossing from the interstitial space into the lymphatic sinus and when you add the antagonist compound W123 it competitively antagonizes the agonist and it opens up the gate again and the lymphocytes are free to move so trapped lymphocytes can do no harm they don't get out there to mediate affective functions the motto is you can check out anytime you like so remember this hotel-California strategy immunosuppression now I'm going to move on to molecular targets that we're interested in and I'll talk about the functional network of ion channels and T cells so starting in 1984 we began to define ion channels and T cells and there are actually six kinds one is an intracellular channel called the IP3 receptor the first channel we characterized as a voltage gated passing channel then we found a swelling-activated chloride channel a channel that's actually activating the cell swells up a channel that's activated by calcium I should say that many of these channels have never been seen anywhere else at any time so it's kind of fun to study them then we found this calcium release activated calcium channel which is really bizarre, crack channel that lets calcium into the cell and we found this magnesium inhibited cation channel called myrrh and then coming around again so here are some phenotypes these are the currents that we record all this is done purely electrically at first so this is the family of currents that represents voltage gated passing channel activity a family of currents for chloro channels another family for calcium activated passing currents we could isolate all these currents simply based on biophysical characteristics of the channels we found this and this with the voltage gated k-channel and the crack current so summarizing 16 years of work we began with the current we quickly showed that low affinity agonists antagonists of the current tetraethylammonium forminoperidine quinine and some calcium antagonists which block this channel in a parallel potency sequence could inhibit the ability of these cells to divide this was the first time any kind of cell division had been associated with any ion channel then I was at a patch clamp course up at Hopkins marine station Stanford University's patch clamp marine station and this friend of mine had these scorpions there and he was studying muscle cells from the scorpion and I said why are you studying these muscle cells most interesting thing about the scorpion is the venom so we actually milked the scorpion ourselves and we applied it to mouse thymocytes and blocked KV 1.3 current so here's a potassium channel being blocked by at this point we got more sophisticated we got a purified toxin and we showed that nanomolar concentrations can block this current then in 1990 we found, George found that KV 1.3 one of the 80 different potassium channels in our bodies is corresponds to this current and we did that by expressing this in oxides and characterizing the potassium currents and they had exactly the right properties that was done along with stuff I'm researching in my lab then from the venom George and I and with Heiko Rauer carried out toxin blocking experiments modeling experiments so this is a KV 1.3 tetramer you can see the symmetrical molecule forms of the pore that goes right down the middle of the channel unfortunately for the channel this toxin which is shown here in the yellow ribbon diagram sits right on top of the pore like a cork in a bottle and blocks conduction through that channel now we've also got toxins that are even more potent so this blocks this is from a cnmine but strangely the toxins are very similar in scorpions and cnmines and this toxin blocks at 10 picovolver very much the same way that the crude toxin does and then we showed in 2001 that the channel phenotype changes from a resting state to an acutely activated state to a chronically activated state so what happens is first the calcium activated potassium channels are upregulated but then they're downregulated as KV 1.3 goes astonishingly high so KV 1.3 in T cells regulates membrane potential calcium signaling in T cells it's blockade and hit its T cell proliferation in vitro ok so what does it work in vivo so we tested this published in 2001 in a rat model multiple sclerosis called adopted transfer E.A.E in this model myelin basic protein specific T cells are injected into the animals these are the Lewis rats and within a few days they get sick and they become paralyzed so myelin based protein T cells are invading the brain and destroying oligodendrocytes that form the myelin sheaths around the neurons and if you inject enough of these nasty cells inside these nasty T cells they kill the rats within about a week however if you inject the peptide C and M and E toxin every day there's only a small phenotype here there's a little bit of a limp tail but you can see these rats are moving around nicely they're even able to sniff each other and socialize and it completely protects the animals from getting this disease now we also showed that it could be used as a treatment mode allow symptoms to develop then treat and ameliorate symptoms so later on in fact more recently with Melanie Matthew again collaborating with Christine Beaton and George and me we showed that these toxins ameliorate not only the MS model but a delayed type hyper sensitivity model and it allowed us to do imaging in the ears of these rats during an inflammatory response so we developed the preparation to do ear imaging and foot pad imaging and V-brow and this is what it looks like so these green cells here are activated effector memory T cells that are crawling around in the skin secreting gobs of cytokines that cause inflammation on the other hand, you can see they're enlarged and they're crawling around in collagen fibers inside the tissue and on the other hand if you apply SHK186 an analog of SHK the parents see an MEPETI toxin what happens is the cells you can still see the same number of cells in the tissue but they're small and they are immotile here's to the smallness they just stay small, they don't activate in the presence of this toxin they're inhibited to calcium signaling in these cells on the other hand, if you're looking the lymph node the same toxin and the same dose does not affect the motility of NaE and CCR7 positive cells motility is just the same unaltered by that we think this corresponds to the selective immunosuppression of a chronic immune response or an