WEBVTT 00:00.000 --> 00:02.000 You 00:31.000 --> 00:42.400 But you can tell if someone's lying, you know, you can sort of feel it in people 00:43.680 --> 00:48.640 And I have lied. I'm sure I'll lie again. I don't want to lie. You know, I don't think I'm a liar 00:48.640 --> 00:54.040 I try not to be a liar. I don't want to be a liar. I think it's like really important not to be a liar 00:54.040 --> 01:04.340 I'm not sure exactly who he is. His name is JJ Cooey, and I believe he's a consultant for CHD 01:04.340 --> 01:12.040 And I believe he has a PhD in some sort of scientific discipline from what I understand 01:24.040 --> 01:31.040 I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. 01:54.040 --> 02:01.040 I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. 02:24.040 --> 02:38.040 I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. 02:38.040 --> 02:46.040 I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. 02:46.040 --> 02:52.040 I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. 02:52.040 --> 03:08.040 I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. I don't want to be a liar. 03:08.040 --> 03:15.040 My friend Denver mixed date. 03:15.040 --> 03:38.600 I wanted to mix it up a little bit, maybe get some other slides out. 03:38.600 --> 03:42.520 We haven't used in a while, I know they're a little more reading. 03:42.520 --> 03:44.720 It's a little more reading. 03:44.720 --> 03:51.520 It's like we got ourselves some readers in the house. 03:51.520 --> 04:01.160 Jimmy Doar made a reference to the reader joke, very, very good Bill Hicks joint, very nice 04:01.160 --> 04:02.160 bit. 04:02.160 --> 04:13.520 Jimmy, Jimmy Doar showed a lot of courage the other night, I'm giving him another shout-out. 04:13.520 --> 04:18.120 It is amazing how many people think they can answer an argument by attributing bad motives 04:18.120 --> 04:23.320 to those who disagree with them using this kind of reasoning you can believe or not believe 04:23.320 --> 04:30.320 anything about anything without having to bother to deal with facts or logic. 04:53.320 --> 05:06.920 It's amazing how many people think they can believe or not believe it or not believe it 05:06.920 --> 05:34.280 or not believe it or not believe it or not believe it or not believe it or not believe 05:34.280 --> 05:36.880 it or not believe it or not believe it or not believe it or not believe it or not believe 05:36.880 --> 05:43.880 it or not believe it or not believe it or not believe it or not believe it or not believe 05:43.880 --> 05:50.880 it or not believe it or not believe it or not believe it or not believe it or not believe 05:50.880 --> 05:57.880 it or not believe it or not believe it or not believe it or not believe it or not believe 05:57.880 --> 06:04.880 it or not believe it or not believe it or not believe it or not believe it or not believe 06:04.880 --> 06:09.880 it or not believe it or not believe it or not believe it or not believe it or not 06:09.880 --> 06:16.880 believe it or not believe it or not believe it or not believe it or not believe it or not 06:16.880 --> 06:22.880 believe it or not believe it or not believe it or not believe it or not believe it 06:22.880 --> 06:28.880 or not believe it or not believe it Or not believe it or not believe it or not believe it 06:28.880 --> 06:33.520 Oh, that was a bad wolf. 06:33.520 --> 06:34.520 That went wrong. 06:34.520 --> 06:35.520 Sorry about that. 06:35.520 --> 06:37.440 We're just going to go through that one. 06:37.440 --> 06:41.440 That was an old slide that didn't need to go that fast, but that's okay. 06:41.440 --> 06:43.560 The stupor is definitely real. 06:43.560 --> 06:45.440 Brick soup is for lunch. 06:45.440 --> 06:48.920 No soup for you there, Mr. Mark. 06:48.920 --> 06:54.760 You had a really good show yesterday reading some kind of document that I actually never 06:54.760 --> 06:58.800 had any faith would actually exist, but you can still learn that biology thanks to 06:58.800 --> 07:05.400 Mark digging up some of these old descriptions of how early vaccine and let's let's call 07:05.400 --> 07:06.400 them what they were. 07:06.400 --> 07:08.040 They were, they were variolations. 07:08.040 --> 07:11.320 They were variolation preparation. 07:11.320 --> 07:16.840 But this is how they addressed smallpox back then and I think we are just up against 07:16.840 --> 07:23.720 one of the most demonic illusions that has ever been perpetrated on humankind. 07:23.720 --> 07:27.400 Very nicely done here in this graphic. 07:27.400 --> 07:32.000 Don't forget that they danced, don't forget that they kept people who had been married 07:32.000 --> 07:35.120 for 50 years apart in their last days. 07:35.120 --> 07:40.480 Don't forget that they, what they did to us, don't forget, you got to understand what 07:40.480 --> 07:41.480 this is about. 07:41.480 --> 07:51.640 This is a battle, you know, it's a battle between good and evil and yeah, we are trying 07:51.640 --> 07:59.800 to put forth some argument about biology, but even this cartoon might be largely a cartoon 07:59.800 --> 08:02.720 imposed upon us by them. 08:02.720 --> 08:09.640 Most of our understanding is dependent on their, on the very science that they have funded 08:09.640 --> 08:13.240 and put in front of us and this illusion is just, you know, it's something that we've 08:13.240 --> 08:15.960 really got to break through for our children. 08:15.960 --> 08:17.760 There are people who are learning biology. 08:17.760 --> 08:21.480 There are people who knew better and we've got to isolate those people who knew better 08:21.480 --> 08:28.320 and make sure that they, they don't rob us at the last little hope that is available. 08:28.320 --> 08:29.640 And that is in our history. 08:29.640 --> 08:35.280 It's our recent history and our more distant history and the people that are involved in 08:35.280 --> 08:38.600 it, what they did, what they said, what they wrote, what they thought. 08:38.600 --> 08:44.280 Those ideas are still the trap within which we find ourselves and the people that wove 08:44.280 --> 08:50.080 that trap and are involved in maintaining it are the people we need to identify. 08:50.080 --> 08:54.680 I don't think if you watch television or skillfully use social media, you're going 08:54.680 --> 08:59.040 to identify any of the people that matter. 08:59.040 --> 09:03.840 Even when paintings like this exist, you're still not quite seeing, you're seeing still 09:03.840 --> 09:06.640 what they want you to see. 09:06.640 --> 09:11.160 And only in a place where your tongues are completely free are we really free and in 09:11.160 --> 09:18.080 this day and age when a room like this could exist, where Twitter could be an illusion 09:18.080 --> 09:24.280 that is entirely created by rooms like this all around the world. 09:24.280 --> 09:26.320 We've got to keep swinging, ladies and gentlemen. 09:26.320 --> 09:27.320 We've got to keep swinging. 09:27.320 --> 09:29.640 Oh, that definitely was Steven Pinker. 09:29.640 --> 09:31.600 That was definitely Steven Pinker. 09:31.600 --> 09:32.600 Yes, sir. 09:32.600 --> 09:34.600 Rebob, it was. 09:34.600 --> 09:35.600 Yes, sir. 09:35.600 --> 09:36.600 Rebob, it was. 09:36.600 --> 09:42.480 Do you remember this song from way back when what's going on? 09:42.480 --> 09:43.480 Oh, sorry. 09:43.480 --> 09:44.480 Hold on. 09:44.480 --> 09:45.480 Let me pause that for a second. 09:45.480 --> 09:50.920 If you remember this song, I think you must remember this song. 09:50.920 --> 09:53.120 It was used in a while ago. 09:53.120 --> 09:54.720 It's a nice one. 09:54.720 --> 09:57.920 It's from the late 80s, I believe, in Australia. 09:57.920 --> 10:04.360 And it was for the Australian Broadcasting Company back in the 80s, I believe. 10:04.360 --> 10:07.200 And it's a really peppy news song. 10:07.200 --> 10:10.120 If you've been here for a while, you're here at the top of the wave. 10:10.120 --> 10:13.720 And if you're joining me for the first time at Gigo and Biological, then you might be 10:13.720 --> 10:18.560 a skilled TV watcher or what we call a skilled social media user. 10:18.560 --> 10:20.680 And you might not be staying focused on the biology. 10:20.680 --> 10:23.760 You might be taking their bait, and you might not love your neighbor. 10:23.760 --> 10:30.200 And we're going to suggest that those strategies need to be reversed. 10:30.200 --> 10:34.600 It could be back when ABC was probably more relevant. 10:34.600 --> 10:36.520 Thanks for spreading the word, ladies and gentlemen. 10:36.520 --> 10:38.160 Thanks to the supporters. 10:38.160 --> 10:40.280 Thanks to the people who are spreading these streams. 10:40.280 --> 10:42.120 This is Gigo and Biological. 10:42.120 --> 10:54.040 A high-resistance, low-noise information brief brought to you by a biologist. 10:54.040 --> 10:57.840 And now, since I haven't used that music in a while, this timing was a little off, and 10:57.840 --> 11:01.120 that thing paused, and who knows why that paused. 11:01.120 --> 11:02.760 I guess I had to tap that one again. 11:02.760 --> 11:05.080 I'm just sorry about that. 11:05.080 --> 11:08.640 There's some old slide combinations here for the beginning. 11:08.640 --> 11:15.120 I just thought I'd break out something new, old, new, old, new kind of thing. 11:15.120 --> 11:17.200 Welcome to the show, ladies and gentlemen. 11:17.200 --> 11:19.920 This is Gigo and Biological. 11:19.920 --> 11:22.720 The paradigm is shifting right now. 11:22.720 --> 11:25.000 There are a lot of moving parts. 11:25.000 --> 11:31.520 It's not something that can be easily understood, but definitely it is responding to our work. 11:31.520 --> 11:36.080 What we do is having a difference on how people try to push this forward. 11:36.080 --> 11:40.760 So let's move the ball forward by not participating in their illusion. 11:40.760 --> 11:44.280 That's really the strategy that I'm advocating for. 11:44.280 --> 11:49.000 This is, again, Gigo and Biological, a high-resistance, low-noise information brief brought to you 11:49.000 --> 11:50.000 by a biologist. 11:50.000 --> 11:53.200 I got my shirt on today. 11:53.200 --> 11:55.160 Welcome to the show. 11:55.160 --> 11:56.920 I look, oh, I know what it is. 11:56.920 --> 12:01.280 These little tiny front lights aren't on, so I look a little better. 12:01.280 --> 12:03.280 It might be a little better. 12:03.280 --> 12:06.800 Somehow I look a little, I don't know, greasy or something. 12:06.800 --> 12:08.480 Anyway, welcome to the show. 12:08.480 --> 12:12.480 This is Gigo and Biological coming to you live from the back of a garage in Pittsburgh, 12:12.480 --> 12:14.480 Pennsylvania. 12:14.480 --> 12:15.680 Thanks for joining me. 12:15.680 --> 12:20.160 Today, we have a little bit of work to do as far as the study hall goes. 12:20.160 --> 12:23.680 It's quite a long video, but I'm going to put it at 1.5 speed and we're going to take 12:23.680 --> 12:28.400 some notes and see what we get from Stanley Prusner's presentation from 2002. 12:28.960 --> 12:34.160 First, I would like to at least comment on the fact that Jessica Hockett, myself, and 12:34.160 --> 12:47.440 a friend of Jessica named John, scanning, hold on, that's the wrong male, why did that 12:47.440 --> 12:57.920 open up, see, I'm starting to get angry at a certain somebody, well, I don't know what 12:57.920 --> 13:01.440 his name is off the top of my head and I'm not trying to be a creep about it, that's 13:01.440 --> 13:02.440 the way it is. 13:02.440 --> 13:08.040 I apologize for that, I should have had that written down, it starts with a K and a very 13:08.040 --> 13:09.040 clever guy. 13:09.040 --> 13:13.200 The three of us presented, Jessica presented the longest in the beginning, about a half 13:13.200 --> 13:17.720 an hour, maybe a little bit longer than that and then John presented it and I presented 13:17.720 --> 13:21.200 very quickly about five or eight minutes each. 13:21.200 --> 13:24.920 I think the message was pretty clear. 13:24.920 --> 13:28.960 The message was a little bit of biology, but just a lot of what the hell happened in 13:28.960 --> 13:34.440 New York City to explain why we didn't investigate it yet, while there isn't a memorial yet, 13:34.440 --> 13:38.320 while the names haven't been released yet, why we haven't explained how this all happened 13:38.320 --> 13:42.760 in such a neat, uniform way and didn't happen in Chicago, even though they had a case a 13:42.760 --> 13:47.880 year or a month before that and so I felt like it was a decent thing, I thought it went 13:47.880 --> 13:48.880 pretty well. 13:49.360 --> 13:57.040 Unfortunately, we were briefing staffers and although I could only see two staffers on 13:57.040 --> 14:05.880 screen, those two staffers were, my best guess is millennials and one of them told us that 14:05.880 --> 14:13.000 she had a master's in public health and that any primary literature I could provide to 14:13.000 --> 14:18.200 back up any of the things that we said would be greatly appreciated and at that stage it 14:18.200 --> 14:22.800 really dawned on me one of the things that has driven me nuts since the beginning of 14:22.800 --> 14:28.360 the pandemic but I haven't really been able to express adequately in words but if anybody 14:28.360 --> 14:34.400 ever says to you to send some primary literature to them which could help them understand something 14:34.400 --> 14:44.640 unless it's in an extremely specific case of misrepresentation of data or a very specific 14:44.680 --> 14:50.920 case of a representation of a very specific situation, there is no combination of primary 14:50.920 --> 14:55.760 literature papers under the number of 5,000 that could be sent to somebody so that they 14:55.760 --> 15:01.720 could get an adequate idea of how distorted biomedical sciences has become as a result 15:01.720 --> 15:08.760 of the application of this p-value ritual over the last 20 or 30 years and with intention 15:08.760 --> 15:09.760 that is. 15:09.760 --> 15:14.440 It's not to say that science can't be productive and can't figure things out but what I'm suggesting 15:14.440 --> 15:24.200 to you is that the distortion, the intentional distortion of certain parts of the general 15:24.200 --> 15:28.640 scientific understanding and our education surrounding it has resulted in the place that 15:28.640 --> 15:34.240 we are, allowed us to be led to a place where we can no longer exercise informed consent 15:34.240 --> 15:46.320 because we simply don't know and trying to hit a home run in that scenario is basically 15:46.320 --> 15:52.320 where I went wrong, I started this, I'm going to show you the slides that I used and try 15:52.320 --> 16:00.040 to give the talk as I intended it but since I felt as though John abbreviated his presentation 16:00.040 --> 16:05.320 greatly in order to facilitate my presentation and I already felt like the staffers were 16:05.320 --> 16:09.280 looking at their watches and deciding whether they could stay any longer or not or whether 16:09.280 --> 16:15.440 they needed to listen a couple times, the kind of the head staffer or the one that was speaking 16:15.440 --> 16:21.000 the most into the microphone said that she's trying to get at our main idea when Jessica 16:21.000 --> 16:27.640 was talking and it's funny because Jessica started her presentation with the main idea 16:27.640 --> 16:31.560 which is that we want you to figure out what the hell happened in New York City because what 16:31.560 --> 16:38.280 happened there was not a natural event, it was like a bomb went off, it was a man-made disaster 16:38.280 --> 16:43.640 and we want you to investigate it and then she explained all the stuff and so it's weird the 16:43.640 --> 16:48.240 staffer was still kind of circling back to that stuff so by the time I got the mic John had 16:48.240 --> 16:54.520 already presented and John had some really nice slides that were actually tacked right on to 16:54.520 --> 17:01.120 the slides that Jessica had so they could flawlessly change to that and he had one or two slides 17:01.120 --> 17:06.320 in particular that I want to get him on my stream to talk about which are really slides 17:06.320 --> 17:11.440 that I've asked actually I believe I've asked Denny Rankor a couple times if he could make 17:11.440 --> 17:24.800 them which would be to let me see if that's the guy yeah that would be the guy if he could 17:24.800 --> 17:32.880 make a slide where you did the American data but you took the New York City event out and he had 17:32.880 --> 17:41.520 that slide and it's actually very striking how intuitively it felt like wow they're really 17:41.520 --> 17:46.960 using that curve to imply something else because of 24,000 people died in that week and he spread 17:46.960 --> 17:52.640 them out around America there would still be kind of a signal especially if you started that signal 17:52.640 --> 17:59.280 from zero right and you know in America 24,000 people have died of COVID so far but the vast majority 17:59.280 --> 18:09.680 are in New York City asterisk so anyway I started off already with a very good background of course 18:09.680 --> 18:16.640 so 45 minutes 40 minutes of Jessica's presentation eight minutes of John's statistical you know look 18:16.640 --> 18:22.080 if you take New York out it's crazy if you look at New York by itself it's so statistically off 18:23.040 --> 18:29.120 off I mean we're not talking about p-values here you know we're talking about z-score and 18:31.120 --> 18:36.880 we are we were way way way way and these nobody making these statistics ever thought that 18:38.480 --> 18:43.040 excuse me ever thought that something like this would be you know a z-score of 200 or something 18:43.040 --> 18:49.040 like that so my presentation couldn't really go too deep into the biology so I started with the 18:49.120 --> 18:54.480 idea that informed consent couldn't be exercised by anyone at the start of the pandemic because 18:54.480 --> 18:59.360 we were misled about the biology and about the whole experience of what was happening 18:59.360 --> 19:03.920 and that New York City as Jessica and John have pointed out was really central 19:05.040 --> 19:08.560 to establishing that there was even a pandemic happening and therefore 19:10.320 --> 19:18.160 I started with again reminding people of this and so we were consciously manipulated into believing 19:18.160 --> 19:22.080 that a crisis was occurring that it was ongoing that something was spreading in the 19:22.080 --> 19:26.960 background this kind of thing and this manipulation was on purpose to get us to accept that this 19:26.960 --> 19:32.800 crisis was occurring but the only numbers we have are the numbers that Jessica just showed you 19:33.680 --> 19:40.240 are almost assuredly fraudulent that John also backed up with statistics that seem to indicate 19:40.320 --> 19:50.560 that it's almost assuredly a man made event and I would argue that that man made event 19:50.560 --> 19:58.320 allowed them to imply the existence of a novel virus that that you and and Senator Ron's staff 19:58.960 --> 20:05.520 are very much in belief existed and the reason why you're in belief of it I would argue is because 20:05.520 --> 20:11.040 they fooled you into thinking that the only question was whether it was a bad cave virus or 20:11.040 --> 20:16.960 whether it was again a function lab league and this mystery that we've been solving over the last 20:16.960 --> 20:22.960 four years has bamboozled all of us into accepting that what we were told in New York City was the 20:22.960 --> 20:31.040 start of something very very terrible was actually a mass casualty event that was misconstrued as this 20:31.360 --> 20:40.480 and the beginning of this the beginning of this whole mystery and I would argue that one of the 20:40.480 --> 20:46.720 ways that they did it and I went from this to this was that they didn't they didn't genuinely 20:46.720 --> 20:52.960 describe what all cause mortality was they didn't actually tell you that you know the excess deaths 20:52.960 --> 20:58.400 where we're experiencing can come from all kinds of things especially if we told doctors with fear 20:58.400 --> 21:04.080 and uncertainty and doubt that there was a new thing that you had to be afraid of yourself 21:05.440 --> 21:09.360 and that if this new thing was there you should treat these people entirely different than you 21:09.360 --> 21:12.480 would have ever treated them had you not been told that this new thing was there 21:14.640 --> 21:21.600 and all of those new things that we were told to do started killing people and nobody acted because 21:21.600 --> 21:26.240 there was this illusion of consensus that it was either a novel virus or a lab leak that we were 21:26.240 --> 21:33.920 responding to and that these people were dying from and the tools that they use to identify it 21:33.920 --> 21:39.600 the financial incentives that they they use to amplify it all of these things are completely 21:39.600 --> 21:47.200 ignored by the people that were just in front of you in that senate committee almost none of this 21:47.200 --> 21:52.640 part of the narrative where hundreds of thousands of Americans were murdered at the beginning of the 21:52.640 --> 22:00.560 pandemic in 2020 and 2021 that's wholly ignored and completely attributed only