auto-anti-immune response so here is another model of EAAE in which the rats are getting sick and then they remain sick but if you inject the toxin on the first day of symptoms yes they get sicker but then the disease is ameliorated over the course of the next few weeks you can even wait until the peak of symptoms and ameliorate the disease on the other hand it doesn't cause the same treatment it does not cause any change in the clearance of a Chlamydia infection or an influenza infection so we think this is a keen approach for a selective immunosuppression of the effector memory cells that are causing chronic damage in inflammatory and auto-immune conditions here are the toxins it's fun to reflect upon animals and what they produce here is a scorpion there is a set of scorpion venoms here that have been crystallized and studied by NMR and they have this kind of structure here is a mapping that we did of the key residues so this is the critical lysine and the C in eminy produces a similar toxin that has the same kind of key lysine that pokes down into the channel and the rest of this molecule stabilizes the binding to the tetramers of KB1.3 and in fact there is a story, an ancient story from Lancet way back in 1983 just after the Almenio when there was a woman in Arizona who was stung by a scorpion this woman had multiple sclerosis and was wheelchair down and then after this scorpion stain which of course is quite painful and disturbing she had a two week period in which she became symptom free she was able to get out of her wheelchair and walk around and then the symptoms kind of gradually returned after that and they noted about why that might be Christine Beaton tested this scorpion toxin in the model of EAE and showed that it was affected in 2001 we followed this up with the study that I showed you earlier in a recent email to George Chandy June 7th Hello, I thought you'd be interested in this I'm a thoracic surgeon who has pancolosine spondylitis for about 8 years my vacation every year with my family in Mexico went again in March and happened to be stung by a scorpion so Google pictures this guy looked it up he captured the scorpion he thought it was the bark scorpion which is centroidis spulturatus after 72 hours again once again I was pain free so today here's another story from another physician I'm a general practitioner in the UK with primary progressive MS I holidayed in the Barbados two weeks ago and accidentally came into contact with fire coral related to the C&M& developed a burning rash and on return to the UK noticed marked improvement in life so these animal toxins out there really have therapeutic potential so I'm now going to review what we knew of the ion channel network as of 2003 so this is what we knew these guys are involved in calcium signaling cable 1.3 KCA 3.1 the crack channel and the IP3 receptor these two mediate volume regulation in the cells which is important to kidney cable 1.3 and the swelling activated chloride channel this one is involved in magnesium homeostasis but what happens is during calcium signaling is encounter with a peptide MAC initiates a proximal signal cascade that gives rise to IP3 and isoglycerol there's calcium release from the IP3 receptor the calcium then depletes inside the ER and that activates this crack channel that causes the calcium signal to go really high from about 15 animal or up to several micromolar and that activates this channel KCA 3.1 and interestingly it activates calcineura in a phosphatase the defosferol is a transcription factor in the cytosol that then is able to accumulate in the nucleus and turn on gene expression cable 1.3 is grammatically upregulated in respect to memory T cells and plays the majority role in balancing off electrically this influx of calcium okay so that's fine we thought we knew a lot but what is the crack channel and how does it work how does it link the status of the store to the influx of calcium so in 2003 we were fortunate to link up with a group in La Jolla that was using bactogargin to under hunt for the crack channel using RNA interference screening so we switched to disavva S2 cells Andy Garment who is here in the audience characterized the crack current in these cells and show that it's biophysically indistinguishable from the T cell it has the same ion selectivity the same rectification properties but these S2 cells in Nusophila are cool because you can do screening by knocking down selectively a single gene using double stranded RNA you don't have to guess about RNAi which RNAi are you going to synthesize they take up full length double stranded RNAs spontaneously from the medium and they chop it into bits with a very small interfering RNA which is very effective in suppressing expression of a particular gene so originally the original screening was done in 96 well placed with 170 candidate genes and this included all the transient receptor potential channel genes that were among the lead candidates we were lucky that it included the one gene that had a strong hit we validated HeriBCI by calcium imaging and patch clamp and quickly extended this HeriBCI including chair cacti cells work that Maria Leodino did and out of that STIM was the only hit STIM and here's some of the data so calcium signals now this is the calcium concentration of the function of time when you take away calcium and re-add it not much happens but if you add that you get a release transient and then here's the signal so when you re-add calcium that causes this rise in calcium up to the micromolar range that's the control treated with an irrelevant double-stranded RNA however if you do it with STIM double-stranded RNA this is what you get everything is normal right up until the calcium re-addition and then there's no signal okay so that says that this molecule STIM is required for this calcium signal and we went on to show with Andy Yeriman that this is the same for the crack current it's quite important because there might be more than one way to suppress that signal but it actually does it by inhibiting the crack channel itself it says that STIM is required for crack channel function this is STIM in the Drosophila format it has an amino terminus inside the lumen of the ER it has an EF hand motif that binds calcium it has two protein-protein interaction domains that span an unremarkable transmembrane segment there are two human proteins that are molecules STIM1 and STIM2 with a shorter end terminus and