to the novel virus 22:00.560 --> 22:05.680 and all of these people's narrative and this is very dangerous because of course as Jessica just 22:05.680 --> 22:12.560 showed you New York City is this bump right there that bump right there is New York City and I was 22:12.560 --> 22:20.480 actually already over here down here and so the mind this bump right here is New York City 22:21.360 --> 22:28.800 and this could just be people acting really really badly this little rise here there's a 22:28.800 --> 22:34.000 lot there's a rise there right you can see that right there's not a dip that dip is missing this 22:34.000 --> 22:38.960 little pattern just completely gets disrupted and now all cause mortality is permanently elevated 22:38.960 --> 22:49.280 by a combination of confusion and stupidity bad ideas and transfection and the initial casualty 22:49.360 --> 22:55.760 event is something that Jessica showed you is not a natural phenomenon does not belie the spread of 22:55.760 --> 23:05.440 a virus but instead suggests a staged event now the reason why I think it's important to understand 23:05.440 --> 23:12.080 this is because these people who were in front of you in the senate not even a month ago and this is 23:12.080 --> 23:18.080 exactly what I said in front of your boss excuse me who are also at an event in Romania in November 23:18.080 --> 23:25.280 of 2023 where they heard Denny Rancor the guy in the square speak about these effects who he 23:25.280 --> 23:31.200 has actually been speaking since 2020 about the fact that the only deaths that are excess are 23:31.200 --> 23:37.040 correlative with poverty level household income serious mental illness obesity and the excess 23:37.040 --> 23:43.600 mortality is a direct correlation with all these things across states irrespective of whether they're 23:43.600 --> 23:51.760 a blue state or a red state and so he suggests that there's no evidence of spread of a particular 23:51.760 --> 23:56.560 novel pathogen there's evidence of bad ideas in the form of protocols and how they treat or don't 23:56.560 --> 24:03.360 treat people and until the vaccines are released the all cause mortality doesn't go up significantly 24:03.360 --> 24:08.800 with any pattern that would indicate a respiratory virus but all of those people when they were in 24:08.800 --> 24:15.760 front of you last month none of them said that they all spoke very very generously about the 17 24:15.760 --> 24:22.480 million people that Denny Rancor said might be or estimated might be having been killed by the 24:22.480 --> 24:28.320 transfections but they conveniently left out that his data also shows that there's no evidence of 24:28.320 --> 24:37.120 real novel spreading pathogen at risk additives spreading pathogen in 2020 and 2021 and this is 24:37.120 --> 24:43.120 very crucial because those same people want us to interpret that what happened in the last 24:43.680 --> 24:50.240 four years well there was an RNA that caused a pandemic but transfection worked pretty well 24:50.240 --> 24:56.800 although we rushed it and so they want justice for the RNA that was released and should never have 24:56.800 --> 25:02.080 been made and they want justice for the people that were injured because we rushed transfection 25:02.080 --> 25:13.520 before it was ready and this illusion is this this this solution is a lie the reality is is 25:13.520 --> 25:20.000 that transfection in healthy humans was criminally negligent all along and that every biologist with 25:20.000 --> 25:26.000 an academic bench in any accredited university in America should have known that transfection is a 25:26.000 --> 25:32.800 long-standing technology even using a product like lipofectamine is a long-standing technology 25:32.800 --> 25:38.080 that's been used on the academic bench for acute expression of proteins in order to manipulate 25:38.080 --> 25:46.160 systems and understand and test hypotheses about different metabolic pathways in any 25:46.720 --> 25:52.000 any biological system however transfection is never shown proven 25:52.960 --> 25:58.000 successful in the treatment of anything that's not a deadly disease like a very specialized 25:58.000 --> 26:04.400 cancer and the fact that they either didn't speak up because they didn't know or didn't speak up 26:04.400 --> 26:11.040 because they were unwilling they did not have the principles to dictate that they should for the 26:11.040 --> 26:17.760 betterment of mankind for the protection of our grandkids from this illusion they're all culpable 26:17.840 --> 26:23.200 they're criminally negligent I lost my job at the University of Pittsburgh School of Medicine 26:23.200 --> 26:28.640 because I spoke out about that specific thing and since then in the four years that I've been trying 26:28.640 --> 26:37.840 to understand what is and what isn't well understood RNA virology I've come to the conclusion that RNA 26:37.840 --> 26:44.080 cannot pandemic and they've always known it in fact it's a it's a central sort of 26:44.400 --> 26:52.400 it's a foundational part of the understanding we have or don't have about RNA viruses that they are 26:52.400 --> 26:59.040 almost intractable in culture because of the nature of how RNA viruses and especially RNA viruses like 26:59.040 --> 27:07.040 coronaviruses are are purported to replicate and those shortcomings are overcome by a technology 27:07.680 --> 27:14.480 called infectious clones and that's why with regard to ignoring all of these things these 27:14.480 --> 27:20.400 same people that were in front of you will never really enunciate a hypothesis which has anything 27:20.400 --> 27:27.840 to do with that that core technology of RNA virology which is infectious clones using DNA to make the 27:27.840 --> 27:35.360 RNA that is infectious and so our hypothesis my hypothesis is that the who declared a particular 27:35.360 --> 27:40.400 a dangerous pandemic of a particular sign and that's not obviously I said it better than that 27:40.400 --> 27:47.120 the the who declared a pandemic of a dangerous novel virus and it was a background signal that 27:47.120 --> 27:55.040 they that they misconstrued as spread they used an event in New York City to to create the illusion 27:55.040 --> 28:01.520 that that an impending crisis was coming and at the same time they already had a plan where they were 28:01.520 --> 28:08.800 ready to invert to change pneumonia and influenza what would have normally been classified as 28:08.800 --> 28:15.440 pneumonia and influenza to a new and novel respiratory pathogen that was a was a national security 28:15.440 --> 28:22.320 priority because this plan was in existence because not any kind of special technology or 28:22.320 --> 28:29.760 decree would be necessary for this to occur and because the vast majority if not the entirety of 28:29.760 --> 28:38.080 the deaths that occurred in 2020 can be explained by the confusion and fear and and 28:38.800 --> 28:45.440 incorrect behavior that resulted from the financial incentives from the testing and from the the 28:45.440 --> 28:53.360 messaging on on television there is no way that an RNA molecule can sustain a pandemic however there 28:53.360 --> 28:59.040 is a way for that sequence to be found in many places around the world and that again is an 28:59.040 --> 29:04.240 infectious clone and that could have been used if that was part of the national security 29:04.240 --> 29:12.160 operative to make operation to make absolutely sure that there was a seamless body of evidence 29:14.720 --> 29:20.720 that would support the idea of a circulating novel pathogen and would support the idea of 29:20.720 --> 29:26.880 rolling out this national security operation en masse would support the idea of co-opting 29:26.880 --> 29:32.080 anybody that ran to New York City and tried to save the day and found out that it wasn't what 29:32.080 --> 29:40.160 they said it was on TV and it would justify the use of several different groups of optoratives 29:40.880 --> 29:46.960 behind the scenes to make sure that this narrative never really got off the rails that nobody ever 29:46.960 --> 29:52.880 questioned a novel virus so that even when we got four years into the pandemic and people were in 29:52.880 --> 29:58.640 front of the senate and speaking about it that the novel virus would never be questioned that 29:58.640 --> 30:03.680 how many people died from it would never be questioned that we would already have moved on to whether 30:03.680 --> 30:08.160 or not the transfection worked or didn't work and how many people were hurt by it 30:10.400 --> 30:15.760 and this theater is actually an orchestrated attack on the united states from the from within 30:16.320 --> 30:21.040 orchestrated by people outside of the united states in cooperation with people inside of the 30:21.120 --> 30:27.200 u.s. government and that's what i think we need to understand most is that this is not an this 30:27.200 --> 30:32.880 is a national security issue for americans and what's happening in canada is separate from us 30:32.880 --> 30:38.720 and their emergency what happens in australia is their emergency and even the people that were in 30:38.720 --> 30:46.880 front of ron johnson in the senate are very very particular in how they express the emergency it's 30:46.960 --> 30:50.880 all of us it's everyone it's the who the globe has threatened no 30:52.960 --> 30:59.040 the only handles of power that we have the only way that we can act is through our government within 30:59.040 --> 31:06.960 our borders within our legislative system that's all we've got once we start trying to organize 31:06.960 --> 31:13.520 with australians or organize with canadians or or or then we're actually doing their work 31:13.520 --> 31:16.240 for them we're we're globalizing in a different way 31:18.160 --> 31:23.680 we need to decentralize that means that we don't need the help of people in other countries they 31:23.680 --> 31:32.400 have their own country to save and i think that's part of the reason why you should be very suspicious 31:32.400 --> 31:36.560 of having a senate hearing where half of the people are from different countries 31:37.360 --> 31:41.280 where half of the people have been on stage with the other half of the people in other countries 31:41.280 --> 31:49.600 multiple times why were they invited them none of this stuff was said to the staff i stopped a 31:49.600 --> 31:54.240 long time ago already but these were the things that i used i had this slide in there but i didn't 31:54.240 --> 32:01.440 really read it because i didn't i just went back after i what after this slide popped up i said i 32:01.440 --> 32:06.720 don't think i should use that slide should leave it here let you read this and then i'll stop for 32:06.720 --> 32:12.640 any questions and there weren't any questions of course but that was already an hour into the 32:12.640 --> 32:19.280 meeting and i think the out the meeting was supposed to be a half an hour and so they were ready to 32:19.280 --> 32:26.880 leave as soon as i quit so some things went really well other things weren't ideal i don't think my 32:26.880 --> 32:34.080 presentation was ideal i was a bit riled up i was going too fast i had already become frustrated 32:34.080 --> 32:38.720 with one of the guys one of the people on the staff because of an earlier comment 32:41.680 --> 32:45.920 i'll just i'll just comment on it now at one point in time when jeff was talking 32:47.040 --> 32:54.880 uh he was saying that um that there is evidence from previous winters and flu seasons that you know 32:54.880 --> 33:01.280 we've had nastier flu seasons in the past that we've not really made any fuss about and so what if 33:01.280 --> 33:06.480 these are actually coronaviruses in the background this background signal is always there and now 33:06.480 --> 33:12.560 they just misconstrued it as something else um of course i'm parsing his words incorrectly but 33:12.560 --> 33:17.840 she said um oh that's really interesting because of course there's lots of evidence or some people 33:17.840 --> 33:23.840 are saying that it was circulating in the background before 2020 and uh we didn't have any PCR testing 33:23.840 --> 33:30.320 and so that's what explains all this and they kind of went on but didn't go on because i think jeff 33:30.400 --> 33:35.760 was just trying to hurry up and he kind of acknowledged it um tried to sort of say 33:35.760 --> 33:42.880 something but it was the way that she trailed off her her comment that made it kind of just kind 33:42.880 --> 33:48.320 of stumbled through and ended up the presentation moving on and then i unmuted really quick and i 33:48.320 --> 33:54.320 was like whoa whoa whoa whoa i just want to go back one step because what um she just said we really 33:54.320 --> 33:59.920 need to point out a little better how ridiculously contradictory that statement would be how holding 33:59.920 --> 34:05.120 those two thoughts in your head shouldn't be possible if you listen to Jessica's presentation 34:05.120 --> 34:13.040 and you heard that New York City is this you know highly isolated event with you know orders of magnitude 34:13.040 --> 34:19.440 more dead bodies than in any other weeks that have ever come before after that and it's a very 34:19.440 --> 34:26.240 strange anomaly that occurs with at-home deaths and also nursing home deaths and all these things 34:26.240 --> 34:38.800 are at the same time um and so it's very strange um to believe that that represents a the spread 34:38.800 --> 34:48.640 of a particular dangerous pathogen those deaths but it was spreading before that and we didn't have 34:48.640 --> 34:56.160 tests and 20 000 people for a week weren't dying because we weren't locked down we weren't aware 34:56.160 --> 35:02.400 we were all just going to restaurants and drinking and and nobody was wearing masks and 35:03.600 --> 35:08.720 and so at some point you know like there was this oh yeah but it's a variant and all it's 35:08.720 --> 35:14.320 this and what about the lockdowns and you know so there was a lot of discussion but the end result 35:14.320 --> 35:22.400 always with Jessica's data is that but you can't explain this with that hand waving because it 35:22.400 --> 35:28.640 essentially death starts out very normal at 4 000 people per week and then goes 35:31.120 --> 35:39.280 or 4 000 people per April and then it goes and there's no biological explanation for that 35:42.240 --> 35:47.120 there would be EMTs that are traumatized from the sheer number of bodies they had to handle 35:48.080 --> 35:53.280 there would be nurses that are traumatized from the sheer number of people 35:56.080 --> 36:00.240 there would be memorials because it's like eight nine eleven events 36:01.760 --> 36:08.320 and so if if it was circulating before then but we didn't know how to track it and we were not 36:08.320 --> 36:14.080 doing anything to close down the borders then how in the hell didn't more bombs go off in other 36:14.080 --> 36:22.960 cities before this and she got very defensive and was like I don't think that's what I said I 36:22.960 --> 36:26.880 don't I don't I don't remember exactly what I said but I don't want to repeat it because I'm 36:26.880 --> 36:35.280 afraid you'll get it I'll get it wrong or I'll you'll take it out of con no you can't say that a 36:36.480 --> 36:41.360 deadly novel pathogen was circulating but it didn't kill anybody before we started testing for it 36:41.360 --> 36:46.960 or we didn't kill anybody before Tony Fauci made the 15 days to stop the spread announcement 36:53.920 --> 36:59.760 so that's where we were um you know that we're still right here right intramuscular injection of 36:59.760 --> 37:04.000 any combination of substances with the intent of augmenting the immune system is dumb 37:04.000 --> 37:09.280 transfection in healthy humans is criminally negligent in RNA cannot pandemic we're also 37:09.280 --> 37:15.440 beyond our own hypothesis here we're also trying um yeah I forgot I wanted to say I'm 37:15.440 --> 37:19.600 wearing Jimmy Dorr's shirt and I saw Jimmy Dorr the other day and I think although he doesn't have 37:19.600 --> 37:23.280 everything right he still thinks there was a novel virus it's probably gained a function and they 37:23.280 --> 37:31.440 lied about it I do think that his courage comes from a place without expertise right so all he 37:31.440 --> 37:35.600 knows for sure is that he was injured by the vaccine and people didn't take him seriously and that 37:35.680 --> 37:40.000 pissed him off and he realized that people were lying to him and now he's people he's realizing 37:40.000 --> 37:45.360 that a lot of people are liars and a lot of people can hold contradictory positions in their head 37:45.360 --> 37:51.120 and so he's as frustrated as I am that at some point in time it seemed like Robert F Kennedy 37:51.120 --> 37:58.800 Jr. was a really legit possibility as a normal everyday well-read smart guy with a lot of 37:58.800 --> 38:04.320 you know forward-looking ideas and tell you to hear him go on about climate change or Israel 38:06.000 --> 38:10.560 and so you know we're all we're all a bit frustrated with the world right now 38:11.760 --> 38:18.800 and so I think it's always good to in those times to to take a chance just kind of listen 38:18.800 --> 38:26.640 and take notes and and learn or maybe not learn from people who like it or not are having a very 38:26.720 --> 38:35.760 big impact on today going forward I have argued that we are probably should be expecting to see 38:36.480 --> 38:44.560 more crowds spelled yachob's disease more more protein misfolding disease is simply because we 38:44.560 --> 38:54.080 are starting to roll out mRNA transfection in old people all around the world and especially in 38:54.080 --> 39:00.480 the United States and in Europe and that rollout as it continues with the flu and RSV and the 39:00.480 --> 39:07.120 pneumonia and whatever else they decide to roll out on people maybe even the shingles shot 39:08.160 --> 39:15.040 you're going to see more and more of these what have already been well-seeded in in the mainstream 39:15.040 --> 39:23.200 narrative as as ramifications of a gain of function virus and potentially a designer spike protein 39:24.800 --> 39:29.440 but actually it's from transfection and they knew this already for a long time they knew 39:30.080 --> 39:35.600 as they moved forward that the vaccine schedule as it stood was probably going to eventually fall 39:35.600 --> 39:41.680 apart that it wasn't going to stand this the the scrutiny of of the citizenry forever as they 39:41.680 --> 39:47.840 ramped it up that's part of the reason why I still find it so amazing that the average young parent 39:47.840 --> 39:52.400 isn't even isn't even curious about how many shots and when they give them in other countries but 39:52.400 --> 39:56.480 if you do that math if you do that research you're going to find out that the American 39:56.480 --> 40:04.960 vaccine schedule is just an exclusively early and dense and so even if some of these purported 40:04.960 --> 40:10.400 intramuscular augmentations of the immune system were somewhat effective the way that they use 40:10.400 --> 40:16.880 them and deploy them in such early lifetime is absolutely absurd and also contradictory to what 40:16.960 --> 40:22.560 we understand about everything to do with neuronal development and and immunological development 40:25.280 --> 40:32.240 in fact it's funny because you can get a you can see a nice talk by Michael Warby from before 40:32.240 --> 40:37.440 the pandemic when he's talking about vaccinating people for flu and given the fact that they have 40:37.440 --> 40:42.480 lifelong immunity to the flu that they're exposed to as a kid we probably have to start vaccinating 40:42.480 --> 40:48.880 people before birth if we really wanted to get ahead of this but of course that's actually the 40:48.880 --> 40:54.000 plan right that's why they want you to think that vaccinating pregnant women's no big deal 40:54.000 --> 41:00.480 yes you shouldn't eat sushi or uh unpasteurized cheese but vaccination is fine intramuscular 41:00.480 --> 41:05.440 injection of any combination of substances is fine for a for a pregnant woman 41:05.760 --> 41:14.000 let's look at Stanley Prusiner and Prions because again I think that this narrative has been 41:14.000 --> 41:18.800 seeded was given a Nobel Prize for goodness sakes I mean let's just talk intellectually a 41:18.800 --> 41:25.840 little bit about a Nobel Prize that I was very very very very very very very very very very 41:25.840 --> 41:34.720 cursorily connected to the Nobel Prize in 2014 was awarded for place cells and grid cells and 41:34.720 --> 41:42.480 spatial coding in in the brain of rodents to John O'Keefe and Edward and Maybert Moser 41:43.680 --> 41:50.720 I was lucky enough to have worked with and studied with and trained with Edward and Maybert Moser and 41:50.880 --> 42:01.040 uh probably more importantly for me Edward uh Menowitter in in Norway um for almost five years 42:01.040 --> 42:07.040 it's where we had our boys um it was some of the most fun that I've ever had um as a young adult 42:07.040 --> 42:13.120 in my life um Norway was a great place to live um and I met a lot of great people there um I 42:13.200 --> 42:21.120 