a longer C terminus each with a polybasic tail and STIM1 can be in the plasma membrane as well but the majority is in the ER so stimulating calcium influx stimulating calcium influx so work by Shenyuan Zhang in my lab showed that this molecule functions but it's not the channel it's the ER calcium sensor that detects the depletion of calcium in the ER to initiate the process of channel activation and it's also the messenger to the plasma membrane so he did this biobutational analysis he mutated the EF hand domain so that it couldn't bind calcium and low and behold when he expressed it compared to the wild type STIM1 he found that it's already right next to the plasma membrane and this EF hand mutant activates the crack channel but without the requirement for stored depletion pretty cool though so sending this protein to the membrane by calcium unbinding activates the crack channel he also showed with native STIM1 protein this is the only time that it's been measured there are dozens of groups now doing this but this is the only time it's been done with a polyclonal antibody generated by Jack Russo of neurogenetics that the native protein translocates from the ER where it co-localizes the bonafide circuit marker of the ER and it forms punta or hotspots right into the plasma membrane you can see them here so normal STIM requires stored depletion to send STIM to the membrane and to activate the channel here's a little cartoon up here here's STIM in the plasma membrane use some kind of channel over there and what happens is when calcium is depleted it comes off the hand and then that causes the STIM1 molecule to move to the plasma membrane where it forms aggregates or clusters and that triggers calcium influx through this channel oh ok we're feeling pretty good and this is as of 2005 now but what is the crack channel big question mark so Shenyuan who had done these mutations went to Harvard I tagged along and saw what was going on there but he spent 3 weeks there screening the entire drosophila genome at their screening center what they give you is 63 384 well plates each pre-allocated with an individual double standard RNA corresponding to every different drosophila gene in the genome 20,000 or more genes and Shenyuan was able to make 3 fluorescence measurements in each well of each one of these 63 plates, 384 balls per plate and to do it twice the data collection phase of this working sort of 16 to 20 hours a day was 3 weeks 150,000 measurements and he came back and we thought about how to analyze it and what we did was we plotted the signal after adding thapsigargan divided by the signal before adding thapsigargan in the two sets of experiments and most of the points here are 23,000 data points on this graph most of them are normal calcium signal on this axis of about 2 but see there are a lot of wells that had a reduced calcium signal however many of those wells would have suppressed cell division or lack of adhesion to the substrate and that could curiously affect the calcium signal and inhibit it so we filtered the data according to no decrease in the maximal fluorescent signal and after we did that we had 75 bits, still sounds like a lot we looked at the sequence of each of the 75 proteins the sequence had been the entire bisophila genome was determined in the early 2000s and we came up with 11 transmembrane proteins 10 out of the 11 proteins including stim so we revalidated the screen we rediscovered stim in this genome and 10 out of the 11 of them had known functions that were clearly not related to ion channels but there was this guy annotated only as old 186F whatever that means we didn't know the function of it so we started exploring it and if you do the same kind of experiments you can show with the neurology you get a nice calcium signal if you knock down old 186F you get no calcium signal in most of the cells at the single cell level what is the two proteins now stim and this old 186F are both required to give you a normal calcium signal so what is old 186F well it's a member of a large gene family that goes here's the bisophila gene back to C. elegans there's even a backyardal homologous in the databases then there's chickens and fish and by the time you get to mammals there are three homologs now named ori or crack am ori 1, ori 2, and ori 3 here's what they look like they're for spanning transmembrane proteins both by hydroxy analysis and by experimental observation ori looks like this ori 1 is bicocellated has a slightly shorter interminous very similar transmembrane 1 ori 2 is like that ori 3 has a longer loop here now we look at the sequence a nice diagram that Karine my lovely Parisian wife made for me and it's proven to be enormously useful so is it really the crack channel so what would you want to do you would want to over express these proteins so if you take the control crack current and you over express stim you don't get an increase in the current but you get a faster activation consistent with it being the messenger but something else being limited if you over express old 186F now without over expressing stim you do get a nice increase in the current but with slow kinetics typical of the control however you express both stim and old 186F you get a monster crack current highly amplified crack current with all of the biophysical characteristics that we can recognize from the native channel and this represents 100,000 functional channels for cell high density of protein 100 functional channels per square micrometer membrane so at this point we really thought we had an old 186F arrived probably the crack channel but is it really forming the pore of the crack channel so we looked at the sequence and analyzed up and down evolution sequence homologies and the yellow shows identity up and down evolution and trans membrane one is highly homologous there's only a couple of little differences and in the loop between trans membrane one and two there's some very conserved residues and among them here comes Korean's diagram again this is the conservation all the way through from fly to the three human homologs in bold there's a difference here but other than that it's conserved from here to right there there's this glue a negative charge residue there's another negative charge residue another negative charge residue this is a negative charge residue in the human homologs so mutated here, here, here and here first to alien and then we hit pater with this conservative