actually got I actually got an email from the universe sorry from the Society for Neuroscience 42:21.120 --> 42:28.400 inviting me to register for the meeting and uh at the bottom was the you know the SFN 42:29.360 --> 42:36.000 meeting committee chair signature is Laura Colgan somebody who I worked with and met in the Moser 42:36.000 --> 42:42.880 lab um in the first couple years I was there a really nice um smart girl I shouldn't say girl 42:42.880 --> 42:50.240 smart woman um who's now I still believe working in New Orleans but I'm I apologize Laura for not 42:50.240 --> 42:59.360 knowing where you are now but being the chairperson for uh for the chairman for the Society for Neuroscience 42:59.360 --> 43:06.080 meeting a meeting of over 35 000 um neuroscientists every year um it's a pretty big step um I guess 43:06.080 --> 43:12.560 that's on the way to being president of Society for Neuroscience um she was uh always gonna be 43:13.200 --> 43:17.280 you know head of her department at some point so I'm really happy I was really happy to get that 43:17.280 --> 43:24.560 letter and just um so anyway what was I saying Norway is a place where Edward and Maybers 43:24.560 --> 43:30.560 Maybers Moser uh work and they in troll time and they actually won the Nobel Prize in 2014 43:31.120 --> 43:38.080 um it was a year so after I had left I moved to Norway the Netherlands in Rotterdam to try and 43:38.080 --> 43:43.120 get tenure there and try to find a permanent job there we had intended to raise our kids um 43:43.840 --> 43:52.080 in in Holland but that didn't work out um and so in 2016 we moved to Pittsburgh um but in 2014 43:52.080 --> 43:56.080 they won that Nobel Prize and so you would have thought that at least that should help a little 43:56.160 --> 44:01.520 bit with my grant applications and with my work but I was doing something in Rotterdam that was so 44:02.160 --> 44:08.400 methodologically close to impossible that I was so obsessed with um that I basically dropped the 44:08.400 --> 44:15.680 ball on getting funding and you know getting any big publications myself and so I found myself 44:15.680 --> 44:20.320 where I was at that time because of you know choices that I had made in mountains that I decided 44:20.320 --> 44:26.480 to climb so I'm not blaming anybody for those years um they were very taxing on our what on 44:26.480 --> 44:34.080 our marriage because I was so obsessed with with making things work at work um the reason why I'm 44:34.080 --> 44:40.080 talking about this is because um the year that they won the Nobel Prize they also won the Nobel Prize 44:40.080 --> 44:46.320 with John O'Keefe who whose first discovery of play cells was actually in the early 70s you might 44:46.400 --> 44:54.160 say 1971 or 1972 is when this happened and then his students actually um two students from Norway 44:54.160 --> 45:00.640 who came to his lab to learn the physical technique of making the electrodes that they recorded these 45:00.640 --> 45:07.440 play cells in the hippocampus from as the animal freely navigated in a small box or maze and uh 45:07.440 --> 45:11.520 they were they were keen on learning those and doing experiments with him and then they recorded 45:11.520 --> 45:18.480 from uh different uh upstream brain region anatomically um giving a lot of input to the 45:18.480 --> 45:24.960 hippocampus as suggested by meno litter and there is where they found their discovery the grid cells 45:24.960 --> 45:31.840 the point is is that that Nobel Prize was awarded to a total of three people essentially a teacher 45:31.840 --> 45:40.160 and his students who had changed the field both several different methodological ways in the 45:40.160 --> 45:45.840 sense of enabling the recording of these these neurons and and and show and developing the 45:45.840 --> 45:52.400 anatomical techniques to follow where you recorded from and and use them for leisure all kinds of 45:52.400 --> 45:57.840 things that essentially change the way that this kind of neuroscience was done these experiments 45:57.840 --> 46:02.080 were done these these methodology spread all around the world and people were putting these 46:02.080 --> 46:08.320 electrodes in all different heads and recording you know uh play cells and then trying to study 46:08.400 --> 46:14.880 how play cells encoded memory and and and how they express themselves in different behavioral 46:14.880 --> 46:21.920 paradigms though I mean they actually changed or even created a field that before them didn't 46:21.920 --> 46:28.400 really exist the hippocampus was originally really exciting because it was this one place where 46:28.400 --> 46:33.600 bliss and lomo were able to create this long-term potentiation that everybody thought was memory 46:33.600 --> 46:39.120 if you could change the electrical signal from a weak signal to a strong signal that was sort of 46:39.120 --> 46:44.720 like this is like the first sort of in vitro preparation of memory but the hippocampus itself 46:44.720 --> 46:51.360 and its functional sort of a model of its functional understanding really comes from this 46:52.560 --> 47:01.280 decades of work that started with the play cells in in in London in John O'Keefe's lab in the 70s 47:02.240 --> 47:10.000 and a and a and a Nobel Prize in medicine wasn't awarded until 2014 Stanley Prouzner got a Nobel 47:10.000 --> 47:17.840 Prize for for prions and the idea of prions before everybody in the field even agreed that prions 47:17.840 --> 47:24.640 were a thing I believe seven years after he coined the term 47:24.640 --> 47:38.880 it should as as Peter Thiel says it should make you suspicious because the potential for people to 47:38.880 --> 47:48.480 lie and exaggerate has been greatly increased and so your default should be yeah your default 47:48.480 --> 47:54.080 assumption should be that there is lie and exaggeration here so let's take a look at this and see what 47:54.160 --> 47:58.320 we can get out of it shall we 47:58.320 --> 48:00.320 you 48:21.680 --> 48:23.360 people who have dementia are demented 48:24.000 --> 48:30.400 and so I think it's okay to talk about demented people what I'm going to tell you about is a 48:30.400 --> 48:35.440 very special journey that happened here at UCSF that begins in 1972 and really is continuing up 48:35.440 --> 48:42.240 to the present so we'll start with this slide and I'm assured the lights will go down so I'm going 48:42.240 --> 48:46.800 to tell you about a saga that represents the triumph of scientific investigation over prejudice 48:46.800 --> 48:51.760 it's really a journey from heresy to orthodoxy it's about prions really a new principle of infection 48:51.760 --> 48:58.320 and disease now the diseases we're going to talk about our crew of New Guinea Natives 48:58.320 --> 49:02.720 Croitesville the Ocob disease called CJD. Curseman stories are shanker disease and fatal insomnia 49:02.720 --> 49:07.520 then scraping of sheep mad cow disease or BSE of cattle and chronic wasting disease of mule deer 49:07.520 --> 49:13.120 and elf now it was 1972 as I mentioned to you a moment ago that I became interested in these 49:13.120 --> 49:17.040 diseases and I had a patient from Marin County a 60 year old woman who was dying of Croitesville 49:17.040 --> 49:21.440 the Ocob disease I was a resident in neurology here and as I began to learn about the animal 49:21.440 --> 49:25.600 disorders scrappy and about the foray people in New Guinea who suffered of crew I thought 49:25.600 --> 49:30.560 this could be really a fascinating area to pursue and really the fascination came from the chemical 49:30.560 --> 49:34.400 point of view because there were a few tidbits of chemistry that suggested that the particles 49:34.400 --> 49:38.480 causing these diseases must be much different than anything that was known this is a slide 49:38.480 --> 49:43.040 by my colleague Steve D. Arman showing you what the brain looks like of a patient who typically dies 49:43.040 --> 49:47.040 of Croitesville the Ocob disease it's a rare disease about one person in a million or one 49:47.120 --> 49:51.360 every 10,000 deaths this is a much less common form with these huge vacuoles that you see 49:53.360 --> 49:58.080 you have to excuse my voice uh that's this is a virus causing it not prions 50:00.240 --> 50:02.160 now for many years scientists thought that scrappy 50:06.480 --> 50:13.040 sorry I got brought brought lunch so I'm eating in the background so first of all let's go back 50:13.040 --> 50:19.440 a little bit here to see that um he doesn't really tell you what this is is his brain or what 50:20.480 --> 50:26.560 um I presume it's brain but it's interesting how quickly you went through it not to tell 50:26.560 --> 50:34.800 you what to look for what to see here there's no not normal form I mean it's again are we 50:34.800 --> 50:41.440 are we here to objectively inform people or are you implanting an idea that you they must accept 50:42.320 --> 50:48.720 and and I see already a problem here and yes it is on 1.5 speed if you want me to slow it down I will 50:48.720 --> 50:53.280 but I think you'll get used to it really quick yes this is a much less common form with these 50:53.280 --> 51:00.800 huge vacuoles that you see you have to excuse my voice uh that's this is a virus causing it not 51:00.800 --> 51:07.920 prions now for many years scientists thought that scrappy was caused by a virus because the 51:08.000 --> 51:12.080 disease is transmissible the agent is small there's a rise in scrappy agent tighter that 51:12.080 --> 51:15.360 precedes disease so that's the concentration or amount of scrappy agent and there are different 51:15.360 --> 51:19.120 strains of scrappy agent that produce different patterns of disease so there was a lot known 51:19.120 --> 51:25.360 when we got into this and what I thought in 1972 was that we really needed to do was to develop 51:25.360 --> 51:29.280 a method to isolate the infectious particles so the infectious particles in this cartoon are 51:29.280 --> 51:33.760 represented by these little red squares and everything else is junk and we needed to separate 51:33.760 --> 51:39.040 these so we could under this is gonna be hard to do while I'm uh I'm gonna try and eat lunch 51:39.040 --> 51:47.440 here and get done so this list of assumptions was gone through way too fast for this to be so 51:47.440 --> 51:55.280 scrappy agent is small and filterable that sounds like you know 1920s virology a rise in scrappy 51:55.280 --> 52:01.680 agent tighter precedes the disease yeah I wonder what that's really based on I wonder what 52:01.840 --> 52:07.360 does that mean that the more you inject in the brain of an animal the quicker it gets sick 52:08.080 --> 52:14.640 you see these assumptions are already sketchy to me the strains of scrappy agent produce 52:14.640 --> 52:20.560 different patterns of disease I'm willing to bet also is based on a very small number of observations 52:21.280 --> 52:26.080 meant to be used to generalize to this extent and to go through this slide this quickly 52:26.320 --> 52:35.120 um well anyway you'll see and we got into this now what I thought in 1972 was that we really 52:35.120 --> 52:39.360 needed to do was to develop a method to isolate the infectious particles so the infectious 52:39.360 --> 52:43.120 particles in this cartoon are represented by these little red squares and everything else is junk 52:43.760 --> 52:48.240 and we needed to separate these so we could understand what they were made of well the problem 52:48.240 --> 52:54.800 became that the particles did not behave very well so instead of having a very steep curve here 52:54.800 --> 52:59.600 the curve was very uh very gradual I won't go into the details of some of the more difficult 52:59.600 --> 53:04.320 pieces of science but this shows it a little more easily so the ideal behavior of a particle 53:04.320 --> 53:07.680 whether it's a virus or a protein or a piece of the classic acid whatever you might want to isolate 53:08.880 --> 53:13.520 it would when we fractionate it had a bit one piece of whatever you might want to get a bite 53:13.520 --> 53:17.120 instead of having a very steep curve that you could understand what they were made of 53:18.080 --> 53:20.000 and so now what we're looking here 53:25.040 --> 53:31.440 log scraping infectivity and so this is a cartoon that's sort of 53:36.160 --> 53:42.400 supposed to show you that somehow or another they've tried different ways to purify 53:43.200 --> 53:50.640 the scraping agent and it doesn't behave the way an agent would be expected to behave 53:52.400 --> 53:58.000 and that for them should be a problem it should indicate that maybe there isn't one 53:58.000 --> 54:05.600 agent but instead they try to stick very hard in fact he will stick to the assumption till the 54:05.680 --> 54:12.160 end of the talk that there is a single causative agent that they just have to develop better 54:12.160 --> 54:18.240 techniques in order to study well the problem became that the particles did not behave very 54:18.240 --> 54:23.680 well so instead of having a very steep curve here the curve was very uh very gradual I won't go 54:23.680 --> 54:27.920 into the details of some of the more difficult pieces of science but this shows it a little more 54:27.920 --> 54:32.240 easily so the ideal behavior of a particle whether it's a virus or a protein or a piece of the 54:32.240 --> 54:38.080 plaque acid whatever you might want to isolate it would when we fractionate it have a bit one peak 54:38.080 --> 54:42.000 it looks like that but instead what we found was that the scrapey agent was spread throughout 54:42.000 --> 54:43.280 the entire uh gradient 54:49.360 --> 54:52.560 so again that's a problem right because it's not one thing 54:53.760 --> 54:58.160 now the argument I believe they're going to use for a little while at least is that the 54:58.160 --> 55:01.760 aggregation of a repetitive protein might produce this kind of smear 55:03.840 --> 55:07.760 however I think that that's you're going to very quickly see that in the end they're going to get 55:07.760 --> 55:15.440 back to it being a single thing and uh so just keep this in mind he's just telling us a story 55:15.440 --> 55:18.960 right now and so we're just listening to his story what we found was that the scrapey agent 55:18.960 --> 55:21.280 was spread throughout the entire uh gradient 55:21.760 --> 55:27.840 now the problem was compounded by the fact that when I started we needed to use 60 mice 55:28.560 --> 55:31.520 and we needed one year to carry out what was called an endpoint titration 55:32.240 --> 55:36.960 and all the red ones are positives and all the white ones are negative so what we would use 55:36.960 --> 55:42.640 are 60 mice as I said uh for a single sample so at each tenfold illusion we would have six mice 55:42.640 --> 55:48.640 and this assay took a year now we were able in the late 1970s to develop a new assay and this was 55:48.720 --> 55:52.400 based on the work of Richard Martian Wisconsin and Richard Kimberlin who was working with Richard 55:52.400 --> 55:56.080 Marsh but he was from England and we turned to the hamsters we followed their lead and we were 55:56.080 --> 56:01.200 able to reduce the number of animals from 60 down to 4 and the time from about 130 days for high 56:01.200 --> 56:06.240 titered samples to 70 days and we did this by constructing a standardized curve and then just 56:06.240 --> 56:10.160 simply reading the dose off of the curve and since we were interested in making more and more 56:10.160 --> 56:13.920 enriched preparations this worked to our advantage because we didn't have to wait to get this endpoint 56:13.920 --> 56:17.520 where we had the negatives and the positives after a whole year we could just simply read off the 56:17.520 --> 56:22.960 titer or the concentration from the standard curve this was a huge step it allowed us to do 56:22.960 --> 56:26.240 more experiments in a period of three years and have been done in the entire field over previous 56:26.240 --> 56:31.280 century now as I said a few moments ago isolating the scraping agent was a nightmare because it 56:31.280 --> 56:39.040 behaved I will admit that I don't really get that I'm gonna play that one more time and see if I 56:39.040 --> 56:47.040 can catch it better a more rapid and economical assay for the scraping agent it seems like to me 56:47.040 --> 56:51.360 what he's saying is if you want to find the scraping agent you have to inoculate 60 animals 56:51.360 --> 56:55.600 and then some of the animals will develop it and then from those animals you can isolate the scraping 56:55.600 --> 57:08.800 agent and with hamsters it's faster or something I don't let's listen the number of animals from 57:08.800 --> 57:14.560 but it was from new assay assay took a year now we were able in the late 1970s to develop a new 57:14.800 --> 57:18.240 assay and this was based on the work of Richard Martian Wisconsin and Richard Kimberlin who was 57:18.240 --> 57:21.840 working with Richard Marsh but who was from England and we turned to the hamsters we followed their 57:21.840 --> 57:26.080 lead and we were able to reduce the number of animals from 60 down to four and the time from 57:26.080 --> 57:32.000 about 130 days for high-tired samples to 70 days and we did this by constructing a standardized 57:32.000 --> 57:36.160 curve and then just simply reading the dose off of the curve and since we were interested in making 57:36.160 --> 57:39.520 more and more enriched preparations this worked to our advantage because we didn't have to wait 57:39.520 --> 57:43.120 to get this endpoint where we had the negatives and the positives after a whole year we could 57:43.120 --> 57:48.320 simply read off the piter or the concentration from the standard curve this was a huge step it 57:48.320 --> 57:52.160 allowed us to do more experiments in a period of three years and have been done in the entire field 57:52.160 --> 57:56.560 over a previous century now as I said to you a moment ago isolating the scraping agent was a 57:56.560 --> 58:01.200 nightmare because it behaved very oddly and also because the method of measurement when we entered 58:01.200 --> 58:09.360 the field was extremely difficult one year and 60 animals so having now moved away from mice 58:09.440 --> 58:13.200 into hamsters able to accelerate our studies by nearly 100 fold so what we could do in one year 58:13.200 --> 58:16.480 would have taken us a century to do and there are few investigators that live a century much 58:16.480 --> 58:22.400 less have a productive lifetime of the century so now what we could do is develop what's called 58:22.400 --> 58:26.720 a purification scheme so we could use detergents and enzymes and back to the centrifuge and these 58:26.720 --> 58:30.880 gradients and now we could follow up the lead that was present in the literature from a woman 58:30.880 --> 58:35.440 named Tig Valper who was a radiobiologist working in England and she had argued that because the 58:35.440 --> 58:39.760 scraping agent was so resistant to UV and ionizing radiation that it did not contain RNA or DNA 58:39.760 --> 58:44.720 nucleic acid and what we found was that all the procedures we use that alter nucleic acids did 58:44.720 --> 58:48.880 not diminish the infectivity and the procedures that modified proteins led to an activation of 58:48.880 --> 58:53.040 the scraping agent but we could only see this after we removed more than 99 percent of the junk 58:53.040 --> 58:58.000 the unwanted molecules and having done that and that's me by the way with brown hair at the time 58:58.640 --> 59:06.160 I thought that by 1982 actually the spring of 81 that we had enough information to say that 59:06.160 --> 59:10.400 this particle was not a virus or a tiny nucleic acid called a viroid discovered by Ted Deener in 59:10.400 --> 59:14.880 the late 1960s and that we needed to give it a new name and so I had the audacity to call these 59:14.880 --> 59:19.120 particles prions that's the long arm of the scientific community pushing on me and people 59:19.120 --> 59:23.840 say well what do those people look like and here they are at the table that's my best slide you can 59:23.920 --> 59:28.880 go home now and it became very very clear that prions eventually became very clear 59:28.880 --> 59:32.640 our infectious proteins and I'm going to take you through a lot more evidence that builds the case 59:32.640 --> 59:39.520 that prions our infectious proteins and then so basically I guess he won the Nobel prize for that 59:39.520 --> 59:48.960 hamster work where the incubation time bioassay right is where they take scraping agent 59:48.960 --> 59:57.920 maybe it's prepared no better than what Marx describes on his stream for the other day 01:00:00.000 --> 01:00:04.640 is then injected directly into the brains of mice and it used to take a year 01:00:06.480 --> 01:00:12.800 but eventually they were able to accelerate that in hamsters I don't know if they altered the 01:00:12.800 --> 01:00:20.480 scraping or if they had to change the PRP protein in a certain way to get that to happen we will 01:00:20.480 --> 01:00:27.680 look at that in the coming journal clubs but in this summary and I apologize for eating lunch 01:00:27.680 --> 01:00:36.080 I'm so hungry the problem with this is though is that I think that we went so quickly to assuming 01:00:36.080 --> 01:00:40.080 that this is the real deal and that more importantly that this recapitulates it 01:00:40.880 --> 01:00:52.080 right that's the problem here is the the animal model of a gain of function virus is one where 01:00:52.080 --> 01:00:59.680 we go into a bat and we swab it we PCR test it we amplify all of the RNA that we see there 01:01:00.800 --> 01:01:05.760 whatever genes are missing we replace by something that we find in a catalog and we rebuild 01:01:06.480 --> 01:01:10.320 the coronavirus that was in that bat based on the fact that well if you assume the spike 01:01:10.320 --> 01:01:15.840 protein is a new spike protein then I guess that's a new virus we can build it we can't grow it 01:01:16.800 --> 01:01:20.480 we can't grow it and make a lot of it we can't culture it and make a lot of it 01:01:22.480 --> 01:01:29.440 but we can use computer algorithms and and nonspecific PCR primers to amplify signals in the 01:01:29.440 --> 01:01:36.000 background that may have already been there that may have nothing to do with the health of the 01:01:36.000 --> 01:01:42.640 animal that may be a a bacteriophage signal for all we know because we do nothing to differentiate 01:01:42.640 --> 01:01:47.040 between them this is very similar to that if we're going to have an animal model 01:01:49.120 --> 01:01:55.040 that has nothing to do with it if we start with humanized mice and infectious clones 01:01:55.680 --> 01:02:01.600 that are purported to be the same as the stuff they found in the bat and now we have this model 01:02:01.600 --> 01:02:09.680 of coronavirus disease where if we squirt this RNA or or the the cell culture products that are 01:02:09.680 --> 01:02:16.080 produced after we transfect a cell culture with this RNA if we put that material into a mouse 01:02:16.080 --> 01:02:24.480 that we call that a model of ARDS or SARS then then then we have a whole industry that can evolve 01:02:24.480 --> 01:02:29.200 from that that has nothing to do with whatever the original natural phenomenon was 01:02:31.040 --> 01:02:37.840 and so it's it's at least worth extreme inspection to find out whether this animal model that's just 01:02:37.840 --> 01:02:43.920 been sort of just presented to us as accepted and and proven and well understood 01:02:45.200 --> 01:02:52.320 is actually an adequate bridge model to come from what happens in scrapie or what happens in 01:02:52.400 --> 01:03:00.080 crowds filled yachab or what happens in kuru and notice that this entire lecture and all the 01:03:00.080 --> 01:03:08.720 lectures about protein misfolding have absolutely never ever mentioned the immune system responding 01:03:08.720 --> 01:03:17.040 to misfolded proteins do you see how significant it is that if you study protein misfolding you 01:03:17.040 --> 01:03:22.160 don't worry about the immune system at all the immune system is not involved this is a completely 01:03:22.160 --> 01:03:26.880 different thing that has to do with aggregation and cell death and you know whatever 01:03:34.880 --> 01:03:40.480 so now he's going to purify the protein and use using sucrose gradient which he showed earlier 01:03:40.480 --> 01:03:46.160 didn't work and so now we're at the development of the prion concept procedures altering nucleic 01:03:46.160 --> 01:03:52.640 acids don't diminish the infectivity so chemicals that are that are thought or assumed to get rid 01:03:52.640 --> 01:04:00.720 of all nucleic act nucleic acid still the infectivity of these preparations is apparently still preserved 01:04:00.720 --> 01:04:06.560 when you do what when you inject them into the brain of an animal or when you use some other 01:04:06.560 --> 01:04:17.280 probably not very adequate experimental correlate for what you what you would think happens in 01:04:17.280 --> 01:04:21.760 these natural forms i'm not suggesting that the natural form of kuru doesn't exist 01:04:22.640 --> 01:04:28.240 crowds felt yachab as a result of using an adenavirus encoding a viral spike protein fusion protein 01:04:28.880 --> 01:04:34.000 could potentially have caused crowds felt crowds felt yachab disease in 28 people like 01:04:34.080 --> 01:04:41.440 luke montanier's paper said i'm suggesting to you that genetic technologies like transformation 01:04:41.440 --> 01:04:49.280 and transfection we're already known a long time ago to interfere with and maybe even induce 01:04:49.280 --> 01:04:58.640 maladaptive states in healthy animals and so what this means is is that we need to be very careful 01:04:58.640 --> 01:05:05.440 about how many of these very recently seeded biological phenomenon biological ideas were 01:05:05.440 --> 01:05:13.440 actually seeded by people who have intention of confusing us and about this sounds very paranoid 01:05:13.440 --> 01:05:21.200 but this is the same year that sars came on the world 2002 when he's given this talk is the year 01:05:21.280 --> 01:05:27.680 that sars is going to be released or the year after i guess it's 2003 really when it happened 01:05:31.120 --> 01:05:36.320 so you can almost imagine in fact a scenario over the last couple decades where this 01:05:37.040 --> 01:05:45.280 this potential bio hazards from mother nature these mythologies were already these were 01:05:45.280 --> 01:05:54.560 mythologies were seeded and while we're being told that these phenomenon are natural we we 01:05:54.560 --> 01:06:01.680 have no way of knowing we have no way of knowing we do have pretty good reason to believe that 01:06:01.680 --> 01:06:06.320 they've been messing around with all sorts of biotechnologies for the last 30 years in attempt to 01:06:06.960 --> 01:06:13.760 figure stuff out and so there's no question that one of the things that could have resulted from 01:06:13.920 --> 01:06:21.120 are these kinds of diseases there's also no question in my mind that that kuru and eating 01:06:21.120 --> 01:06:25.600 your dead relatives might be a bad idea that would involve your immune system going a little 01:06:25.600 --> 01:06:29.120 going a little haywire can imagine that i can 01:06:33.600 --> 01:06:38.720 but i do think that an infectious protein is much more interesting from a bioweapon perspective 01:06:38.720 --> 01:06:43.920 than it is from a biological perspective not diminishing the infectivity and the procedures 01:06:43.920 --> 01:06:47.520 that modified proteins led to an activation of the scrapey agent but we could only see this 01:06:47.520 --> 01:06:52.320 after we removed more than 99 percent of the junk the unwanted molecules and having done that 01:06:53.200 --> 01:07:00.480 and that's me by the way with brown hair at the time i thought that by 1982 actually the spring 01:07:00.480 --> 01:07:05.280 of 81 that we had enough information to say that this particle was not a virus or a tiny nucleic 01:07:05.360 --> 01:07:09.760 acid called a viroid discovered by Ted Deener in the late 1960s and that we needed to give it a 01:07:09.760 --> 01:07:13.920 new name and so i had the audacity to call these particles prions that's the long arm of the 01:07:13.920 --> 01:07:17.600 scientific community pushing on me and people say well what do those people look like and here 01:07:17.600 --> 01:07:23.760 they are at the table that's my best slide you can go home now and it became very very clear 01:07:23.760 --> 01:07:27.760 that eventually became very clear our infectious proteins and i'm going to take you through a 01:07:27.760 --> 01:07:32.800 lot more evidence that builds the case that prions are infectious proteins and then as we move through 01:07:32.880 --> 01:07:37.440 this we'll get to some very new data and we'll talk about bse and we'll talk about other prions 01:07:37.440 --> 01:07:40.960 diseases of humans and then at the end we'll talk about therapies and there'll be a lot of chemistry 01:07:40.960 --> 01:07:45.600 at the end but it won't be complicated because i put the chemistry in the beginning you'll all 01:07:45.600 --> 01:07:51.600 leave so here we have there is there is something to be said about too many jokes 01:07:52.800 --> 01:07:57.120 have this purification scheme where we remove 99 percent of the unwanted particles we then knew 01:07:57.120 --> 01:08:01.120 that a protein was important and we went on to identify and find this protein and then the next 01:08:01.200 --> 01:08:05.040 thing we did was to try to determine whether we could separate this protein from the infectivity 01:08:05.040 --> 01:08:08.800 and so we used a lot of approaches so we're now measuring the biological activity enhancers 01:08:08.800 --> 01:08:12.080 and we're looking at the protein by physical methods and we tried to separate it and what we 01:08:12.080 --> 01:08:15.600 found is that every time we destroyed the protein we destroyed the infectivity or every time we 01:08:15.600 --> 01:08:22.800 destroyed the infectivity the protein was removed or destroyed and when we eventually figured out 01:08:22.800 --> 01:08:26.560 what was going on we found that there was a normal form of the protein in all of us now this is a 01:08:26.560 --> 01:08:30.240 gel and the proteins migrate and the smaller they are the faster they migrate through this gel this 01:08:30.320 --> 01:08:35.360 porous material sort of like jello and then we can stain them with antibodies and so what we found 01:08:35.360 --> 01:08:39.920 was that the protein was a protein in all of us which is a normal form of the prion protein 01:08:39.920 --> 01:08:43.920 and when we treat with enzymes that destroy proteins the protein was completely destroyed 01:08:43.920 --> 01:08:48.400 but in the brains of these hamsters that became ill with scrapi what we found was that there was 01:08:48.400 --> 01:08:51.760 both a normal form of the protein and another form of the protein that when we treated with 01:08:51.760 --> 01:08:55.760 enzymes that destroy proteins there was a residual piece and here they're higher-ordered multiverse 01:08:56.720 --> 01:09:02.400 they're larger forms that are aggregated of the scrapi causing protein and eventually 01:09:02.400 --> 01:09:06.320 the protein that I just showed you in the gel after we treated with proteases what we did was to use 01:09:06.320 --> 01:09:11.600 a lot of very complicated equipment in collaboration with a man named Leroy Hood who was at Caltech at 01:09:11.600 --> 01:09:18.480 the time and Darlene Groff who's been working with me for 25 years went to Caltech over a period 01:09:18.480 --> 01:09:23.040 of a year about seven different times and we finally were able to work out what's called the 01:09:23.120 --> 01:09:27.360 amino acid sequence the N terminal 15 amino acids so right in this region now this was more 01:09:27.360 --> 01:09:30.960 complicated because it was ragged because we'd thrown in these proteases but what we had found 01:09:30.960 --> 01:09:35.840 of course is that if we threw the proteases into the normal preparations there was nothing well 01:09:35.840 --> 01:09:39.680 now we had the possibility of doing what's called reverse genetic engineering and we went backwards 01:09:39.680 --> 01:09:43.680 and with Charles Weisman and Zurich we found the gene that codes for the prion protein and we found 01:09:43.680 --> 01:09:47.280 that all of us have this gene and it became very clear that all of us have the normal form of the 01:09:47.280 --> 01:09:51.360 prion protein and then by a process that we still don't understand in great detail it could be 01:09:51.440 --> 01:09:55.760 converted into a protein which is infectious and which can eventually kill us as human beings or 01:09:55.760 --> 01:10:02.000 kill animals like the cattle in Europe so now we knew that there was a gene for the protein of the 01:10:02.000 --> 01:10:09.280 prion and the protein of the prion we called PRP scrapey and it's and that this gene encodes 01:10:09.280 --> 01:10:12.960 the precursor protein PRPC there's a mistake here it's not found it's only found in six mammals 01:10:12.960 --> 01:10:18.640 but the gene is found in all mammals so now we had two proteins the precursor protein PRPC that 01:10:18.720 --> 01:10:23.440 we all have a normal protein and the protein found in okay so just notice the wording that 01:10:23.440 --> 01:10:28.320 he's using here a precursor protein we have to assume that it does something normally 01:10:29.280 --> 01:10:35.440 and that's the normal protein and that the the prion scrapey protein has to be the abnormal 01:10:35.440 --> 01:10:42.480 protein so it's really weird that he doesn't say it in the way that his disease model implies it 01:10:42.480 --> 01:10:50.320 should be said um as if the protein only exists to cause the disease in its precursor form it's 01:10:50.320 --> 01:10:56.640 fine in its functional form it causes disease that's really not the way we should be thinking of this 01:10:57.520 --> 01:10:58.480 especially if 01:11:02.240 --> 01:11:07.760 we have to work from the assumption that the that the that the prion protein does something 01:11:08.640 --> 01:11:14.560 otherwise what is it there for on the membrane a protein that's on the memory that doesn't do 01:11:14.560 --> 01:11:20.880 anything sounds like a very bad idea to me had two proteins the precursor protein PRPC that we 01:11:20.880 --> 01:11:25.680 all have a normal protein and the protein found in the animals that were ill with prion disease 01:11:26.400 --> 01:11:29.760 we did another set of experiments and we asked what about the messenger RNA so the messenger 01:11:29.760 --> 01:11:34.080 RNA is the intermediate between the gene and the protein and we said well the messenger RNA ought 01:11:34.080 --> 01:11:39.120 to rise as the as these animals go out toward the these are now mice that get sick at 130 days 01:11:39.120 --> 01:11:44.320 as incubation time gets longer and longer and so the n-terminal amino acid sequence of of 01:11:45.520 --> 01:11:54.960 prion protein 27 to 30 is determined isocoding DNAs used to retrieve PRPC DNA clones 01:11:55.520 --> 01:12:01.600 so my guess is is that they used 01:12:04.320 --> 01:12:13.520 DNAs that would encode for the amino acid sequence of the n-terminal then they use those DNA probes 01:12:13.520 --> 01:12:23.600 and they hybridized against the DNA of a human or a mouse or something like my human I guess 01:12:23.600 --> 01:12:34.320 and found a genomic DNA to which that n-terminal DNA hybridized to 01:12:40.240 --> 01:12:46.400 expected the prion protein mRNA levels were similar in uninfected controls with animals with 01:12:46.400 --> 01:12:54.880 scraping so when they look for the mRNA for the for the protein they find it and two forms are 01:12:54.880 --> 01:13:05.600 identified cellular and pathogenic and is derived from pr by n-terminal truncation let's see how 01:13:05.600 --> 01:13:10.400 he explains this hider the prions goes up the messenger RNA ought to rise but in fact the messenger 01:13:10.400 --> 01:13:14.000 RNA state exactly the same it never changed and this worried us a little bit where we're looking 01:13:14.000 --> 01:13:18.480 at the right protein but we got lucky because using that same technology genetic engineering 01:13:18.480 --> 01:13:23.360 we now looked at black mice or brown mice and we looked at these white mice the white mice had 01:13:23.360 --> 01:13:29.280 long incubation times about 225 days the short ones about 130 days and what we found was that this 01:13:29.280 --> 01:13:33.360 gene the prion protein gene that i've been talking about determined whether the mice had a short 01:13:33.360 --> 01:13:38.320 incubation time or a long incubation time and the mice with long incubation times had two amino 01:13:38.320 --> 01:13:43.680 acids out of 250 that were different and so this began to cement the idea that this protein PRP 01:13:43.680 --> 01:13:49.120 that we discovered was key in the disease process wow so that's a really key thing to realize because 01:13:49.120 --> 01:13:53.920 what is this established this is we need to find this paper so i'm going to write this one down 01:13:53.920 --> 01:14:02.640 because this is a really important one it's 86 through 87 and this is brown and white mice huh 01:14:09.360 --> 01:14:13.120 the reason why this is important is because this whole thing would have been established 01:14:13.120 --> 01:14:19.520 by a p-value test right if the p-value between the incubation time and the brown mice and the 01:14:19.520 --> 01:14:26.240 incubation time and the white mice in the experiment was significant as determined by whatever probability 01:14:26.240 --> 01:14:33.200 test they use that ritual would be enough to establish this as fact and then he would go on to 01:14:33.200 --> 01:14:40.080 say that these two amino acid differences between those mice and those mice are the reason why the 01:14:40.080 --> 01:14:46.800 incubation time is different and then that whole field moves on based on the idea that well i mean 01:14:46.800 --> 01:14:56.640 obviously think about how that that could potentially set up years and years of being wrong 01:14:59.040 --> 01:15:02.560 that's not definitive proof that this protein's involved 01:15:04.320 --> 01:15:09.680 how can that be even if there is a p-value difference there how can that be assumed 01:15:09.760 --> 01:15:13.360 is that the only difference between the brown mice and the white mice 01:15:15.440 --> 01:15:17.280 wow isn't that special 01:15:19.360 --> 01:15:24.000 isn't that special the lady from mit told us that all those heat shock proteins are really 01:15:24.000 --> 01:15:28.960 responsible for protein folding and misfolding and that they're like chaperone proteins and 01:15:28.960 --> 01:15:34.240 it's so cool because yeasts are eukaryotes and were eukaryotes and we have the same proteins 01:15:34.240 --> 01:15:37.920 that chaperone are protein folding and prevent them from misfolding 01:15:39.520 --> 01:15:45.120 and so it doesn't matter that the brown mice might have different heat shock protein variants 01:15:45.120 --> 01:15:51.520 than the white mice do it doesn't matter at all i think the lady from mit who's dead now would 01:15:51.520 --> 01:15:59.040 probably disagree but i don't know what's going on here that's how p-values are used to create 01:15:59.040 --> 01:16:03.760 the distortion of information and knowledge from noise 01:16:06.640 --> 01:16:08.240 it's a single experiment 01:16:10.480 --> 01:16:17.280 where the difference between incubation times to injection of material in their brain 01:16:17.280 --> 01:16:22.800 has nothing to do with looking at what was injected or where it was injected or how much 01:16:22.800 --> 01:16:29.360 exactly was injected it has to do with those two amino acids in that one protein that has to 01:16:29.360 --> 01:16:33.440 be i mean look i mean it has to be look at the other experiments we did there was like a gel 01:16:33.440 --> 01:16:33.840 and stuff 01:16:38.160 --> 01:16:42.880 out of 250 that were different and so this began to cement the idea that this protein 01:16:42.880 --> 01:16:45.840 PRP that we discovered was key in the disease process 01:16:47.360 --> 01:16:50.480 so what i've told you is that not only did the pre-on protein track with scraping 01:16:50.480 --> 01:16:53.680 infectivity so every time we tried to alter the protein we altered the infectivity and vice versa 01:16:53.680 --> 01:16:58.880 but also amino acid substitutions changed the incubation times so these findings link the phenotype 01:16:58.880 --> 01:17:02.320 which is called the disease characteristics in this case to the genotype that changes 01:17:02.320 --> 01:17:07.520 their mutations in the DNA okay so to a certain extent this is interesting because what it now 01:17:07.520 --> 01:17:14.320 implies of course is that function is related to structure which is one of the real overarching 01:17:14.320 --> 01:17:19.520 principles of reductionist biology one of the things that we all agree on as we move through 01:17:19.520 --> 01:17:23.680 the world trying to understand what's going on if you want to understand how a tree works 01:17:25.280 --> 01:17:29.840 it's probably related to its structure it's its structure in the macro sense and also structure 01:17:29.840 --> 01:17:37.600 at the cellular level and the molecular level but structure begets function is something that holds 01:17:37.600 --> 01:17:45.280 at the protein level from everyone's perspective the the amino acid sequence determines its structure 01:17:46.240 --> 01:17:58.160 the the RNA sequence that encoded the amino acid is to a large extent synonymous 01:17:59.600 --> 01:18:05.600 but we know that synonymous mutations that don't change the amino acid for which they code 01:18:06.160 --> 01:18:10.960 can still be highly correlated with genetic disease 01:18:11.760 --> 01:18:20.320 and so clearly the cartoon about yes we have DNA we translated it to RNA the RNA translates 01:18:20.320 --> 01:18:25.600 to amino acids and the amino acids arrange in a certain way because there are three-dimensional 01:18:25.600 --> 01:18:32.240 electrostatic shape inside of a three-dimensional electrostatic solvent called water and so they 01:18:32.240 --> 01:18:40.720 form a particular shape to try and best optimize their energy distribution in a stable way 01:18:44.240 --> 01:18:48.080 but that's a variable process a process