point mutation glutamate to a spartate and what that does is it keeps it being a store operator channel but now instead of seeing an inward calcium current we see an outward current why is that? Investigating the ion selectivity of this and we showed that instead of carrying calcium into the cell it's carrying monovalent cations with this weird looking IV shape if we could explain by calcium blocking the monovalent current and negative membrane potentials so that clinches the identification of ORI as the poor forming subunit of the channel it is the bona fide ion channel it's the crack channel itself which is activated by stem or stem 1 E 180 ORI E 106 in ORI 1 controls calcium selectivity these two residues have minor effects they actually attract cations toward the poor but this one represents the site of the first known channelopathy of the immune system and to the Rouse group, Stefan Feske had been following these patients for years from Germany, their Turkish origin these patients have severe combined immune deficiency disorder fabulous study and he was able to show that arginine is mutated by the tryptophan in this ORI 1 on chromosome 12 a single point mutation that makes it impossible for these kids to mount an immune response and they die unless they get bone marrow transplantation and then it completely rescues them so now what are we working on we're working on choreography, molecular choreography with Obam Pena and Lo de Moreau we teamed up to show these kinds of things calcium story depletion calcium story depletion first the calcium comes off stim then it aggregates and forms into these junctions which are pre-existing junctions that come within 20 nanometers of the plasma membrane and my former postdoc which Louis was able to show with EM ORI starts out being a dimer but once stim is in the active state it forms a tetramer that lets the calcium come into the cell a dimer of dimers forming the conducting channel furthermore Maria Liudino with a lot of help from our friends in the lab was able to show that stim 1 and ORI 1 form at the immunological synapse between the T cell and the degenerative cell so there's molecular choreography at the molecular level of stim interactive of ORI and there's also molecular choreography at the molecular level of these proteins migrating to the synapse targets for immunosuppression so I've talked briefly about calcineurin cyclosporin A is a commonly used immunosuppressant to suppress the immune system unfortunately it and other strategies to inhibit calcineurin are pretty toxic to delivering the kidney this is severe combined immune deficiency in the native natural immunosuppression of people with this ORI 1 patient Stefan Festi has now found that there are three stim opethies patients in which the stim molecule the messenger molecule is mutated and it results in life threatening disease SHK peptide we've talked about as a selective immunosuppressant for affected memory cells and FTY 720 calcineurin inhibitors toxic side effects high dose corticosteroids are even worse egress block the hotel california strategy agonist trap the lymphocytes in the lymph node adverse events are prompting concern as they did with the anti alpha 4 integrin to saliv kb 1.3 blockade human trials set to begin fall 2010 this has been a dream of George and me since 1984 we're still searching for a low molecular weight early bio available kb 1.3 blockade but we're going to use SHK peptide as an injectable to begin to do safety trials and hopefully to treat patients within the near future Target and crack ORI 1 or stim is in an early state we just discovered the molecules within the last three years four years potent and specific blockers are needed there are a whole host of people out there in pharmaceutical lamp screening for these blockers the patients are profoundly immunosuppressed which means it's going to work if you can find a blocker but they do have normal cns function kids are coordinated they're just immunosuppressed and if you transplant bone marrow cells there's a cure so it's a promising target for acute immunosuppression now let me reminisce a little bit so from Seattle to Woods Hole to Philadelphia to UCI here we are with my postdoctoral supervisor, Bernal Hillow at the University of Washington in front of Clay Armstrong's house in Woods Hole all of us guys are working on squid except for Chuck Stevens who's just visiting playing frisbee with us and as you can see me and Paul Fulmer some culture visiting and some other folks here's Brian Salisberg looking like a Yeti and there's Barry Cohen his collaborator and Clay Armstrong my postdoctoral supervisor Summer of 76 now in that summer I was really lucky to be coordinated by Steve White who is chairman of the Physiology and Biophysics department here and Steve came to to Woods Hole to visit me kind of a reverse site visit back when it was called that and I took him out sailing on this little tiny boat that I had and I didn't know too much about sailing we got out there in the hole itself between home and massive land and the current was going 7 knots and the wind was going 20 knots the other direction and I took the boat over and Steve went kind of floating away and I looked for him and spite of all that he managed to hire me it's just a miracle it's really a miracle that I got hired at all I'm so lucky here we are in 1985 just after the nature paper in the JVM paper I'll already show the slide here's our running scoreboard Roger Chen visited us that year and he was so in coordinated he threw a dart into the ceiling so in 1985 you can see the t-shirts was the time that the Lakers first beat the Celtics this is an auspicious slide to show I hope because of what's going to happen later on tonight so I actually wound up here at Irvine and was exploring out of Catalina Island discovered some amazing good fishing out there there are big populations of local of the lessons of giant axons out there in winter they're good to eat they have good electrophysiological properties here we are on an expedition to the Catalina Island Marine Laboratory so we got a boat we got a captain of the boat Chris Rhodes we were in the squid business we were catching squid out there having a great time with the barbecues and visitors and all that and they all disappeared because of El Nino and so here is the function of the giant axon you can see the squid it's got this siphon here this is a jet propulsion escape