that isn't understood a process that 01:18:48.080 --> 01:18:54.560 occurred to the dead lady from MIT actually goes wrong an awful lot and we waste a lot of energy 01:18:54.560 --> 01:19:03.360 misfolding proteins all the time and that prion protein now is a very special protein that has 01:19:03.360 --> 01:19:10.000 the propensity apparently as we move forward we're going to hear how it can can cause other 01:19:10.000 --> 01:19:16.080 proteins with a similar sequence to misfold as well so prion protein misfolds other prion protein 01:19:16.080 --> 01:19:20.400 even though we don't know what prion protein really does in the in the healthy animal it's 01:19:20.480 --> 01:19:28.320 there it's mRNA is there all the time and in in some animals like hamsters it develops faster 01:19:28.320 --> 01:19:34.400 than other animals like mice and in some brown mice it develops longer faster than in white mice 01:19:38.800 --> 01:19:42.080 now we come to a whole different approach and we spent six years here at UCSF 01:19:43.600 --> 01:19:47.760 trying to identify the difference between PRPC the normal form of the protein that all of us have 01:19:47.760 --> 01:19:52.000 and the scrapey form of the protein that's found only in the disease and we looked and looked so 01:19:52.000 --> 01:19:58.560 far the only evidence that they have of a different protein is that gel keep that in mind right do 01:19:58.560 --> 01:20:02.400 we need to go back to that a little bit if I just go back to the gel 01:20:09.120 --> 01:20:14.880 this is the gel right this this is the gel that he explained is the evidence that he has for 01:20:14.960 --> 01:20:21.600 prion protein being different this is the normal protein this is the lane with the normal protein 01:20:21.600 --> 01:20:30.480 after they digested it with proteases this is the protein apparently from the the scrapey infected 01:20:30.480 --> 01:20:36.160 animal i don't know what all this other crap is that didn't show up in this line which is also 01:20:36.160 --> 01:20:43.360 very frustrating how am i supposed to compare this experiment if this lane is supposed to be 01:20:43.360 --> 01:20:47.920 equivalent to that lane except this lane has four more bars in it than this one does 01:20:47.920 --> 01:20:54.240 what kind of shit experiment is this and how is he able to just get through the all 01:20:54.240 --> 01:21:00.480 gel it moves uh the smaller fragments move faster than the the heavy fragments and uh so uh yeah 01:21:01.360 --> 01:21:06.560 so what are these fragments well anyway his argument is is that this is the protease 01:21:06.560 --> 01:21:13.840 digested side and here they still have a protein whereas here it's all gone and so in that in the 01:21:13.840 --> 01:21:19.040 in the normal functioning prion protein format it can be completely digested by protease and here 01:21:19.040 --> 01:21:25.920 it can't that is the only evidence that they have for an alternate form of a protein that's it 01:21:27.360 --> 01:21:29.200 because the sequence is the same 01:21:31.520 --> 01:21:34.800 as best they can tell it's just one gene 01:21:37.520 --> 01:21:45.120 these are not as related complementary experiments as he's implying and without careful 01:21:46.240 --> 01:21:54.000 and maybe even several months of hard studious work you can't claim to be an expert on prions 01:21:54.000 --> 01:22:00.320 because nobody explains it well enough to be this is not a good enough explanation of prions 01:22:00.400 --> 01:22:04.080 for me to say oh yeah i guess i believe it this is hand waving 01:22:07.280 --> 01:22:13.200 this is how they explain to you how pandemics work and why masks work and why lockdowns if done 01:22:13.200 --> 01:22:18.880 correctly work and why zero virus after the end of a pandemic is a useful goal 01:22:20.080 --> 01:22:24.480 it's that kind of it's that kind of explanation on protein track with scraping 01:22:24.480 --> 01:22:27.760 infectivity so every time we try to alter the protein we alter the infectivity and vice versa 01:22:27.760 --> 01:22:32.960 but also amino acid substitutions change the incubation times so these findings link the phenotype 01:22:32.960 --> 01:22:36.480 which is called the disease characteristics in this case to the genotype the changes are 01:22:36.480 --> 01:22:42.400 mutations in the DNA thank you solar flare i'm clicking a whole different approach and we spent 01:22:42.400 --> 01:22:47.840 six years here at UCSF trying to identify the difference i'll take it between prpc the normal 01:22:47.840 --> 01:22:51.360 form of the protein that all of us have and the scraping form of the protein that's found only 01:22:51.360 --> 01:22:54.800 the disease and we looked and looked for a chemical difference because if we could have found a 01:22:54.800 --> 01:22:58.960 chemical difference then it would be very easy to make that chemical change on prpc 01:22:58.960 --> 01:23:03.680 and convert it into prpc but we never found it this was a work of a young postdoc Neil Stahl 01:23:03.680 --> 01:23:07.440 who was working with Mike Baldwin who's still here and who's a professor in the school of pharmacy 01:23:07.440 --> 01:23:13.840 and mass spectrometry so then Fred Cohn became involved and we began to think about models 01:23:13.840 --> 01:23:17.360 and the first thing that Fred did he said i wonder if this isn't really the case that 01:23:17.360 --> 01:23:21.360 the normal form of the prion protein is full of these helical structures called alpha helices 01:23:21.440 --> 01:23:26.080 and that some of these are reduced and that there's a lot of beta sheet these beta strands 01:23:26.080 --> 01:23:31.520 these blue ribbons and at that point what we did was we knew that there was a lot of beta structure 01:23:31.520 --> 01:23:35.760 from other studies from our won't work and from that of other people we went it off and purified 01:23:35.760 --> 01:23:39.520 out of brains of animals the normal form of the prion protein and we found that Fred in fact was 01:23:39.520 --> 01:23:43.600 correct that there was very little beta structure and then more recently Tom James and his colleagues 01:23:43.600 --> 01:23:47.600 here with the help of peter right now i thought it was really hard to purify the prion proteins 01:23:47.680 --> 01:23:53.520 from animals with the disease and now he just says we're going to go do it and we found out 01:23:53.520 --> 01:24:01.440 that there's more beta sheets i don't know pain dyson scripts uh from protein that was made by 01:24:01.440 --> 01:24:05.520 genetic engineering and bacteria and determined its structure so here we're sort of halfway 01:24:05.520 --> 01:24:09.760 through the protein around residue 90 and then you see this long unstructured region then one the 01:24:09.760 --> 01:24:13.520 first alpha helix then more of this unstructured region with a small amount of beta structure 01:24:13.520 --> 01:24:18.320 than the second helix and the third helix and what's called the c-terminus now what we 01:24:18.320 --> 01:24:22.000 hypothesized was that the first helix and in fact we thought the first helix was made up of two 01:24:22.000 --> 01:24:25.840 this whole region was made up of two helices and we were wrong and Kurt Rudrich and Zurich showed us 01:24:25.840 --> 01:24:32.080 that we were wrong much to our dislike and unhappiness and so this was the model that we had for a number 01:24:32.080 --> 01:24:37.440 of years so what i've just told you is that when prpc is converted into prp scraping the protein 01:24:37.440 --> 01:24:42.560 undergoes a profound change in shape or as scientists call it confirmation prpc is rich in alpha helix 01:24:42.560 --> 01:24:50.480 whereas prp scraping is rich in beta structure now if you believe alpha fold and you believe 01:24:52.080 --> 01:24:58.480 people who study proteins alpha helix is something that you can predict from the sequence 01:24:59.040 --> 01:25:04.320 and a beta pleated sheet is something you can predict from the sequence and it's not very clear 01:25:04.320 --> 01:25:12.640 to me that those two are are not mutually exclusive i i was i was taught that those are kind of 01:25:12.640 --> 01:25:17.120 mutually exclusive in the sense of we don't really understand how proteins fold but we do 01:25:17.120 --> 01:25:23.280 understand that they have two general motifs that of an alpha helix and that of a beta pleated sheet 01:25:23.280 --> 01:25:29.520 and these two general motifs of course have very specific expression but these general motifs are 01:25:29.600 --> 01:25:34.320 used to construct or or or or build out some of these protein machines 01:25:36.160 --> 01:25:42.960 so the idea that one part of a protein could be both an alpha helix and a beta pleated sheet 01:25:42.960 --> 01:25:48.800 is already a very novel idea in my humble opinion and that i believe is what he's implying here 01:25:50.240 --> 01:25:53.680 and we went on to do some studies in collaboration with Dennis Burton at Scripps 01:25:54.240 --> 01:25:58.720 and this is the work of David Parrots who's still here at UCSF and what we did was to make 01:25:58.720 --> 01:26:02.320 antibodies that reacted to different regions of the prion protein so this is the three helix 01:26:02.320 --> 01:26:07.440 protein now what's really incredible about this is that antibodies are reacting to a three-dimensional 01:26:07.440 --> 01:26:12.880 static shape they don't necessarily react to only one and antibodies are very 01:26:15.520 --> 01:26:20.480 non-specific so it's very very would be very interesting to go back 01:26:21.360 --> 01:26:27.520 and see how these antibodies were raised they're raised to different epitopes as you can see here 01:26:27.520 --> 01:26:36.160 they are raised to epitopes that are about 22 amino acids this is 23 right this is no that's 30 01:26:36.880 --> 01:26:40.880 30 28 i don't know i can't do that math it's not very many 01:26:42.320 --> 01:26:47.120 so they're small epitopes presumably then what injected into a rabbit or something and then 01:26:47.120 --> 01:26:51.680 they take the plasma out and they get those antibodies and those antibodies although they 01:26:51.680 --> 01:26:56.880 were raised to that epitope aren't necessarily all going to be the same antibodies with the same 01:26:56.880 --> 01:27:02.720 specificity so unless they used a monoclonal antibody as they were made in the 90s that would be one 01:27:02.720 --> 01:27:10.240 thing to look at here and what they're doing is trying to identify using antibody binding whether 01:27:10.320 --> 01:27:14.000 or not particular structures are present or not present 01:27:21.200 --> 01:27:27.360 so i i just wanted to be sure you knew what they were doing with these antibodies 01:27:28.480 --> 01:27:32.640 let's see what he says hey this is helix a helix b and helix c that i showed you before 01:27:32.640 --> 01:27:41.360 and this is residue 90 and in this first region the the prion protein binds antibodies and the 01:27:41.360 --> 01:27:45.600 native form of the prion protein binds them quite well but the binding region or epitope disappears 01:27:45.600 --> 01:27:50.000 when prp scrape is formed so that was an important discovery and we use that information and we 01:27:50.000 --> 01:27:57.920 contrasted that with the green where but remember that when prion protein scrape is formed they can't 01:27:57.920 --> 01:28:05.200 separate it from the native prion so again these pluses and minuses are binding strength and whenever 01:28:05.200 --> 01:28:14.720 you have one key tip i would say that whenever you have a paper that wants to basically 01:28:16.160 --> 01:28:21.040 say take your word for it that okay we rate this as a three and that one is a two and this one is a 01:28:21.040 --> 01:28:27.360 one and that's a you know you should take it with a grain of salt here both the native form of PRPC 01:28:27.360 --> 01:28:31.680 and the native form of PRPC mine antibodies that's down here 01:28:32.160 --> 01:28:39.440 so these epitopes are regions so that's the second basic prince proof that the scrappy protein in 01:28:39.440 --> 01:28:46.240 its in its infectious form is a different shape than this one but in this preparation we're not 01:28:46.240 --> 01:28:53.120 sequencing a protein we're not starting with a pure protein in my i i don't think i don't know 01:28:53.120 --> 01:28:56.800 what we're doing here we got to we got to find that out we got to know we hope we're doing a 01:28:56.800 --> 01:29:04.720 pure protein here but is it a synthetically pure made protein is it a so that's again we have we 01:29:04.720 --> 01:29:10.560 have a lot of work to do here to find out where p values are essentially holding up what is a what 01:29:10.560 --> 01:29:17.360 is a what is a house that shouldn't be holding up we already have seen a couple examples of this 01:29:17.360 --> 01:29:22.880 like the brown and white mice thing with two amino acids difference and this protein means that 01:29:22.880 --> 01:29:27.440 the difference that we saw in the experiment had to do with those two amino acids that's the 01:29:27.440 --> 01:29:35.200 kind of assumptions that we're always talking about here these numbers plus two plus three is that 01:29:35.200 --> 01:29:42.720 really is he really saying that the native and the denatured pre-on scrappy protein are different 01:29:42.720 --> 01:29:47.520 by a factor of one out of three that's their scale i think that's down here 01:29:50.000 --> 01:29:54.640 so these epitopes are regions of the alpha helix rich precursor protein p rpc of 01:29:54.640 --> 01:29:59.360 prions are exposed but become buried when the protein is refolded into an infectious form 01:29:59.360 --> 01:30:08.000 p rpc scraping so why didn't they make um anybody see this is the problem i have with that 01:30:08.080 --> 01:30:16.720 they have the the protein here native pre-on protein scrappy so why don't they make antibodies 01:30:16.720 --> 01:30:25.600 to that why don't they make antibodies to the pre-on protein scrappy part and then show me 01:30:25.600 --> 01:30:31.360 that those antibodies bind pre-on scrappy but they don't bind this one why don't they do the 01:30:31.360 --> 01:30:37.680 reverse experiment apparently they have a pure form of this right otherwise you couldn't do this 01:30:38.640 --> 01:30:45.920 this assay so they raised antibodies to these epitopes but they don't raise antibodies to the 01:30:45.920 --> 01:30:50.560 epitopes of the pre-on scrappy because it's so hard to isolate or something i don't know 01:30:51.440 --> 01:30:55.920 they can't get it to form badly in a in a dish or using the right ribosome or what 01:30:57.280 --> 01:31:00.560 you see the problem here here 01:31:01.040 --> 01:31:08.480 so these epitopes are regions of the alpha helix rich precursor protein p rpc of prions are 01:31:08.480 --> 01:31:13.120 exposed but become buried when the protein is refolded into an infectious form p rpc scraping 01:31:15.680 --> 01:31:21.120 now when we treat with proteases and we cleave off the n terminal region and we form the ragged 01:31:21.120 --> 01:31:25.920 n terminus that i showed you that we sequenced and we call this prp 2730 this protein and this 01:31:25.920 --> 01:31:29.040 is the one that i showed you before where we've treated with protease so it's a little bit shorter 01:31:29.840 --> 01:31:33.440 that protein polymerizes into these rod shape structures that you see here 01:31:33.440 --> 01:31:40.400 so after they treat it with an enzyme it forms rod shape structures there's nothing to do with 01:31:40.400 --> 01:31:45.280 the physiology of it at all right this is an in vitro preparation with an in vitro result 01:31:46.400 --> 01:31:53.040 and yet somehow or another we got here by him saying something about the refolding 01:31:53.520 --> 01:32:00.480 now when we treat with proteases and we cleave off the n terminal region and we form the ragged 01:32:00.480 --> 01:32:05.280 n terminus that i showed you that we sequenced and we call this prp 2730 this protein and this 01:32:05.280 --> 01:32:08.400 is the one that i showed you before where we've treated with protease so it's a little bit shorter 01:32:09.680 --> 01:32:13.440 that protein polymerizes into these rod shape structures that you see here and there was a very 01:32:13.440 --> 01:32:17.360 famous man at Berkeley who died about 15 years ago roblie Williams was really the father of the 01:32:17.360 --> 01:32:20.960 electron microscopy of viruses and he was working with us at the time because i really thought we 01:32:21.040 --> 01:32:24.560 were going to find an interesting virus not a protein and we would see these rod shape structures 01:32:24.560 --> 01:32:29.280 and he called them rods some people would call them fibers and one day i guess we'd been working 01:32:29.280 --> 01:32:32.800 together for almost a year and a half i was looking at a book on the electron microscopy of proteins 01:32:32.800 --> 01:32:37.360 and all this looking like amyloid proteins now amyloid is a mammalian protein these are usually 01:32:37.360 --> 01:32:42.240 cathologically laid down in big fibers and there are many many diseases like alzheimer's disease 01:32:42.240 --> 01:32:47.200 where there are amyloid plaques of many of these proteins but so the first connection here so far 01:32:47.200 --> 01:32:52.400 seems to be that one microscopist called it a fibro and now they the people that study alzheimer's 01:32:52.400 --> 01:32:57.680 disease have also called the proteins involved in alzheimer's disease fibers and so now that's 01:32:57.680 --> 01:33:06.400 okay now they must be the same thing it turned out that this broke that what we were seeing was that 01:33:06.400 --> 01:33:10.720 the protein of the prion was forming amyloid and that's what these rods represent and then it formed 01:33:10.720 --> 01:33:14.640 plaques in the brain and i'll show you some of those plaques a little later now we also found 01:33:14.640 --> 01:33:18.480 and this is the work of holder willy who's here at UCSF along with the help of david agar and 01:33:18.480 --> 01:33:24.080 many others uh hana serven uh darlene groth that the protein forms these 01:33:25.680 --> 01:33:30.320 rods that the plaques a little later amyloid and that's what these were this broke that 01:33:30.320 --> 01:33:34.080 what we were seeing was that the protein of the prion was forming amyloid and that's what 01:33:34.080 --> 01:33:39.680 these rods represent the protein of the prion was forming amyloid so what i hear here is that prion 01:33:40.640 --> 01:33:47.440 protein is the sub component of amyloid is that what i'm hearing is that right 01:33:53.680 --> 01:34:00.320 hmm that's interesting that's that i didn't think that they were the same i didn't think 01:34:00.320 --> 01:34:05.280 they were the same protein i thought amyloid was a different protein um i didn't know that it 01:34:05.280 --> 01:34:09.360 was prion protein that was folded differently without an n-terminus because that's what he just 01:34:09.360 --> 01:34:15.760 said these are prion rods that are formed by the prion protein after you cleave its n-terminal 01:34:15.760 --> 01:34:22.960 off with proteases and these form under the microscope and now he seems to be indicating 01:34:22.960 --> 01:34:31.280 in 2002 that the amyloid plaques are actually prion protein did i not hear that did i not hear that 01:34:33.840 --> 01:34:38.560 on the electron microscopy of proteins and all this look like amyloid proteins now amyloid 01:34:38.560 --> 01:34:42.080 was is a mammalian protein these are usually depend logically uh laid down 01:34:42.080 --> 01:34:46.240 in big fibrils and there are many many diseases like Alzheimer's disease where there are amyloid 01:34:46.240 --> 01:34:57.520 many many diseases like Alzheimer's disease where amyloid plaques are laid down i don't 01:34:57.520 --> 01:35:02.000 don't know many many many diseases where amyloid plaques all this look like amyloid proteins 01:35:02.000 --> 01:35:06.480 now amyloid was is a mammalian protein these are usually Happyl logically uh laid down in 01:35:06.480 --> 01:35:08.640 is like Alzheimer's disease, where there are amyloid plaques, 01:35:08.640 --> 01:35:09.560 so many of these proteins. 01:35:09.560 --> 01:35:12.480 But it turned out that this broke that what we were seeing 01:35:12.480 --> 01:35:15.120 was that the protein of the prion was forming amyloid, 01:35:15.120 --> 01:35:16.720 and that's what these rods represent. 01:35:16.720 --> 01:35:18.120 And then it formed plaques in the brain, 01:35:18.120 --> 01:35:20.640 and I'll show you some of those plaques a little later. 01:35:20.640 --> 01:35:22.760 Now, we also found, and this is the work of Holger-Willy, 01:35:22.760 --> 01:35:25.200 who's here at UCSF, along with the help of David Agard 01:35:25.200 --> 01:35:28.080 and many others, Hana Sermon, Darlene Groth, 01:35:28.080 --> 01:35:33.040 that the protein forms these rods, 01:35:33.040 --> 01:35:34.240 and then at one end, we sometimes 01:35:34.240 --> 01:35:36.000 see these two-dimensional crystals. 01:35:36.000 --> 01:35:37.920 And we don't know whether the crystals form first, 01:35:37.920 --> 01:35:39.760 and from that, the rods form, or the rods 01:35:39.760 --> 01:35:43.840 begin to dissolve, and we form the crystals on the surface. 01:35:43.840 --> 01:35:46.160 But we were able to use those images of the crystals 01:35:46.160 --> 01:35:48.600 and then do what's called correlation averaging. 01:35:48.600 --> 01:35:50.800 Now, keep in mind, this is what Robert Malone 01:35:50.800 --> 01:35:53.680 purports to have simulatedly done at the beginning 01:35:53.680 --> 01:35:58.240 of the pandemic for the three CL protease of the coronavirus. 01:35:58.240 --> 01:36:04.480 He made a virtual X-ray crystallography model of that enzyme 01:36:04.480 --> 01:36:07.920 and then used that three-dimensional X-ray crystallography 01:36:07.920 --> 01:36:12.400 model to scan all known pharmaceuticals and nutraceuticals 01:36:12.400 --> 01:36:15.640 in the FDA catalog in less than three weeks 01:36:15.640 --> 01:36:21.240 to identify ivermectin, fluvoxamine, silicoxib, 01:36:21.240 --> 01:36:28.280 and remdesivir has four potential repurposed drugs 01:36:28.280 --> 01:36:30.760 to respond to the pandemic. 01:36:30.760 --> 01:36:39.320 He spun up his volunteer team and developed an X-ray crystallography 01:36:39.320 --> 01:36:42.160 model, computer-generated, similar to what 01:36:42.160 --> 01:36:46.880 is being described here, but just a computer-generated version 01:36:46.880 --> 01:36:50.480 of it, of the three CL protease and then 01:36:50.480 --> 01:36:55.320 scanned all known drugs and pharmaceuticals 01:36:55.320 --> 01:36:58.920 using the domain program, which was a DITRA program run 01:36:58.920 --> 01:37:04.680 by his friend, David Hone, and came up 01:37:04.680 --> 01:37:07.720 with four candidate drugs, of which at least one of them 01:37:07.720 --> 01:37:14.400 was tested by Steve Kirsch, through his early treatment 01:37:14.400 --> 01:37:18.720 fund, something something before he started his VRSF fund. 01:37:23.000 --> 01:37:25.960 I mean, that's neither here nor there with regard 01:37:25.960 --> 01:37:27.320 to what's happening here. 01:37:27.320 --> 01:37:30.160 This is just to say that when you can crystallize a protein, 01:37:30.160 --> 01:37:33.160 then you can take high-resolution pictures of it 01:37:33.160 --> 01:37:37.440 and get some idea of the structure of the protein 01:37:37.440 --> 01:37:43.240 because the crystal is a ever-repeating lattice of a pure protein. 01:37:43.240 --> 01:37:47.480 So this is a ever-repeating lattice of the prion protein, 01:37:47.480 --> 01:37:50.720 not the prionscrapi protein, which they still 01:37:50.720 --> 01:37:54.240 can't produce separately from the prion protein, which 01:37:54.240 --> 01:37:57.040 is interesting because the prion protein is not 01:37:57.040 --> 01:37:59.200 an infectious protein. 01:37:59.200 --> 01:38:00.600 And then we can average some more. 01:38:00.600 --> 01:38:02.880 So these are all imaging techniques. 01:38:02.880 --> 01:38:04.240 And we can develop these power spectra 01:38:04.240 --> 01:38:06.200 and get very, very detailed images. 01:38:06.200 --> 01:38:08.160 And so we went through a lot of this. 01:38:08.160 --> 01:38:09.880 And eventually, we're able to show 01:38:09.880 --> 01:38:12.480 that a piece of the prion protein in the middle of the protein 01:38:12.480 --> 01:38:14.680 is found in the center in these black regions 01:38:14.680 --> 01:38:18.160 and that the sugar chains are found around the edges 01:38:18.160 --> 01:38:19.160 of the black regions. 01:38:19.160 --> 01:38:20.960 And that gave rise to two different models. 01:38:20.960 --> 01:38:23.200 What we call a trimer of dimers, so there 01:38:23.200 --> 01:38:26.560 are six here, or simply a trimer, as shown here. 01:38:26.560 --> 01:38:28.560 And this is the right-handed model. 01:38:28.560 --> 01:38:31.520 This is the left-handed model. 01:38:31.520 --> 01:38:33.080 And here, you simply see one of these now. 01:38:33.080 --> 01:38:34.440 It's a much different-looking structure 01:38:34.440 --> 01:38:35.840 for the model of prp-scrapi. 01:38:35.840 --> 01:38:37.080 It has what are called beta helices. 01:38:37.080 --> 01:38:39.080 So this beta structure is now in a helix. 01:38:39.080 --> 01:38:40.080 This is an alpha helix. 01:38:40.080 --> 01:38:41.200 This is a beta helix. 01:38:41.200 --> 01:38:43.740 This is PRPC, as I talked about earlier, with helix A, 01:38:43.740 --> 01:38:46.400 helix B, and helix C. Here, you see part of helix B 01:38:46.400 --> 01:38:48.240 preserved, and all of helix C preserved. 01:38:48.240 --> 01:38:50.840 And the rest of this now is changed into beta helix. 01:38:50.840 --> 01:38:51.800 This is a current model. 01:38:51.800 --> 01:38:52.920 This is what I was thinking about this. 01:38:52.920 --> 01:38:56.360 It may or may not be right. 01:38:56.360 --> 01:38:58.280 In the way we obtained these images 01:38:58.280 --> 01:38:59.040 was using what we call it. 01:38:59.040 --> 01:39:03.120 Now remember, he doesn't have the crystal of this. 01:39:03.120 --> 01:39:07.880 So I don't really know what their molecular basis for assuming 01:39:07.880 --> 01:39:08.840 there's beta. 01:39:08.840 --> 01:39:10.160 That's a beta-pleated sheet. 01:39:10.160 --> 01:39:11.520 He called it a beta helix. 01:39:11.520 --> 01:39:13.000 I don't know. 01:39:13.000 --> 01:39:14.920 I thought you called that a beta-pleated sheet. 01:39:14.920 --> 01:39:15.640 I could be wrong. 01:39:15.640 --> 01:39:17.880 There could be also a beta helix. 01:39:17.880 --> 01:39:19.720 I mean, I'm not a protein guy. 01:39:19.720 --> 01:39:20.800 This is the current model. 01:39:20.800 --> 01:39:22.000 This is what I was thinking about this. 01:39:22.000 --> 01:39:25.360 It may or may not be right. 01:39:25.360 --> 01:39:27.320 In the way we obtained these images 01:39:27.320 --> 01:39:29.440 was using what we call the old electron microscope, which 01:39:29.440 --> 01:39:30.600 means it's no longer in service. 01:39:30.600 --> 01:39:32.000 But I'd simply show you this. 01:39:32.000 --> 01:39:33.720 And now what we're doing is working 01:39:33.720 --> 01:39:36.640 with a new microscope, which was purchased 01:39:36.640 --> 01:39:38.480 with the help of the Fairchild Foundation. 01:39:38.480 --> 01:39:40.240 This is a $2 million instrument. 01:39:40.240 --> 01:39:42.000 And you can maybe understand all this new detail that 01:39:42.000 --> 01:39:44.280 wasn't there in the last slide. 01:39:44.280 --> 01:39:46.120 So you can see that it's really essentially blank. 01:39:46.120 --> 01:39:48.200 And now we begin to see all kinds of information. 01:39:48.200 --> 01:39:51.240 So we're very optimistic that this will yield much more 01:39:51.240 --> 01:39:53.120 structure in the near future. 01:39:53.120 --> 01:39:54.680 So prions are composed of prion proteins 01:39:54.680 --> 01:39:55.920 that are rich in beta structure. 01:39:55.920 --> 01:39:58.040 And we don't know whether this is beta helix or beta sheet. 01:39:58.040 --> 01:39:59.320 But all the evidence so far argues 01:39:59.320 --> 01:40:02.320 for this new model of beta helix. 01:40:02.320 --> 01:40:04.960 So there are beta helix and beta sheet, according to that, 01:40:04.960 --> 01:40:07.560 which is good to know. 01:40:07.560 --> 01:40:10.000 I still don't think he's provided a lot of evidence 01:40:10.000 --> 01:40:15.040 that prion in an alternate form can cause other proteins 01:40:15.040 --> 01:40:16.840 to form that way yet, right? 01:40:16.840 --> 01:40:18.640 We haven't even been able to establish 01:40:18.640 --> 01:40:21.160 that prion scrappy exists as a separate protein. 01:40:21.160 --> 01:40:24.520 He's only shown as a crystal of prion protein 01:40:24.520 --> 01:40:25.920 in its natural form. 01:40:25.920 --> 01:40:27.680 Keep that in mind. 01:40:27.680 --> 01:40:29.480 When the prion protein PRPC is synthesized, 01:40:29.480 --> 01:40:30.640 it's made deep in the cell in what 01:40:30.640 --> 01:40:31.800 is called the endoplasmic reticulum. 01:40:31.800 --> 01:40:32.320 Here we go. 01:40:32.320 --> 01:40:34.120 It travels through what is called the Golgi apparatus 01:40:34.120 --> 01:40:35.160 out to the surface. 01:40:35.160 --> 01:40:36.920 And on the surface in these what are called 01:40:36.920 --> 01:40:39.360 rafts or cholesterol rich microdomains, 01:40:39.360 --> 01:40:41.040 where there's a lot of cholesterol on the surface 01:40:41.040 --> 01:40:43.960 in these little regions, the normal form of the prion 01:40:43.960 --> 01:40:46.400 protein PRPC is converted into PRP scrappy. 01:40:46.400 --> 01:40:48.120 And then it travels deep into the cell again 01:40:48.120 --> 01:40:49.600 and all the way down into what are called bicosomes. 01:40:49.600 --> 01:40:52.840 So does this look like anything that you've seen before, 01:40:52.840 --> 01:40:56.640 another cartoon of some intracellular biological process 01:40:56.640 --> 01:41:00.960 is just to be assumed to work exactly like it's drawn 01:41:00.960 --> 01:41:03.840 because there's a guy with a laser pointer shining on it? 01:41:06.480 --> 01:41:08.880 Because nothing that we've talked about so far 01:41:08.880 --> 01:41:13.560 in this discussion has led us to believe 01:41:13.560 --> 01:41:16.680 that this part of this thing is like real. 01:41:16.680 --> 01:41:18.280 It's an assumption. 01:41:18.280 --> 01:41:20.760 The cartoon is an assumption. 01:41:20.760 --> 01:41:22.920 And they're not going to do very many experiments 01:41:22.920 --> 01:41:26.320 to test any predictions that this thing might make. 01:41:26.320 --> 01:41:29.320 They're going to test to see if they can find data 01:41:29.320 --> 01:41:31.160 which fits this cartoon. 01:41:31.160 --> 01:41:32.960 See, it's converted into PRP scrappy. 01:41:32.960 --> 01:41:34.680 And then it travels deep into the cell again 01:41:34.680 --> 01:41:36.400 and all the way down into what are called bicosomes. 01:41:36.400 --> 01:41:40.000 And of course, this is a part of the biology 01:41:40.000 --> 01:41:42.000 that they want you to ignore that there 01:41:42.000 --> 01:41:45.400 are these lipid rafts that are in our membrane 01:41:45.400 --> 01:41:47.440 which are cholesterol-rich regions 01:41:47.440 --> 01:41:52.360 where most of the intracellular extracellular communication 01:41:52.360 --> 01:41:54.560 and trafficking occurs. 01:41:54.560 --> 01:41:56.320 And so it's one of the reasons why, 01:41:56.320 --> 01:41:58.840 besides the fact that myelin in your brain 01:41:58.840 --> 01:42:00.720 is made up of a lot of cholesterol, 01:42:00.720 --> 01:42:04.280 it's one of the wonderful reasons why statins are a terrible 01:42:04.280 --> 01:42:08.280 and that reducing cholesterol is just a terrible health motive 01:42:09.800 --> 01:42:11.520 that could have only been put out there 01:42:11.520 --> 01:42:14.400 by people who are idiots or people who are malevolent. 01:42:16.400 --> 01:42:19.400 And this is really feeling like covering up 01:42:19.400 --> 01:42:22.360 for people that are malevolent more than anything else. 01:42:22.360 --> 01:42:26.480 This doesn't feel like a legitimate biological story 01:42:26.480 --> 01:42:28.840 but it feels like mythology to me. 01:42:28.840 --> 01:42:30.720 Where proteins are normally degraded 01:42:30.720 --> 01:42:32.040 but PRP scrappy persists. 01:42:32.040 --> 01:42:33.400 But it doesn't persist forever as I'll show you 01:42:33.400 --> 01:42:35.000 a little bit later and it's changed the way 01:42:35.000 --> 01:42:36.000 we think about this. 01:42:39.000 --> 01:42:40.280 So the precursor of the prion 01:42:40.280 --> 01:42:42.000 is an alpha helix-rich protein PRPC 01:42:42.000 --> 01:42:43.520 that's synthesized in what's called the ER 01:42:43.520 --> 01:42:45.240 and a plasma-criticulum of the cell. 01:42:45.240 --> 01:42:47.200 The infectious form of the prion protein PRP scrappy 01:42:47.200 --> 01:42:48.880 of the prion is rich in beta structure 01:42:48.880 --> 01:42:51.680 and it is formed from PRPC on the surface of the cell 01:42:51.680 --> 01:42:53.360 in these cholesterol-rich micro domains. 01:42:53.360 --> 01:42:55.120 So this, all of this right here, 01:42:55.120 --> 01:42:57.840 besides the cholesterol-rich micro domains, 01:42:57.840 --> 01:43:00.360 all of this here is based on very, very little evidence, 01:43:00.360 --> 01:43:02.840 sometimes only one paper, just keep that in mind. 01:43:05.520 --> 01:43:07.760 Now recently we discovered that the prion protein 01:43:07.760 --> 01:43:09.680 was around a long, long time ago 01:43:09.680 --> 01:43:12.880 and that it has a cousin, actually a sister or a brother. 01:43:12.880 --> 01:43:14.200 It was an ancient gene duplication 01:43:14.200 --> 01:43:16.640 before men and mice diverged. 01:43:17.720 --> 01:43:19.960 And with the help of Lee Hood and David Westaway 01:43:19.960 --> 01:43:20.760 who was here at the time 01:43:20.760 --> 01:43:22.320 and later Richard Moore who came from Edinburgh 01:43:22.320 --> 01:43:25.000 and Patrick Tremblay who came from Montreal, 01:43:25.000 --> 01:43:26.560 we were able to unravel this mystery. 01:43:26.560 --> 01:43:28.240 So here's the prion protein gene 01:43:28.240 --> 01:43:30.320 and 16,000 bases downstream, 01:43:30.320 --> 01:43:31.680 we predicted there was a new gene 01:43:31.680 --> 01:43:33.520 which would be for the doppel protein. 01:43:34.600 --> 01:43:36.480 And when we began to look at this gene, it became... 01:43:36.480 --> 01:43:37.440 And so what are they doing? 01:43:37.440 --> 01:43:39.240 They're looking for gene homology, right? 01:43:39.240 --> 01:43:40.800 And so this could be a duplication, 01:43:40.800 --> 01:43:42.080 this could be any number of things 01:43:42.080 --> 01:43:43.800 that happens in our genome. 01:43:43.800 --> 01:43:46.040 But they're starting to make an argument 01:43:46.040 --> 01:43:50.360 that some of these motifs may or may not be other proteins 01:43:50.360 --> 01:43:55.040 in the genome with potential to be infectious. 01:43:55.040 --> 01:43:57.120 And clear that there were many common features 01:43:57.120 --> 01:43:58.800 and that helix A existed in both, 01:43:58.800 --> 01:43:59.920 helix B existed in both, 01:43:59.920 --> 01:44:01.560 and helix C existed in both. 01:44:01.560 --> 01:44:02.760 But there were differences. 01:44:02.760 --> 01:44:05.000 When we looked at individual amino acids one by one, 01:44:05.000 --> 01:44:08.240 there were only 25% that shared sequence homology 01:44:08.240 --> 01:44:09.600 that were identical. 01:44:09.600 --> 01:44:10.920 But when we looked at the structure, 01:44:10.920 --> 01:44:13.160 and this is with the work of Jane Dyson and Peter Wright 01:44:13.320 --> 01:44:14.240 and they're post-cycled. 01:44:14.240 --> 01:44:16.840 It's one existed in both and helix C existed in both. 01:44:16.840 --> 01:44:17.640 But there were differences. 01:44:17.640 --> 01:44:22.000 So helix C and helix B existed in both. 01:44:22.000 --> 01:44:23.480 Listen to him say it. 01:44:23.480 --> 01:44:25.440 And when we began to look at this gene 01:44:25.440 --> 01:44:27.800 it became clear that there were many common features 01:44:27.800 --> 01:44:29.480 and that helix A existed in both, 01:44:29.480 --> 01:44:30.560 helix B existed in both, 01:44:30.560 --> 01:44:32.040 and helix C existed in both. 01:44:32.040 --> 01:44:33.360 Okay, they exist in both. 01:44:33.360 --> 01:44:36.600 So both of these proteins apparently have the same sequence 01:44:36.600 --> 01:44:38.560 because otherwise that alpha helix, 01:44:38.560 --> 01:44:40.800 you wouldn't call it alpha helix A 01:44:40.800 --> 01:44:43.040 if it's not the same sequence of amino acids 01:44:43.080 --> 01:44:45.680 because it would be a different alpha helix, right? 01:44:45.680 --> 01:44:46.840 But there were differences. 01:44:46.840 --> 01:44:49.080 When we looked at individual amino acids one by one, 01:44:49.080 --> 01:44:52.480 there were only 25% that shared sequence homology. 01:44:52.480 --> 01:44:54.800 So if there are only 25 amino acids 01:44:54.800 --> 01:44:56.320 that share sequence homology, 01:44:56.320 --> 01:45:00.200 then how can you call it sequence the same sequence? 01:45:00.200 --> 01:45:02.840 It's not alpha helix A. 01:45:02.840 --> 01:45:04.160 It's not alpha helix B. 01:45:04.160 --> 01:45:05.780 It's not alpha helix C. 01:45:07.520 --> 01:45:10.560 Because we know that proteins use these motifs, 01:45:10.560 --> 01:45:14.880 alpha helix, beta pleated sheet, beta helix, 01:45:14.880 --> 01:45:17.040 as standard motifs. 01:45:17.040 --> 01:45:19.680 They're not so different these 20 amino acids 01:45:19.680 --> 01:45:22.200 that they don't fold similarly 01:45:22.200 --> 01:45:25.000 depending on their main attribute, 01:45:25.000 --> 01:45:29.440 which is hydrophobicity or hydrophilic. 01:45:29.440 --> 01:45:32.440 They either like to be near the polar solvents of water 01:45:32.440 --> 01:45:34.160 or they wanna fold away from it. 01:45:37.320 --> 01:45:39.520 And so again, those amino acids 01:45:39.520 --> 01:45:41.520 make a huge difference to the propensity 01:45:41.520 --> 01:45:45.120 by which that alpha helix curls up tightly away from water 01:45:45.120 --> 01:45:47.200 and bends to the left or the right. 01:45:47.200 --> 01:45:49.520 These differences are huge. 01:45:49.520 --> 01:45:53.240 And he says there's only 25% homology between these, 01:45:53.240 --> 01:45:57.200 but I mean, we'll still call them alpha helix A, B and C. 01:45:57.200 --> 01:46:00.880 Holy cow, this goes against the actual principle, 01:46:00.880 --> 01:46:03.920 the very foundational understanding that we have 01:46:03.920 --> 01:46:08.000 that the amino acid sequence has something directly to do 01:46:08.000 --> 01:46:12.760 with the potential structure and function of the protein. 01:46:12.760 --> 01:46:14.080 We're identical. 01:46:14.080 --> 01:46:15.400 But when we looked at the structure, 01:46:15.400 --> 01:46:17.680 and this is with the work of Jane Dyson and Peter Wright 01:46:17.680 --> 01:46:19.920 and their postdoc, Wapping Mo, at Scripps, 01:46:19.920 --> 01:46:21.480 comparing this to the structure done in Zurich 01:46:21.480 --> 01:46:23.000 by Kurt Vootrick that I mentioned earlier. 01:46:23.000 --> 01:46:25.440 So this is mouse doppel and mouse PRP. 01:46:25.440 --> 01:46:27.440 So what you see is that over time, 01:46:27.440 --> 01:46:30.760 the structures were preserved, here's helix A, helix A, 01:46:30.760 --> 01:46:34.040 helix B, helix C, helix B, and helix C. 01:46:34.040 --> 01:46:36.200 And yet, the sequence is diverged enormously. 01:46:36.240 --> 01:46:38.040 75% of the residues are different, 01:46:38.040 --> 01:46:40.000 but the overall structures are extremely similar. 01:46:40.000 --> 01:46:42.320 They're not arguing that those overall structures 01:46:42.320 --> 01:46:46.120 would suggest that their function is identical, but they are. 01:46:46.120 --> 01:46:47.640 Don't you see they are? 01:46:50.400 --> 01:46:52.480 Even though they know so little, 01:46:52.480 --> 01:46:55.360 they are already making that argument. 01:46:55.360 --> 01:46:56.840 We don't know what either of these proteins does. 01:46:56.840 --> 01:46:58.400 We don't know the function of either protein. 01:46:58.400 --> 01:46:59.960 Ha, ha, we don't know the function 01:46:59.960 --> 01:47:02.880 of either of these proteins in 2002. 01:47:02.880 --> 01:47:05.440 Now let's turn to the human preon diseases for a moment. 01:47:07.200 --> 01:47:08.720 The sporadic form of these diseases 01:47:08.720 --> 01:47:09.960 is called Croites Valley Aqua disease. 01:47:09.960 --> 01:47:12.760 And this accounts for 85% of all preon disease. 01:47:12.760 --> 01:47:15.480 The inherited forms account for 10% to 15% 01:47:15.480 --> 01:47:17.120 and are called Gerson stories are shanker disease, 01:47:17.120 --> 01:47:19.200 familial CJD, and fatal familial insomnia. 01:47:19.200 --> 01:47:20.520 These are autosomal dominant diseases, 01:47:20.520 --> 01:47:22.000 so half of the family members are afflicted 01:47:22.000 --> 01:47:23.080 if they live long enough. 01:47:23.080 --> 01:47:25.880 And the infectious forms of these diseases are very rare. 01:47:25.880 --> 01:47:28.120 And they include kuru among new-getting natives. 01:47:28.120 --> 01:47:31.080 It was transmitted by ritualistic cannibalism. 01:47:31.080 --> 01:47:33.240 Iatrogenic Croites Valley Aqua disease caused by growth hormone 01:47:33.240 --> 01:47:34.680 derived from human to doitaries. 01:47:34.680 --> 01:47:37.520 And I'll talk much more about new variant CJD in Britain. 01:47:37.520 --> 01:47:39.760 See, so they gave growth hormone to people 01:47:39.760 --> 01:47:42.440 and then they got CJD. 01:47:42.440 --> 01:47:45.120 So is that an immune reaction or is that a protein 01:47:45.120 --> 01:47:46.720 that you can only have one of? 01:47:53.800 --> 01:47:57.360 I don't know, I'm still skeptical 01:47:57.360 --> 01:47:59.600 of these things all being one phenomenon 01:47:59.600 --> 01:48:02.800 that is protein misfolding out of control. 01:48:02.800 --> 01:48:04.520 Hormone derived from human to doitaries. 01:48:04.520 --> 01:48:08.680 And I'll talk much more about new variant CJD in Britain. 01:48:08.680 --> 01:48:10.760 Sporadic CJD is a disease of older people. 01:48:10.760 --> 01:48:12.320 So the peak is around 70 years of age. 01:48:12.320 --> 01:48:14.080 This is Larry Schoenberger's data from the CDC. 01:48:14.080 --> 01:48:15.960 It's a 20-year experience in the United States. 