response the only one in the animal kingdom there he goes with the giant axon causing the squid to disappear just as they did in El Nino so I turned to cells that I could carry with me everywhere, namely my own blood cells so some credits the crack team and the imaging team this is my lab I'm very proud of these guys Shenyuan is now an assistant professor at Texas A&M University Melanie Matthew has I'm so thankful Melanie that you decided to stay here as a postdoc she's done a lot of imaging work she's the best experimentalist on our two-photon imaging Kim Garrett is now at Pasteur Institute working with Philippe Bousseau over there his Lou who's been working with me for the last 20 years Maria Leodino who's now working with Jim Hall Andy Yarrowman who did all the nice electrophysiology and continues to do so Debussy Sen is now left the lab he's doing a postdoc up at UCSF here's Olga Sifrino still in the lab doing great experiments Oban Pena still here looking for a job in France back in France and here is Julian Yu who's now moved to take a permanent job in Texas and I'd like to credit Mark Milner and Cindy Wei who were pioneers in developing the immuno-imaging methods that I showed you in the beginning of the talk here are my collaborators Ian Parker and Angelo here's Ian finishing the Badwater Ultra Marathon which he plans to run again for the eighth time this is the race from the depths of Death Valley in the middle of the summer 135 miles halfway up Mount Whitney an insane achievement okay and then there's George Chandy my long-term collaborator and friend we've worked on everything related to these potassium channels with Christine and Heiken a whole bunch of other people money from NIH collaborations with Hugh Rosen Jermaine Osana and my son Stuart Cowan who was a graduate student of Hugh Jason Cister and Tak Okada we've collaborated with them on the T-cell B-cell cooperation I also want to thank everybody in the Physiology and Biophysics Department all my colleagues I want to thank Bruce Tromburg for some key collaborative efforts in the mid-90s Frank LaFerreira for collaborative efforts later on on Alzheimer's related issues and I want to thank you for staying long and hope we have a good time watching the Lakers tonight thank you very much happy to answer some questions Sid and as always just one small point really for one of the assassinations by NK cell when NK cell was first described it was said to be a blood spleen department cell that seldom went into them well yes what were the circumstances that you found them there and that they were so active well Kim Garrett started studying this and yes they're there they're recruited during inflammatory response and if you're on gets involved and other side kinds that help to recruit them but there is a population of NK cells in the lymph nodes and it is a fact that they can move around and they can control in the lymph nodes they also are in the blood and spleen but we found them in the lymph nodes in the NK cells and studied NK cells in the past I appreciate your question way in the back so why do you think it differs to mind and efficiency is that just because of lack of T cell health and T cells or the crack channels crack channels probably throughout T cells not just the helper T cells but the cytotoxic T cells probably also in B lymph sites so their function is probably pretty well it's arrived one there are two other arrives to complicate matters the two other arrives do not seem to compensate for the deficit so an arrive one specific locker might be a superb immunosuppressant if you want to just wipe out the acute immune response thanks very much


All right, and if you want me to write you to our next talk with Christy Lee Manhattan from Core Scientific, she's going to be speaking with us about my opinion, which is a narrow hatch, so it's up. It is of interest to some of us here. Please, while we each come in and take a seat, we ask that if you're looking out the back, please keep the volume down as a courtesy to those who are planning to listen to the talk. I'd greatly appreciate it. But in the conversations that I've already had, I've been speaking about high, that's going to be very good to you. It's a courtesy to the people that are listening to the talk. Thank you very much, and with that, I'm going to hand it over to you for a hand. Tessie, Tessie, there you go. Nah. Well, I love the narrow hatch. Have you been having a good death ball? Good to hear. This year, I'm going to have to drop a leg. A death ball. You know, volume smells like a certain well-darned day about this morning. Speaking of hatches, today I'm going to talk to you about access to lining. What is it? Why? Why does it impact you now? Well, except for the lining, this is a cool notion. It's the concept that any human being around the world can jump in and start to do the good to do the bad. We can do what we're supposed to require. See, this is your group when you start to do the strap and ever, or especially when lining is in the lower up, in the top 10 parentheses, and it's trying to launch a lot of adoption in most areas of this analyzation. That low battery is critical to mass adoption. The video that you can see here is out quite quickly. That's why a game like Fortnite can have 250 million players versus the five of them players that game like Warped Warcraft. When you, as a human, have a very little risk to try something new, you're more likely to be game-ish. It's instinct. And the one and only top product is about taking advantage of the risk. In fact, that's all the reasons I'm so good at changing technology, gentlemen. It's one of the only industries in the world right now in our psychology, economics. Infrastructure, hardware, software, and security are so highly important. In other industries, a group of one of those players can still have the function of product. In blockchain, if you move the single part, it comes back. So today, some of the things I've worked out about our engagement of the day is making crypto cards and how we generate more users through the use of crypto for our money. How is that going to contribute to decentralization in a narrow state of the day to go? A little bit about energy consumption. Because this is one of the things that you're going to need to go a lot as developers when you're talking to outside participants, as well as outside companies. Specifically, we're going to try to stop you and prove to our algorithm. Regardless of whether it's TV-based, TV-based, or S-based. And also, our next is a solution that doesn't need a state of the almost all-facing existence. A consensus algorithm is key to the journey. User-related is how to look at your product and substitute a great group of products. Well, many part of that for the state is the features of cryptocurrency scalability and for that, the biggest trade-off for companies where a purely good group of products is symbols. And in networks, they'll have a very mature ecosystem of very cheap use of this. Think about a brand whose move has a limited standard for the products. How likely are they to purchase points with their hard data and statistics? How likely are they? The generation can ask for a morning to be carefully decided. See, the hard-working part of it is critical to use integration. Many like the part of the game of mining hardware and this is an ad for security, do you just see them again? Or are they not supposed to stick with us? But if you get that on some of the worst of the data you get in hardware, that's why it's very delicate, healthy supply chains, and a minimal power to enter. An ASIC algorithm is the acquisition of users who may have been catmined as much as their GPUs or CPUs, or participants who want to get engaged with a mineral investment. Let me show you. So this slide comes with a lot of minus signs, particularly public hardware. See, any account that has a strict card should be in the ASIC state of the network, probably, or rather, really the main ASIC supply chain. And with both of the users in confidence in a network slowed down. We can see the correlation of actual global prices. An ASIC mining factory is going to use some of the largest mining costs. They're going to tell about how low the price is set in how it creates the second correlation. But the reality is that on some of the top areas in the world, they want to see what everything is to ensure how the hardware is lagged or is not generous. This creates the longing incentives of a network participant. It creates the longing incentives for a rise in quality. Contrast is a synthesis of solid and non-superior hardware. Their data can be such as hardware or is only reserved for all of these cases. It's going to get into the CPI of the new physical logic scan to human operators, to video games and streams of the home user. Now for the CPUs, most likely, a CPU is going to have the GPU in your box, but you're limited to one, if you're a home user or have a memory device, or two, if you're a server operator, or four cases, if you're bad at this. Now, I'm going to make a pause at the time by investing to a whole system of my colleagues. Christy, Christy, what about those big, scary server calls that ends on the Microsoft app? What if I am taking trouble currently at work? Here's today. If there's a money to be made in any way, shape, or form, you are going to be incentivized by authorization.  They include authority, every single consensus algorithm. And that's okay. All you want to do is ensure that you have set yourself up for success and you're putting out your success to get as much user interaction by our needs. A participant like Amazon or Microsoft is also going to update his network of science and be open on that in later. And I was actually surprised by the people that didn't realize there are two types of miners that are going to spread around each of them today. So the math and miners are kinds of miners that are going to be going out, seeking profit. They're not going to do the $1,800 committee, but won't be able to spot the wrong comments and don't get out of the test of the miner. They are sitting aside and generally switched to one of the most popular. Now, sometimes the math and miner will follow along with the committee for operation and transition to open global, a tribalistic miner. By contrast, tribalistic miners stay open to my network, regardless of the press. These people are being human in country years when it comes to security, optimizations, applications, and spread awareness. Even during the bare walk, there are a subset of miners that stay open to both the miner and the miner, the Ethereum and the miner, and even the lack of a network. Even today with the lack of a point of habit, there is still a double limit that should pay calls at the start of the year. It is a point in our responsibility to do whatever it takes to capture as many tribalistic miners as possible. Much like that this season of traditional era, a tribalistic miner has to settle with some part of the strategy, whether it's a vision of the team, the product community, or the code. Now, a majority of miners, a majority of them are user-based by converting using the mining miners to tribalistic miners. The only way to do this without massive mining budgets is that any woman or woman can save any of the future of strategy or simply win a move. So if the miner is going to treat it to heat up the universe. Now, surprisingly, I also deal with this data all the time on here the second day, whether it calls for new internals and participants, clients, all of the generally used clients. Yes, there are some of the most 670 volts of energy currently consumed by the bit that we're going to have right now. To which we should probably win out the backing system right now through some of the current systems. Now, if we're going to find out the global climate change system, yes, power consumption is probably going to be involved. Actually, if we're going to deal with kind of the heating system, power is going to be involved. But zoom out on the big picture. See, we've got to put it into comparison. Bitcoin right now represents 0.25% of global electricity production in the world. It consumes less than 0.29% of the total electricity in the world. Now, we're going to have around electricity that we could produce for the Bitcoin network apart of 66 times the hydrogen, 90 times the volume of its waste, 22 times the solar room and the gas. See, current and current in the market is the only activity right now in the world that can turn wasted energy into cash. Funnily enough, it's funnily enough gas and solar and energy. Honestly, that energy in the market is actually incredibly expensive and a bit of a special thing. And that's the ability of this energy. Also, it's a suitable friend, so if you see more of the hardware, whatever you can think, if you are used to how much