01:48:15.960 --> 01:48:19.400 So very few young people develop sporadic CJD. 01:48:19.400 --> 01:48:20.880 Now, the genetic forms of this disease 01:48:20.880 --> 01:48:23.000 have a fascinating history because the first reports 01:48:23.000 --> 01:48:25.360 of Croites Valley Aqua disease were in 1921. 01:48:25.360 --> 01:48:26.760 And 10 years later, the first pedigree 01:48:26.760 --> 01:48:28.560 was brought of familial CJD. 01:48:28.560 --> 01:48:30.840 And in 1973, Ray Rus was currently 01:48:30.840 --> 01:48:32.720 chair in neurology at the University of Chicago, 01:48:32.720 --> 01:48:34.280 working with Carlton Gajosecond Joe Gibbs 01:48:34.280 --> 01:48:36.600 at the NIH, who did all the early work on Kuru. 01:48:36.600 --> 01:48:40.160 And all the transmission work on Kuru and CJD into apes and monkeys. 01:48:40.160 --> 01:48:41.720 They three of them wrote a paper. 01:48:41.720 --> 01:48:43.400 And they reported the first familial cases 01:48:43.400 --> 01:48:45.840 transmitted into non-human primates. 01:48:45.840 --> 01:48:46.960 They offered three explanations. 01:48:46.960 --> 01:48:49.520 First, that the CJD virus, as they thought it was in 1973. 01:48:49.520 --> 01:48:50.520 They thought it was a virus. 01:48:50.520 --> 01:48:53.000 Was transmitted among family members living in close proximity. 01:48:53.000 --> 01:48:54.560 Second, there was a genetic predisposition 01:48:54.560 --> 01:48:57.600 to a ubiquitous CJD virus that was everywhere, like the ether. 01:48:57.600 --> 01:48:59.320 And third, like in AIDS, there was 01:48:59.320 --> 01:49:00.840 vertical transmission of the CJD virus 01:49:00.840 --> 01:49:02.160 from parent to offspring. 01:49:02.160 --> 01:49:04.760 So it turned out that all three explanations were wrong. 01:49:04.760 --> 01:49:07.320 And in 1989, Karen Chow, who had just 01:49:07.320 --> 01:49:08.520 finished her neurology residency here 01:49:08.520 --> 01:49:10.560 and came to work with me as a postdoctoral fellow, 01:49:10.560 --> 01:49:13.160 identified first gene mutation in the PRPG 01:49:13.160 --> 01:49:17.080 in a patient who had died of GSS at UCLA. 01:49:17.080 --> 01:49:18.960 And from that, it became very clear 01:49:18.960 --> 01:49:24.320 that this mutant form of PRPC refolds into PRP scraping. 01:49:24.320 --> 01:49:26.400 It became readily apparent that it 01:49:26.400 --> 01:49:29.440 misfolds into PRP scraping. 01:49:29.440 --> 01:49:31.040 That's a very bold statement. 01:49:31.040 --> 01:49:34.960 And we're going to have to find that paper. 01:49:34.960 --> 01:49:36.520 It's a 1989 paper. 01:49:47.120 --> 01:49:52.160 That this mutant form of PRPC refolds into PRP scraping. 01:49:52.160 --> 01:49:54.320 Now, subsequent to this, this became an industry. 01:49:54.320 --> 01:49:56.320 And there are more than 30 gene mutations 01:49:56.320 --> 01:49:58.240 that have been identified, all of them above the line here, 01:49:58.240 --> 01:50:01.360 that cause these inherited forms of the disease. 01:50:01.360 --> 01:50:04.120 We've also identified what are called polymorphisms. 01:50:04.120 --> 01:50:07.080 And those are shown below the line. 01:50:07.080 --> 01:50:10.280 This one right here, and this one right here in sheep, 01:50:10.280 --> 01:50:13.160 protect people from CJD or from scraping. 01:50:13.160 --> 01:50:14.880 And we've been doing a lot of drug development 01:50:14.880 --> 01:50:17.120 with Fred Cohn and one of his postdoctoral fellows, 01:50:17.120 --> 01:50:17.880 Marty May. 01:50:17.880 --> 01:50:22.040 So he's suggesting that some of these single point mutations 01:50:22.040 --> 01:50:25.080 here can protect people from the misfolding disease, which 01:50:25.080 --> 01:50:27.600 is a pretty remarkable statement that I 01:50:27.600 --> 01:50:29.160 can't necessarily dispute. 01:50:29.160 --> 01:50:32.760 And he's suggesting that these are all associated 01:50:32.760 --> 01:50:35.880 with familial disease. 01:50:35.880 --> 01:50:43.880 I'm still at least skeptical. 01:50:43.880 --> 01:50:45.480 And we've been doing a lot of drug development 01:50:45.480 --> 01:50:48.920 with Fred Cohn and one of his postdoctoral fellows, Marty May. 01:50:48.920 --> 01:50:50.680 Now, let me turn to Prian's and Mad Cows 01:50:50.680 --> 01:50:52.000 and the transmission to people, which 01:50:52.000 --> 01:50:53.800 has really created a public health crisis that threatens 01:50:53.800 --> 01:50:56.920 the food supply as well as the blood supply worldwide. 01:50:56.920 --> 01:51:00.800 Just yesterday, Poland joined the food and blood supplies 01:51:00.800 --> 01:51:02.240 an interesting statement to make. 01:51:02.240 --> 01:51:04.880 It's definitely relevant for public health, then, isn't it? 01:51:04.880 --> 01:51:07.800 Countries of the BSE club, the first case. 01:51:07.800 --> 01:51:10.440 Now, we believe that BSE arose through industrial cannibalism 01:51:10.440 --> 01:51:11.840 in the late 1970s. 01:51:11.840 --> 01:51:13.800 And there's an argument whether it arose spontaneously 01:51:13.800 --> 01:51:16.920 in a cow, perhaps a mutation, or it came from sheep 01:51:16.920 --> 01:51:18.600 where scrapies endemic, but nevertheless, 01:51:18.600 --> 01:51:22.120 it recycled through cows and then into humans. 01:51:22.120 --> 01:51:24.680 Here's a typical newspaper clipping. 01:51:24.680 --> 01:51:26.320 And it's not only a British problem. 01:51:26.320 --> 01:51:28.720 This is also a problem throughout the con. 01:51:28.720 --> 01:51:30.040 And while there are over 100 cases, 01:51:30.040 --> 01:51:32.000 a new variant CJD in young people in Britain, 01:51:32.000 --> 01:51:35.360 there are now four cases in France. 01:51:35.360 --> 01:51:37.520 This is the BSE epidemic that begins in 1986 01:51:37.520 --> 01:51:39.800 with the discovery of the disease by Gerald Wells. 01:51:39.800 --> 01:51:40.560 It takes around 19 years. 01:51:40.560 --> 01:51:43.240 So cannibalism would imply something 01:51:43.240 --> 01:51:45.840 other than protein misfolding, right? 01:51:45.840 --> 01:51:49.240 Because if you feed healthy cattle, 01:51:49.240 --> 01:51:51.560 the remains of healthy cattle, that 01:51:51.560 --> 01:51:54.200 doesn't mean that you're giving them an infectious disease 01:51:54.200 --> 01:51:56.080 unless they already had it. 01:51:56.080 --> 01:52:00.160 So for me, this implies a autoimmune reaction. 01:52:00.160 --> 01:52:04.200 It implies autoimmunity caused by eating stuff 01:52:04.200 --> 01:52:05.720 that's too closely related to you 01:52:05.720 --> 01:52:08.240 and having that go through your payer's patches 01:52:08.240 --> 01:52:10.880 and cause a four alarm fire. 01:52:10.880 --> 01:52:13.840 That seems a lot more understandable 01:52:13.840 --> 01:52:17.080 and more likely than the idea that, 01:52:17.080 --> 01:52:18.680 again, we're dealing with something 01:52:18.680 --> 01:52:21.360 where you get the wrong amino acids in the wrong order 01:52:21.360 --> 01:52:24.200 and it's just one of those is all it's needed 01:52:24.200 --> 01:52:26.680 and you're eventually gonna be dead. 01:52:26.680 --> 01:52:29.440 That's an amazing mythology that I don't think 01:52:29.440 --> 01:52:32.880 any of this past talk has done anything to substantiate. 01:52:32.880 --> 01:52:35.640 92 with almost 40,000 cases 01:52:35.640 --> 01:52:37.400 and then the number of cases keep declining. 01:52:37.400 --> 01:52:38.600 But we keep finding more and more cases 01:52:38.600 --> 01:52:40.520 because more and more sensitive assays are being used 01:52:40.520 --> 01:52:42.480 or measurement techniques to detect the prions. 01:52:42.480 --> 01:52:44.600 All of these animals were clinically ill 01:52:44.600 --> 01:52:46.720 than at least 36 months of age. 01:52:46.720 --> 01:52:50.800 This represents, and these little balls or circles 01:52:50.800 --> 01:52:53.120 are represent one case, whereas each square 01:52:53.120 --> 01:52:56.360 represents a thousand cases on the y-axis. 01:52:56.360 --> 01:52:59.920 So what we're seeing is that there is this continuous 01:52:59.920 --> 01:53:01.440 increase in the number of cases per year, 01:53:01.440 --> 01:53:03.600 this is the yearly numbers of new variants, CJD, 01:53:03.600 --> 01:53:05.600 except in 2001, the numbers down here. 01:53:05.600 --> 01:53:08.200 What it'll be in 2002, we don't know. 01:53:08.200 --> 01:53:10.000 So we see that this disease appears about a decade 01:53:10.000 --> 01:53:11.160 after the beginning of BSE, 01:53:11.160 --> 01:53:13.240 so we think the incubation time exceeds one decade 01:53:13.240 --> 01:53:15.720 and in from crew studies, we know it can be up to four decades. 01:53:15.720 --> 01:53:18.080 Wow, so we could be waiting right now 01:53:18.080 --> 01:53:20.240 for it to happen to all of us is what is impre- 01:53:20.240 --> 01:53:21.800 what is implication is. 01:53:21.880 --> 01:53:23.920 That's pretty cool because if it happens 01:53:23.920 --> 01:53:26.720 from a transfection that's 20 years in the future, 01:53:26.720 --> 01:53:29.320 he will have already predicted it's gonna occur. 01:53:33.080 --> 01:53:36.360 Boy, that's creating a pretty wide envelope 01:53:36.360 --> 01:53:39.760 for naturally occurring prion disease 01:53:39.760 --> 01:53:43.820 to just show up and mass across the population, isn't it? 01:53:44.760 --> 01:53:47.960 Oh man, we shouldn't have been eating those cows 20 years ago. 01:53:47.960 --> 01:53:51.160 Right, we shouldn't have been eating all that beef 20 years ago. 01:53:52.800 --> 01:53:54.760 Wow, I can't believe it. 01:53:54.760 --> 01:53:56.640 We better stop eating beef all together. 01:53:56.640 --> 01:53:58.560 Maybe we should just eat fake beef 01:53:59.520 --> 01:54:02.280 because I mean, prions take 40 years to incubate. 01:54:02.280 --> 01:54:05.440 Obviously, the reason why everybody's dying of prions now 01:54:05.440 --> 01:54:07.640 is because we've been eating meat for 40 years. 01:54:07.640 --> 01:54:09.200 Just listen to this talk. 01:54:13.360 --> 01:54:16.000 This is an MRI scan and it shows, 01:54:16.000 --> 01:54:17.120 if we didn't have that light on over there, 01:54:17.120 --> 01:54:18.280 you could see this much better, 01:54:18.280 --> 01:54:19.880 this hyper-density is what it's called ate 01:54:19.880 --> 01:54:21.240 and all the way down into the pulvinar, 01:54:22.080 --> 01:54:24.280 which is this dorsal nucleus of the thalamus 01:54:24.280 --> 01:54:26.680 and this is called a hockey stick sign. 01:54:26.680 --> 01:54:29.160 And that's very typical of patients with new variant CJD. 01:54:29.160 --> 01:54:32.120 Patients with sporadic CJD have all these hyper-intensities 01:54:32.120 --> 01:54:32.960 that are seen bilaterally, 01:54:32.960 --> 01:54:35.360 but they don't have the lighting up of the pulvinar. 01:54:37.680 --> 01:54:39.400 I said I would show you some of these analog plaques 01:54:39.400 --> 01:54:40.880 and in patients with new variant CJD 01:54:40.880 --> 01:54:42.720 and this is the work of Steve D'Armond again 01:54:42.720 --> 01:54:43.960 with slides sent to, I should say, 01:54:43.960 --> 01:54:45.840 sessions sent to us by Bob Will and Jim Ironside 01:54:45.840 --> 01:54:46.680 and Edinburgh. 01:54:46.680 --> 01:54:48.040 So these are the plaques around by this halo 01:54:48.040 --> 01:54:49.360 of sponge-formed generation. 01:54:49.360 --> 01:54:52.680 They stain with antibodies to the prion protein very intensely. 01:54:55.800 --> 01:54:59.480 So which antibody, how do you raise an antibody 01:54:59.480 --> 01:55:01.640 to the prion protein if it's in that form? 01:55:01.640 --> 01:55:04.480 You reuse the one that binds to the parts 01:55:04.480 --> 01:55:05.280 that are still there. 01:55:05.280 --> 01:55:08.160 You don't have to be specific about that at all right now. 01:55:08.160 --> 01:55:10.320 Given the fact that just 20 minutes ago 01:55:10.320 --> 01:55:12.400 you were using antibodies as an indicator 01:55:12.400 --> 01:55:15.240 of whether it was prion protein in a natural form 01:55:15.240 --> 01:55:16.760 or in the infectious form, 01:55:16.760 --> 01:55:19.200 don't you think you could have used that same technique 01:55:19.200 --> 01:55:22.560 to show what fraction of that protein was in 01:55:22.560 --> 01:55:24.720 in a badly folded form 01:55:24.720 --> 01:55:27.720 or in a dangerous folded form? 01:55:27.720 --> 01:55:29.840 Or do you just think that he wasn't clever enough 01:55:29.840 --> 01:55:32.840 to ask that question with the tools that he already had? 01:55:32.840 --> 01:55:35.840 BUZZER. 01:55:35.840 --> 01:55:38.520 This is why they hate me to be in the audience of these talks, 01:55:38.520 --> 01:55:41.160 why they hated me in the audience of these talks. 01:55:41.160 --> 01:55:42.360 Oh, very intensely. 01:55:45.840 --> 01:55:47.400 I've already said this seven times. 01:55:49.960 --> 01:55:52.200 Now, one wanted to try to figure out 01:55:52.200 --> 01:55:53.360 where did all of this start? 01:55:53.360 --> 01:55:54.560 Did it really start in sheep? 01:55:54.560 --> 01:55:55.760 Did it start in cows? 01:55:55.760 --> 01:55:57.240 And did it come from the cows to the humans? 01:55:57.240 --> 01:55:58.440 And if we were looking for a virus, 01:55:58.440 --> 01:55:59.920 we could do this very easily. 01:55:59.920 --> 01:56:01.080 The virus would be simple to track 01:56:01.080 --> 01:56:02.320 because the virus would have a nucleic acid, 01:56:02.320 --> 01:56:03.720 a piece of DNA or RNA, 01:56:03.720 --> 01:56:07.000 and we could easily track it into the sheep and see. 01:56:07.000 --> 01:56:11.000 Viruses are easy to track according to Stanley Prusen 01:56:11.000 --> 01:56:14.200 or easy, I mean, come on, that's easy work. 01:56:14.200 --> 01:56:16.040 Easy work. 01:56:17.040 --> 01:56:20.440 BUZZER. 01:56:20.440 --> 01:56:22.200 And it had nothing to do with the sheep, 01:56:22.200 --> 01:56:24.400 but that same piece of DNA or RNA 01:56:24.400 --> 01:56:26.600 appeared in the cow and then appeared in the human. 01:56:26.600 --> 01:56:27.480 We can't do that. 01:56:27.480 --> 01:56:28.920 So what we had to do was really begin 01:56:28.920 --> 01:56:30.640 to look at what are called prion strains. 01:56:30.640 --> 01:56:32.120 And what's happening is that PRP scrape 01:56:32.120 --> 01:56:33.280 is signified by a square. 01:56:33.280 --> 01:56:35.600 It's stimulating the conversion of bovine PRPC. 01:56:35.600 --> 01:56:38.920 This is from the sheep into PRP scrapey of the cow. 01:56:38.920 --> 01:56:41.200 And the same thing, we can reiterate this into the human. 01:56:41.200 --> 01:56:42.400 Now, there's a lot of data, but I'm 01:56:42.400 --> 01:56:43.840 only going to show you very little. 01:56:43.840 --> 01:56:45.360 And what we did was to use transgenic mice 01:56:45.440 --> 01:56:47.240 to investigate the origin of BSE 01:56:47.240 --> 01:56:48.720 and the transmission of prions to humans. 01:56:48.720 --> 01:56:50.680 Oh, so we're going to use an animal model 01:56:50.680 --> 01:56:54.280 to understand the cartoon that he says definitely happens, 01:56:54.280 --> 01:56:58.840 that the sheep protein causes the refolding of the cow protein 01:56:58.840 --> 01:57:01.800 that causes the refolding of the human protein. 01:57:01.800 --> 01:57:03.920 There's three assumptions there on top 01:57:03.920 --> 01:57:05.920 of three different cartoons. 01:57:05.920 --> 01:57:07.320 And yet somehow or another, we're 01:57:07.320 --> 01:57:09.800 going to move right on to a transgenic animal model. 01:57:09.800 --> 01:57:10.440 Let's hear it. 01:57:10.440 --> 01:57:11.440 Let's hear it. 01:57:11.440 --> 01:57:12.400 Now, there's a lot of data, but I'm 01:57:12.400 --> 01:57:13.920 only going to show you very little. 01:57:13.920 --> 01:57:15.400 And what we did was to use transgenic mice 01:57:15.400 --> 01:57:17.760 to investigate the origin of BSE and the transmission 01:57:17.760 --> 01:57:18.960 of prions to humans. 01:57:18.960 --> 01:57:20.480 There's now compelling evidence that indicates 01:57:20.480 --> 01:57:22.720 that prions and beef products were ingested by humans, who 01:57:22.720 --> 01:57:26.480 later developed variant CJD. 01:57:26.480 --> 01:57:29.000 What we began with, and this is Jim Mastrioni's work, 01:57:29.000 --> 01:57:31.920 Glenn Telling's work, was what we call a humanized mouse. 01:57:31.920 --> 01:57:34.800 So now this mouse behaves toward prions like a human being. 01:57:34.800 --> 01:57:37.320 And when we take sporadic CJD or familial CJD 01:57:37.320 --> 01:57:38.880 and transmit into the mice brain extracts 01:57:38.880 --> 01:57:40.520 from people who have died of these diseases, 01:57:40.520 --> 01:57:42.520 all of the mice get sick in about 200 days. 01:57:42.520 --> 01:57:45.000 But when we took new variant CJD from young people in England, 01:57:45.000 --> 01:57:46.880 people in their early 20s or late teens, 01:57:46.880 --> 01:57:49.920 we found that about 25%, this is wrong, it's not 60%, 01:57:49.920 --> 01:57:53.080 but about 25% of the mice became ill by 500 days. 01:57:53.080 --> 01:57:56.320 And when we took brain extracts from cows with BSE, 01:57:56.320 --> 01:57:59.120 none of the mice developed disease by 600 days. 01:57:59.120 --> 01:58:00.240 Now, Mike Scott did an experiment 01:58:00.240 --> 01:58:01.760 where he created bovinized mice. 01:58:01.760 --> 01:58:03.440 So these are mice that behave like cows 01:58:03.440 --> 01:58:04.760 with respected prions. 01:58:04.760 --> 01:58:07.800 And when he took mad cows, brain, 240 days, 01:58:07.800 --> 01:58:08.840 and all the mice were sick. 01:58:08.840 --> 01:58:10.560 With new variant CJD, what he found 01:58:10.560 --> 01:58:13.080 was that 100% of the mice developed disease 01:58:13.080 --> 01:58:15.240 at 260 days after inoculation. 01:58:15.240 --> 01:58:16.360 So this was quite a shock. 01:58:16.360 --> 01:58:18.440 And that when we took sheep's scrapie from the United States, 01:58:18.440 --> 01:58:23.200 the mice were even more susceptible, 210 days, 100% were sick. 01:58:23.200 --> 01:58:27.400 Now, we would have to look at what this mouse model is. 01:58:27.400 --> 01:58:32.120 I don't know if they have overexpression of the bovine protein 01:58:32.120 --> 01:58:34.440 in their brain or the human protein in their brain. 01:58:34.440 --> 01:58:35.920 That's my guess. 01:58:35.920 --> 01:58:40.160 My guess is that this time, I'm sure 01:58:40.160 --> 01:58:41.000 I'm right. 01:58:41.000 --> 01:58:43.800 Okay, I would bet my, I bet everything on it. 01:58:43.800 --> 01:58:46.160 In 2002, and when this was done, 01:58:46.160 --> 01:58:48.160 it would have been before 2002, 01:58:48.160 --> 01:58:50.440 there would have been no way for them to knock out 01:58:50.440 --> 01:58:53.520 the native gene and replace it with the bovine gene. 01:58:53.520 --> 01:58:56.040 So what they're most likely working with here 01:58:56.040 --> 01:58:58.840 is a mouse that also expresses the bovine 01:59:00.440 --> 01:59:05.320 prion protein driven by some genetic technology. 01:59:05.320 --> 01:59:06.880 And then when they inject that animal 01:59:06.880 --> 01:59:08.320 in the brain, it gets sick. 01:59:11.160 --> 01:59:15.960 And so we would have to, again, look into these papers 01:59:15.960 --> 01:59:16.800 and figure it out. 01:59:16.800 --> 01:59:21.000 But my guess is that this isn't gonna be as clean a result 01:59:21.000 --> 01:59:24.160 as what he's suggesting it is here, 01:59:24.160 --> 01:59:27.800 simply because the experimental model is not as clean 01:59:27.800 --> 01:59:29.320 as he wants you to believe it is. 01:59:29.320 --> 01:59:34.320 It's a bovine, it responds like a cow with respect to prion, 01:59:37.000 --> 01:59:39.120 it responds like a human with respect to prion. 01:59:39.120 --> 01:59:41.640 You can hear him say it again if you want to. 01:59:41.640 --> 01:59:43.040 Into the mice, brain extracts from people 01:59:43.040 --> 01:59:44.320 who've died of these diseases. 01:59:44.320 --> 01:59:46.320 All of the mice get sick in about 200 days. 01:59:46.320 --> 01:59:48.800 But when we took new variants, CJD from young people in England, 01:59:48.800 --> 01:59:50.720 people in the early 20s, late teens, 01:59:50.720 --> 01:59:53.760 we found that about 25%, this is wrong, it's not 60%, 01:59:53.760 --> 01:59:56.880 but about 25% of the mice became ill by 500 days. 01:59:56.880 --> 02:00:00.120 And when we took brain extracts from cows with BSE, 02:00:00.120 --> 02:00:02.960 none of the mice developed disease by 600 days. 02:00:02.960 --> 02:00:04.120 Now Mike Scott did an experiment 02:00:04.120 --> 02:00:05.600 where he created bovineized mice. 02:00:05.600 --> 02:00:07.280 So these are mice that behave like cows 02:00:07.280 --> 02:00:08.120 with respect to prions. 02:00:08.120 --> 02:00:10.080 See bovineized mice and then again, 02:00:10.080 --> 02:00:11.640 they're injecting into the brain. 02:00:11.640 --> 02:00:16.640 Again, how does that equivalent to eating your dead relative? 02:00:17.080 --> 02:00:20.920 How does that equivalent to eating another cow? 02:00:20.920 --> 02:00:24.480 It's just all not the experimental model that you want it to be 02:00:24.480 --> 02:00:26.400 if you want to understand what he was showing 02:00:26.400 --> 02:00:28.040 in the previous slides. 