you're training and all, well, that creates incredibly powerful incentives with participants in the ecosystem right now to think about a bigger scale. Take Bitcoin out of the picture. I started with zero, so we got this problem. There was a lot of energy around the world. New coins would get a lot of power. I've tried to get it in as far as the miners. And essentially, a lot of the miners kind of can have an industry. See, there's nothing I can do to help Bitcoin at all, simply because of how much it costs to be out of what it was designed. Any sort of consumer device will not catch up with this decade of hard work. So, two things happen during the period of socialization. One is that I happened to start with more of the NLPC, the National Social Defense Council. They did a study on how I evolved in America. Basically, I learned how many devices will withstand energy and they found that access are costing Americans $19 million and so these are devices like a TV that are consuming energy all around the world, but all around the world. See, doing a lot of work out and we're down to get some devices is actually hurting all of us. So, it's getting in the industry and it's just one of the things there right now. So, there's only power leakage. This is a fact of the embedded power of our ecosystem. So, these were things like fish bombs. They found, consumed, and tolerated. Set up boxes, fans. So, I thought I'd come to mind with an aquarium and then I thought I'd still be able to do this real work.  These drones meant that I came to power set up for a day, just five. Now, Project Platinum wouldn't have worked without a target and an algorithm like MMS that was dedicated to poles on this world-wide user distribution. The economics wouldn't work. They'd have never gone funded. The hardware would never have evolved. All of a sudden, through this one simple act chasing the right to my narrow room, we managed to convert a whole new generation of people to start to get back to cars and expose them to narrow pole points and cause my pets to also come in the car. See, that's the important thing to understand. Your algorithm is just one piece of the saturation model. It's also about how many of those are just going to get adopted into your network. How many of these users came through for our aim to travel as the clients? Enter ready-nice. So, ready-nice provision. CPUs, one-to-one, as opposed to some pushing up on those vision as well. There's a little bit of a problem with that. Ready-nice is targeted towards devices that consume forward device with a narrow room. Now, this might be very popular on home desktop, but the reality is that most home desktops are more actively engaged in line. Most of the participants in the mining ecosystem now do not have home desktop anymore to sell or some sort of hand-held device they have a gaming device. Not all of these kinds of, not all of this put in action has sufficient amount. For project planning, we're working the end of the term of about 616 gigabytes of DDR3 on the board that gets into, that gets into our raw cache. So, we personally don't have a lot of problems with that project. But I started to realize that's when we started to talk to a lot of participants in core scientific and how we started to spread. We wanted to start all of this out, all of the buses. We've engaged with a lot of these, some of which I wish I had told about today, but a lot of it for a few weeks, that I will always want to see the benefits of proof of work mining. See, Rennix is very required again. The Rennix, I think the Rennix bale for embedded devices is going to be 256 megabytes, just. Now, while a CPU target is great and perfect, the memory requirement will benefit the hardware and the memory parts, such as cameras. So prior to what I found, the majority of the facial recognition cameras in places such as China, also the Asia Contents, and so Germany, this is the perfect target for grandmasse. And then I start thinking, hmm, is that really the use of participants in one hour an hour? Because that's what they're going to attract. See, that's like a lot of thinking about how it's going to be consensus measures. This subtle change tweaks the user base that they're going to attract. Now, the future of mining involves the mass consumer hardware. I know it was CPUs to GPUs to basics, and I was starting to see that there's this trend of serving mass consumer hardware on the back. It's going to come in full with embedded devices. Why? Again, it is those incentives that they made to protect. Let's just give it a little exercise for a moment. Imagine what happened in the day of the refresh, tokenizing it, and starting all the memory objects all the time. So I was wondering what you needed for that day, just depending on how long did I have access? Why did they do that? Well, and I just learned that I don't know if they're actually just high level, so, you know, maybe it's the experiment that doesn't work for you to keep on. Now, all of a sudden, I was all who was paying you for an active subscription to the machine to launch it from the fridge, sent it over to her. They got the first chance to send you an application. They say, hey, what the hell is that? Here's a special universal language. Right now, who's running on the sets? Take a step further. Imagine you have a subscription service where the woman, your machine will report a failure, and it goes up to the chain, replacing the parts of the issue. See, you're watching those ends of the line as it goes up, and it's all over. There's a sense that you have to go that way, and then it starts using your fridge and money to pay back for the cost of the life of all. You get an application and a phone and a place to call. Take the final step further. Imagine you want to buy a fridge. You can see at the start of the report, a long-duration and company life that replaces this fridge needed over the years of operation and how long it takes to see the service, how long it is over here, all of this data will report a failure system. See, these things are already inactive, actively being built behind the scenes. Small update to your proof-of-work algorithm has changed the kinds of users that are starting to engage now. And again, that will allow you to step forward right now. See, I'm not going to show you an error right now. I've been engaged by the end of the year, and an error is going to have at least two accesses and these results will go there. Simply because of the amount of devices that are going to be involved into the ecosystem. These devices we're watching on, these devices will be dedicated contributing steady cash flow. And what's going to end up happening is a failure. This always happens with mining networks. We've seen it a little bit with the Bitcoin, where you can correlate the net hash drops to actually the price. See, when you have steady, actually and steady user engagement, your price can be steady, say, sell by yours, your market price stays consistent. This attracts a lot more investment, a lot more, I guess, confidence in the network, so on and so forth. And here's who the people are. People that's only going to understand what kind of narrow it is, start working from the rates where they can actually spend, ways where they can start using that currency. They don't need to understand what the currency vision is. All they need to know is how they can residualize it into an actual piece of data.  And so, I have a urge to the community to start thinking a little bit about how this includes the randomness. That's the less it's going to demand on incentivizing the wrong kind of participants, at least I believe. But hey, who knows, I could also be wrong. So if you have any questions about my randomness, or if you have any questions about what's the work that I'm going to do, or what I hope for scientific is working on, feel free to ask me. Thank you very much. So with the project live, there's a research channel to the full security here. There's also some research specific group in there about how people can help those work. My answer is simply people who engage in the narrow community of people that make sure that we do all school, school, and incentivize the action. That is something that I would have to spend a lot of time thinking and researching. When we've talked to manufacturers, most of them are okay. They need a little care here, the device is fine to do, as long as it's consistent revenue in some shape or form. And as long as they can ensure that they have the ability or they can track, hey, if I send three shares to the pool, did I pay for three shares? So my answer is, no, it's civilization. The other way to play this is by having an algorithm which distributes percentage of the action to different pools. So say you have a hundred money for games that allocate a hundred five to pool X, a hundred five to pool Y, and so on and so forth. That's also what I'm going to play. You're going to lose. Excellent. Smartpapers or smart TVs? You'd be surprised by them. And then I'm very, very proud of them today. So most of the top of the line is Samsung Smartpapers. Actually, not a very good device. Same as previous. Now, the workers' rooms. I told the government, here's what I did in general, see a play out of key tabs. If all of a sudden the company that Samsung gets a great deal, where they can sell and take tangles or pay back or they can give tangles and look towards to the consumer, that is the one they have or they can have theirs at Sony. Then, Sony is going to be incentivized by open purchase remembering and operating their new TV to forward the back to the managers. We saw this actually happening in 2017 in the great, my perfect brush, working in a new process. So, which was, which was crazy. And I looked at that, and I looked at all the projects as well. And that told me a lot about how manufacturers get directly tied to people for their incentives, even though not included. So, that's what I imagine is going to happen there. Now, the whole user is going to get easier. See, there are probably FPGAs and ASICs on the sustaining their own hardware system. A few of the participants here also know this. So, this is going to be all ASICs and hardware. I am plus to the surprise, aside from the fact that Monero has one, one somewhere on our building where we can put our own framework to the ASIC and bypass the JET. Other than that, Monero is going to be forward. Meaning, I could do an FPG that's straight forward right now and it would be only about 1.3X more format on a GPU you're going to get in the most general. So, it's going to be fairly, fairly down there. So, very exciting. Mm-hmm, exactly. So, here's the extra part of that. For a bigger company based in the US, they're going to be requiring the entire lens off, meaning that it would have to be used as an option to opt in to this service. And then it's entirely up to the kinds of user, meaning that they control all of the actual product keys. Now, when it's a company outside of the US, most of them are going to take a wide set of ties to actually directly control the line and directly control the rules to stop them. So, with a larger company, you naturally need a wide set of ties to whatever makes a company mine. So, it is a way. Now, the way to flip this around, is to require it to work with what's already going on, where the lining rules are abstracted. In a company like India, where the lining is actually legal right now, and for the company, it is a bit sketchy. How do we be able to directly convert the mining and narrowing to via without any, and possibly not able to be to the owner of the order, or to be, or any form of local points or local points, if you're on a gaming system, except now, might be able to work with everybody to allow that. Thank you, everybody. You're welcome. The developers have looked at the blockchain ecosystem as the whole. You don't pay attention to us, and that's okay. Well, I can do it with the put out of this case. I can contact people, comment on of course, scientific submission is with the blockchain space, yeah. But we can depend to keep providing infrastructure and back home services. And one of the things that's very interesting to realize now is that most of the AC manufacturers are the developers that are doing that. So AC manufacturers right now are spending significant resources in investing in the FPGA and GPU technology. We've seen this with Renaissance from Minnesota, which are essentially just GPUs. We're seeing this in the area of PGA's, they're currently only, and we're starting to see this with the developers in the research of FPGA technology. So there are some things that are going to be closely tied to the internet, and that's something that we need to watch out for, because as we have more distance, the network will more power over the years and more risks are being covered.