02:00:28.040 --> 02:00:31.080 And when he took mad cows, brain, 240 days 02:00:31.080 --> 02:00:32.120 and all the mice were sick. 02:00:32.120 --> 02:00:35.040 With new variants, CJD, what he found was that 100% 02:00:35.040 --> 02:00:37.360 of the mice developed disease at 260 days 02:00:37.360 --> 02:00:38.480 after inoculation. 02:00:38.480 --> 02:00:39.600 So this was quite a shock. 02:00:39.600 --> 02:00:41.680 And that when we took sheep's scrapie from the United States, 02:00:41.680 --> 02:00:45.080 the mice were even more susceptible, 210 days, 100% were sick. 02:00:46.480 --> 02:00:47.600 And we looked at the neuropathology 02:00:47.600 --> 02:00:49.040 against Stevie Arman's work. 02:00:49.040 --> 02:00:51.480 What Steve found was that if we began with sheep's scrapie, 02:00:51.480 --> 02:00:53.360 there was very little of the prion protein deposited 02:00:53.360 --> 02:00:54.480 in what's called the corpus callosum, 02:00:54.480 --> 02:00:56.520 the structure that connects both sides of your brain. 02:00:56.520 --> 02:00:58.400 And some people with intractable epilepsy, 02:00:58.400 --> 02:00:59.480 this structure is cut. 02:01:01.520 --> 02:01:04.080 When we looked, if we began with BSE in these bovineized mice, 02:01:04.080 --> 02:01:05.680 we saw large amounts of the prion protein 02:01:05.680 --> 02:01:07.160 deposited in these big plaques. 02:01:07.160 --> 02:01:08.640 And if we took new variant CJD, 02:01:08.640 --> 02:01:10.040 the image was indistinguishable. 02:01:11.040 --> 02:01:12.360 So when we put all this together, 02:01:12.360 --> 02:01:14.040 the indistinguishable neuropathology, 02:01:14.040 --> 02:01:15.440 the very similar incubation times, 02:01:15.440 --> 02:01:17.200 it became very clear that there was little doubt 02:01:17.200 --> 02:01:19.400 that the people had become sick from the cows. 02:01:19.400 --> 02:01:20.960 And that's in addition to all the human epidemiology 02:01:20.960 --> 02:01:23.080 showing the disease has confined largely to Great Britain 02:01:23.080 --> 02:01:24.680 with four cases in France, as I said, 02:01:24.680 --> 02:01:26.280 one in Italy and one in Ireland. 02:01:27.680 --> 02:01:29.480 And now there's actually one in the United States, 02:01:29.480 --> 02:01:31.640 but this is a woman who, 22 years old, 02:01:31.640 --> 02:01:33.640 22 years old, living in Florida, 02:01:33.640 --> 02:01:36.200 who lived in Great Britain for nearly 10 years. 02:01:36.200 --> 02:01:37.560 And it's a British citizen. 02:01:39.080 --> 02:01:40.560 So all of this put together, 02:01:40.560 --> 02:01:41.840 Mike Scott began to think about this 02:01:41.840 --> 02:01:42.760 in a slightly different way. 02:01:42.760 --> 02:01:44.640 And he's begun to wonder if, in fact, 02:01:44.640 --> 02:01:46.840 all sheep with screpy prions, the blue ones, 02:01:46.840 --> 02:01:50.080 also have a few BSE prions that multiply more slowly. 02:01:50.080 --> 02:01:51.720 And that what happened in the late 1970s 02:01:51.720 --> 02:01:53.080 was that when they reduced the temperature 02:01:53.080 --> 02:01:55.560 and also the process, they did less severe, 02:01:55.560 --> 02:01:57.280 what they call the rendering process, 02:01:57.280 --> 02:02:00.040 the screpy prions were still destroyed, 02:02:00.040 --> 02:02:01.720 but the BSE prions survived. 02:02:01.720 --> 02:02:03.760 And now they multiplied in cattle 02:02:03.760 --> 02:02:05.360 where they became pathogenic for humans. 02:02:05.360 --> 02:02:06.640 And there were no more screpy prions, 02:02:06.640 --> 02:02:08.000 the blue ones, the whole down the amount 02:02:08.000 --> 02:02:09.240 of the red ones that could be made, 02:02:09.240 --> 02:02:11.240 because whether that's true or not, I don't know. 02:02:11.240 --> 02:02:12.240 So this is a hypothesis. 02:02:12.240 --> 02:02:14.160 But there's no way for that to happen 02:02:14.160 --> 02:02:17.280 unless you're talking about prions moving separately 02:02:17.280 --> 02:02:22.040 as animals breed over time and building up separately 02:02:22.040 --> 02:02:24.600 in the population, even though they're not passed on 02:02:24.600 --> 02:02:25.680 through birth. 02:02:25.680 --> 02:02:27.600 That's incredible. 02:02:27.600 --> 02:02:29.760 Or maybe they are passed, I mean, wow. 02:02:30.760 --> 02:02:32.360 That's a pretty funny model. 02:02:32.360 --> 02:02:34.120 It makes a lot of predictions that I bet 02:02:34.120 --> 02:02:35.680 they won't bother testing. 02:02:35.680 --> 02:02:37.680 We're trying to pursue this. 02:02:37.680 --> 02:02:39.360 So variant CJD is caused by prions 02:02:39.360 --> 02:02:41.080 in beef products from mad cows. 02:02:41.080 --> 02:02:43.040 Over 85% of all human prion diseases 02:02:43.040 --> 02:02:45.000 are sporadic 10 to 15% are inherited. 02:02:45.000 --> 02:02:47.200 So I just want to point out that while there are about 5000 02:02:47.200 --> 02:02:49.000 cases of CJD across the planet each year, 02:02:49.000 --> 02:02:51.760 we're talking about 25 cases of variant CJD so far. 02:02:51.760 --> 02:02:54.800 So just to give you a little perspective on the numbers. 02:02:54.800 --> 02:02:58.880 Let's now turn to therapeutic approaches to prion diseases. 02:02:58.880 --> 02:03:00.240 So there are a large number of approaches 02:03:00.240 --> 02:03:01.600 that we've taken here at UCSF. 02:03:01.600 --> 02:03:03.440 And I won't talk about rational drug design based 02:03:03.440 --> 02:03:05.080 upon what is called dominant negative inhibition 02:03:05.080 --> 02:03:05.880 or gene therapy. 02:03:05.880 --> 02:03:07.360 And I'm also not going to talk about what 02:03:07.360 --> 02:03:08.280 is called enhanced clearance. 02:03:08.280 --> 02:03:10.640 But I come back to talk a little bit about clearance of prions. 02:03:10.640 --> 02:03:11.760 I am going to talk about antibodies 02:03:11.760 --> 02:03:13.920 that were used to inhibit prion replication. 02:03:13.920 --> 02:03:16.160 And then I'm going to talk about a drug called quinacrine 02:03:16.160 --> 02:03:18.080 that we're giving to patients in a study with Bruce Miller 02:03:18.080 --> 02:03:22.240 and Michael Geshwin and many, many other people here at UCSF. 02:03:22.240 --> 02:03:24.320 So this is David Parris' work using these antibodies produced 02:03:24.320 --> 02:03:27.040 by Dennis Burton and Anthony Williamson at Scripps. 02:03:27.040 --> 02:03:29.200 And again, the same blue antibodies react out here, 02:03:29.200 --> 02:03:30.520 more toward the N-terminus, the green ones 02:03:30.520 --> 02:03:33.000 at the C-terminus, and the red ones in the middle region. 02:03:33.000 --> 02:03:36.880 That's Helix A, Helix B, Helix C. 02:03:36.880 --> 02:03:38.600 All this looks a little complicated. 02:03:38.600 --> 02:03:40.360 But let's just look over here at this graph. 02:03:40.360 --> 02:03:42.040 So we're adding increasing amounts of antibody 02:03:42.040 --> 02:03:43.040 to cultured cells. 02:03:43.040 --> 02:03:44.800 And we're seeing the decrease in the amount of PRP 02:03:44.800 --> 02:03:46.280 scrapey, the protein of the prion. 02:03:46.280 --> 02:03:48.240 And the red antibodies work better than the blue antibodies 02:03:48.240 --> 02:03:49.080 but only a little bit. 02:03:49.080 --> 02:03:52.440 But both red and blue work much better than the green antibodies. 02:03:52.440 --> 02:03:54.040 And if we look here, now instead of having 02:03:54.040 --> 02:03:56.280 the concentration of antibody, it's the duration of antibodies. 02:03:56.280 --> 02:03:59.080 Are you at all surprised that one 02:03:59.080 --> 02:04:00.840 of the therapies he wanted to talk about 02:04:00.840 --> 02:04:04.200 was using monoclonal antibodies to fight prion disease? 02:04:04.200 --> 02:04:05.880 Are you at all surprised? 02:04:05.880 --> 02:04:08.240 This is at a time when monoclonal antibodies 02:04:08.240 --> 02:04:15.680 are right at the hottest biologic there is to make. 02:04:15.680 --> 02:04:19.680 And the IP law is ripe for the writing. 02:04:19.680 --> 02:04:20.800 Antibody treatment. 02:04:20.800 --> 02:04:22.960 So we picked one concentration of antibody. 02:04:22.960 --> 02:04:25.600 And now what we've done, and you see that the red ones 02:04:25.600 --> 02:04:27.720 work quite rapidly compared to the blue ones. 02:04:27.720 --> 02:04:29.800 And what this allows us to do is to look at when 02:04:29.800 --> 02:04:32.400 we see a half maximal decrease, when we've gone down 50%. 02:04:32.400 --> 02:04:36.000 And that's about at this point at about 30 hours right in here. 02:04:36.000 --> 02:04:38.840 It was actually IMM, UNITY. 02:04:38.840 --> 02:04:41.880 That's what I got, bodies anti. 02:04:41.880 --> 02:04:45.840 He spelled immunity not by antibodies. 02:04:45.840 --> 02:04:48.720 And in 1990, Bruce Cheesebrow and Byron Coe 02:04:48.720 --> 02:04:50.240 working at the Rocky Mountain Laboratory 02:04:50.240 --> 02:04:52.280 did a lot of work on the synthesis of PRPC 02:04:52.280 --> 02:04:54.040 and defined what is called the Half-Life for formation. 02:04:54.040 --> 02:04:55.480 Very rapid. 02:04:55.480 --> 02:04:58.360 And David Borchelt working here in San Francisco confirmed 02:04:58.360 --> 02:05:00.560 that and then showed that the Half-Life time for degradation 02:05:00.560 --> 02:05:01.600 is about six hours. 02:05:01.600 --> 02:05:03.880 And that PRPC was made even more slowly 02:05:03.880 --> 02:05:05.560 with a half-time of formation of about three to 10. 02:05:05.560 --> 02:05:08.400 OK, so here they're transfecting these two. 02:05:08.400 --> 02:05:12.880 And now PRPC grapey actually must have a different sequence 02:05:12.880 --> 02:05:16.360 because otherwise you're expressing the same protein. 02:05:16.360 --> 02:05:17.960 It gets folded in two different ways. 02:05:17.960 --> 02:05:19.600 So either you're trans, how can you 02:05:19.600 --> 02:05:23.120 transfect neuroblastoma cells with this protein 02:05:23.120 --> 02:05:24.640 unless it has a different sequence? 02:05:25.480 --> 02:05:27.200 What's going on here? 02:05:27.200 --> 02:05:30.400 What is going on here? 02:05:30.400 --> 02:05:33.720 David Borchelt working here in San Francisco confirmed that 02:05:33.720 --> 02:05:35.480 and then showed that the Half-Life time for degradation 02:05:35.480 --> 02:05:36.600 is about six hours. 02:05:36.600 --> 02:05:38.800 And that PRPC grapey was made even more slowly 02:05:38.800 --> 02:05:41.080 with a half-time of formation of about three to 10 hours. 02:05:41.080 --> 02:05:45.000 Now, I had thought that PRPC grapey was complete granite 02:05:45.000 --> 02:05:46.560 and that it was never degraded. 02:05:46.560 --> 02:05:47.960 But in the last slide that I showed you, 02:05:47.960 --> 02:05:48.960 I don't get that at all. 02:05:48.960 --> 02:05:50.880 This is really represents the degradation of PRPC 02:05:50.880 --> 02:05:53.800 the loss of prions from the culture. 02:05:53.880 --> 02:05:56.000 They couldn't separate the two 02:05:56.000 --> 02:05:57.920 because they're the same weight. 02:05:59.080 --> 02:06:00.400 They can't separate the two 02:06:00.400 --> 02:06:03.320 because they exist together in their cartoons. 02:06:06.120 --> 02:06:08.440 But then they shouldn't be able to exist together 02:06:08.440 --> 02:06:11.000 because the one causes the other to become it. 02:06:13.640 --> 02:06:16.080 And yet they have the same amino acid sequence. 02:06:16.080 --> 02:06:20.800 So how would you express one or the other in a cell culture? 02:06:20.800 --> 02:06:22.440 You'd have to use the same RNA. 02:06:22.440 --> 02:06:23.440 I don't get it. 02:06:23.440 --> 02:06:24.320 I don't get it. 02:06:24.320 --> 02:06:25.880 I don't get it. 02:06:25.880 --> 02:06:27.280 And so we were able to calculate a number, 02:06:27.280 --> 02:06:28.240 as I mentioned before, 02:06:28.240 --> 02:06:31.640 where 50% of the PRPC grapey disappears of about 30 hours. 02:06:32.600 --> 02:06:33.680 This has important implications 02:06:33.680 --> 02:06:34.680 for thinking about all the purposes. 02:06:34.680 --> 02:06:35.520 I'm just an idiot then. 02:06:35.520 --> 02:06:36.360 I got more reading to do. 02:06:36.360 --> 02:06:37.200 That's all. 02:06:37.200 --> 02:06:39.120 See, this is the reason why I haven't started teaching it yet 02:06:39.120 --> 02:06:40.120 because I've got more read. 02:06:40.120 --> 02:06:41.400 I don't understand that. 02:06:42.600 --> 02:06:44.600 And that contradiction seems pretty big. 02:06:44.600 --> 02:06:46.960 The whole talk has been explaining 02:06:46.960 --> 02:06:49.200 about how difficult it is to sort this out 02:06:49.200 --> 02:06:50.520 because it is a pre-... 02:06:50.520 --> 02:06:52.840 It is a protein that has alternate forms 02:06:52.840 --> 02:06:55.480 that alternate forms are hard to study. 02:06:55.480 --> 02:06:57.000 And yet now we can express them 02:06:57.000 --> 02:06:59.480 in different models when we want them. 02:07:01.920 --> 02:07:03.120 Tells us that cells are capable 02:07:03.120 --> 02:07:05.640 of degrading both PRPC and PRPC grapey. 02:07:05.640 --> 02:07:07.400 And it raises the question whether PRPC grapey 02:07:07.400 --> 02:07:09.720 is normally found at very low levels in normal cells 02:07:09.720 --> 02:07:11.840 and has a physiological function. 02:07:11.840 --> 02:07:13.880 And that is a very appealing way of thinking about all this 02:07:13.880 --> 02:07:15.120 because it makes much more sense 02:07:15.120 --> 02:07:18.040 than thinking that PRPC grapey is something totally apparent. 02:07:18.040 --> 02:07:19.360 It raises the question of whether or not 02:07:19.360 --> 02:07:20.200 we really have an issue. 02:07:20.200 --> 02:07:22.520 It's a kinetic race between the formation of PRPC grapey 02:07:22.520 --> 02:07:24.960 and the cell's ability to clear PRPC grapey. 02:07:24.960 --> 02:07:27.240 And that when the cell can keep up with the formation 02:07:27.240 --> 02:07:30.720 and clear it like it does with all other proteins in fact, 02:07:30.720 --> 02:07:33.280 when that happens everything is functioning fine. 02:07:33.280 --> 02:07:35.320 But when the cell gets out of balance, 02:07:35.320 --> 02:07:39.200 it can no longer clear PRPC grapey at the rate that it's formed. 02:07:39.200 --> 02:07:40.360 I'm talking about very, very low levels 02:07:40.360 --> 02:07:42.880 that we can't detect even by these animal assays. 02:07:42.880 --> 02:07:45.000 Then something goes awry and we begin to accumulate 02:07:45.000 --> 02:07:47.440 more and more PRPC grapey and eventually the animal gets sick 02:07:47.440 --> 02:07:49.320 and goes on to die or the human being. 02:07:50.200 --> 02:07:53.000 Now, I promised you a little chemistry at the end. 02:07:53.000 --> 02:07:54.480 And if you just look down here at chloropromacy 02:07:54.480 --> 02:07:55.920 and this is thoracine, this is one of the first 02:07:55.920 --> 02:07:57.600 antipsychotic drugs and this is the structure of it 02:07:57.600 --> 02:07:59.200 and it has these three rings. 02:07:59.200 --> 02:08:02.240 And when Karsten Korth added one micromolar, this amount, 02:08:02.240 --> 02:08:04.880 he still saw these protease resistant bands. 02:08:04.880 --> 02:08:07.400 So the three bands are the ones with no sugars, 02:08:07.400 --> 02:08:08.800 one sugar chain and two sugar chains, 02:08:08.800 --> 02:08:09.800 they're shed a little better here. 02:08:09.800 --> 02:08:11.320 So two sugar chains, one and none. 02:08:11.320 --> 02:08:12.160 I'm gonna go back. 02:08:12.160 --> 02:08:13.320 Or even up in here. 02:08:13.320 --> 02:08:15.000 It's a very appealing way of thinking about all this 02:08:15.000 --> 02:08:16.640 because it makes much more sense than thinking 02:08:16.640 --> 02:08:19.160 that PRPC grapey is something totally apparent. 02:08:19.200 --> 02:08:20.520 It raises the question of whether or not 02:08:20.520 --> 02:08:22.120 we really have an issue, it's a kinetic race 02:08:22.120 --> 02:08:23.680 between the formation of PRPC grapey 02:08:23.680 --> 02:08:26.120 and the cells of the clear PRPC grapey. 02:08:26.120 --> 02:08:28.360 And that when the cell can keep up with the formation 02:08:28.360 --> 02:08:31.880 and clear it like it does with all other proteins, in fact, 02:08:31.880 --> 02:08:34.440 when that happens, everything is functioning fine. 02:08:34.440 --> 02:08:36.440 But when the cell gets out of balance, 02:08:36.440 --> 02:08:40.360 it can no longer clear PRPC grapey at the rate that it's formed. 02:08:40.360 --> 02:08:41.520 And we're talking about very, very low levels 02:08:41.520 --> 02:08:43.840 that we can't detect even by these animal assays. 02:08:43.840 --> 02:08:46.480 So very, very low levels, kind of like asymptomatic. 02:08:46.480 --> 02:08:50.560 You're asymptomatic for CAD, all your life. 02:08:50.560 --> 02:08:53.320 And if you lose the race, then you become, 02:08:53.320 --> 02:08:57.160 so it's actually not what a lot of these worst case scenario 02:08:57.160 --> 02:09:00.520 people said, which is you just need one molecule. 02:09:00.520 --> 02:09:04.160 You have one molecule all the time by this hypothesis. 02:09:04.160 --> 02:09:06.400 And it's just a question of whether you fall behind 02:09:06.400 --> 02:09:10.280 with degradation or whether you inherited too much 02:09:10.280 --> 02:09:13.680 to be ever catch up in the beginning or something like that. 02:09:14.680 --> 02:09:17.680 It's extraordinary what is being said here. 02:09:17.680 --> 02:09:19.680 Then something goes awry, and we began to accumulate 02:09:19.680 --> 02:09:21.680 more and more PRPC grapey, and eventually the animal gets sick. 02:09:21.680 --> 02:09:22.680 And it goes on to die. 02:09:22.680 --> 02:09:25.680 And still we're talking about a very specific protein 02:09:25.680 --> 02:09:29.680 with a very specific sequence in a very specific structure. 02:09:29.680 --> 02:09:32.680 And then sometimes we're not. 02:09:32.680 --> 02:09:34.680 Sometimes we're talking about a very specific protein 02:09:34.680 --> 02:09:37.680 with a very specific sequence in a very specific structure 02:09:37.680 --> 02:09:39.680 that has two structures. 02:09:39.680 --> 02:09:42.680 And those two structures, apparently, one of them 02:09:42.680 --> 02:09:45.680 is more stable than the other, so much so that it can cause 02:09:45.680 --> 02:09:48.680 the good one to form the bad one. 02:09:48.680 --> 02:09:53.680 But that part of this cartoon is almost wholly missing 02:09:53.680 --> 02:09:56.680 from the explanation. 02:09:56.680 --> 02:09:58.680 And instead we have incubation times 02:09:58.680 --> 02:10:01.680 and p-value differences between brown mice and white mice 02:10:01.680 --> 02:10:04.680 with two amino acids difference in this protein. 02:10:04.680 --> 02:10:08.680 And then that being a sign that the protein is involved 02:10:08.680 --> 02:10:12.680 in whatever happens. 02:10:12.680 --> 02:10:14.680 Okay, it is 335. 02:10:14.680 --> 02:10:16.680 I need to go to basketball. 02:10:16.680 --> 02:10:20.680 I'm going to play the funny opening that I started with again 02:10:20.680 --> 02:10:24.680 as a way out in case you didn't see it because it's just funny. 02:10:24.680 --> 02:10:29.680 I got a new toy, which is actually a new toy for 02:10:29.680 --> 02:10:36.680 it is a new toy, which I am planning to use on the wheel. 02:10:36.680 --> 02:10:40.680 And also maybe on my motorcycle if we get a little better weather. 02:10:40.680 --> 02:10:45.680 And that toy, of course, was a little camera 02:10:45.680 --> 02:10:47.680 that allowed me to make this video. 02:10:47.680 --> 02:10:49.680 But anyway, thank you very much for joining me. 02:10:49.680 --> 02:10:52.680 This has been Giga Ohm Biological, a high-resistance, 02:10:52.680 --> 02:10:55.680 low-noise, oh, that's not the right song. 02:10:55.680 --> 02:10:57.680 You funny man, that's not the right song. 02:10:57.680 --> 02:10:59.680 This is the right song. 02:10:59.680 --> 02:11:01.680 That's it. 02:11:01.680 --> 02:11:07.680 This has been a production of Giga Ohm Biological. 02:11:07.680 --> 02:11:11.680 And this was, of course, not meant to make you take me seriously 02:11:11.680 --> 02:11:15.680 as a basketball player, but at least to let you know that I do 02:11:15.680 --> 02:11:17.680 actually go to the gym with my kids. 02:11:17.680 --> 02:11:23.680 And I'm going to try and figure out a way to make neurobiology 02:11:23.680 --> 02:11:27.680 shorts and immunology shorts while I'm shooting around. 02:11:27.680 --> 02:11:29.680 And I thought this camera would kind of help me. 02:11:29.680 --> 02:11:32.680 It actually works surprisingly well. 02:11:32.680 --> 02:11:36.680 It also works surprisingly well on the one wheel. 02:11:36.680 --> 02:11:39.680 So I think I'm going to be able to make some shorts 02:11:39.680 --> 02:11:43.680 that I haven't been able to make before because I just don't like 02:11:43.680 --> 02:11:49.680 putting three GoPros on something and having to edit for hours afterwards. 02:11:49.680 --> 02:11:52.680 And that thing seems to have solved that problem a little bit. 02:11:52.680 --> 02:11:55.680 I can give a GoPros to my wife for her yoga. 02:11:55.680 --> 02:11:58.680 Anyway, thanks very much for joining me. 02:11:58.680 --> 02:12:03.680 And when Giga Ohm Biological, we are trying to distribute the knowledge 02:12:03.680 --> 02:12:07.680 that can dispel this big E enchantment. 02:12:07.680 --> 02:12:10.680 Thanks very much, and we'll see you again soon. 02:12:28.680 --> 02:12:31.680 Thank you very much. 02:12:58.680 --> 02:13:01.680 Thank you very much.