WEBVTT

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You

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But you can tell if someone's lying, you know, you can sort of feel it in people

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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

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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

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I'm not sure exactly who he is. His name is JJ Cooey, and I believe he's a consultant for CHD

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And I believe he has a PhD in some sort of scientific discipline from what I understand

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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.

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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.

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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.

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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.

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I don't want to be a liar. I don't want to be a liar. I don't want to be a liar.

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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.

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My friend Denver mixed date.

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I wanted to mix it up a little bit, maybe get some other slides out.

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We haven't used in a while, I know they're a little more reading.

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It's a little more reading.

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It's like we got ourselves some readers in the house.

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Jimmy Doar made a reference to the reader joke, very, very good Bill Hicks joint, very nice

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bit.

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Jimmy, Jimmy Doar showed a lot of courage the other night, I'm giving him another shout-out.

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It is amazing how many people think they can answer an argument by attributing bad motives

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to those who disagree with them using this kind of reasoning you can believe or not believe

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anything about anything without having to bother to deal with facts or logic.

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It's amazing how many people think they can believe or not believe it or not believe it

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or not believe it or not believe it or not believe it or not believe it or not believe

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it or not believe it or not believe it or not believe it or not believe it or not believe

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it or not believe it or not believe it or not believe it or not believe it or not believe

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it or not believe it or not believe it or not believe it or not believe it or not believe

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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

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it or not believe it or not believe it or not believe it or not believe it or not

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believe it or not believe it or not believe it or not believe it or not believe it or not

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believe it or not believe it or not believe it or not believe it or not believe it

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or not believe it or not believe it Or not believe it or not believe it or not believe it

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Oh, that was a bad wolf.

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That went wrong.

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Sorry about that.

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We're just going to go through that one.

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That was an old slide that didn't need to go that fast, but that's okay.

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The stupor is definitely real.

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Brick soup is for lunch.

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No soup for you there, Mr. Mark.

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You had a really good show yesterday reading some kind of document that I actually never

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had any faith would actually exist, but you can still learn that biology thanks to

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Mark digging up some of these old descriptions of how early vaccine and let's let's call

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them what they were.

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They were, they were variolations.

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They were variolation preparation.

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But this is how they addressed smallpox back then and I think we are just up against

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one of the most demonic illusions that has ever been perpetrated on humankind.

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Very nicely done here in this graphic.

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Don't forget that they danced, don't forget that they kept people who had been married

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for 50 years apart in their last days.

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Don't forget that they, what they did to us, don't forget, you got to understand what

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this is about.

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This is a battle, you know, it's a battle between good and evil and yeah, we are trying

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to put forth some argument about biology, but even this cartoon might be largely a cartoon

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imposed upon us by them.

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Most of our understanding is dependent on their, on the very science that they have funded

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and put in front of us and this illusion is just, you know, it's something that we've

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really got to break through for our children.

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There are people who are learning biology.

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There are people who knew better and we've got to isolate those people who knew better

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and make sure that they, they don't rob us at the last little hope that is available.

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And that is in our history.

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It's our recent history and our more distant history and the people that are involved in

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it, what they did, what they said, what they wrote, what they thought.

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Those ideas are still the trap within which we find ourselves and the people that wove

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that trap and are involved in maintaining it are the people we need to identify.

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I don't think if you watch television or skillfully use social media, you're going

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to identify any of the people that matter.

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Even when paintings like this exist, you're still not quite seeing, you're seeing still

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what they want you to see.

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And only in a place where your tongues are completely free are we really free and in

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this day and age when a room like this could exist, where Twitter could be an illusion

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that is entirely created by rooms like this all around the world.

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We've got to keep swinging, ladies and gentlemen.

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We've got to keep swinging.

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Oh, that definitely was Steven Pinker.

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That was definitely Steven Pinker.

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Yes, sir.

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Rebob, it was.

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Yes, sir.

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Rebob, it was.

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Do you remember this song from way back when what's going on?

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Oh, sorry.

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Hold on.

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Let me pause that for a second.

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If you remember this song, I think you must remember this song.

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It was used in a while ago.

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It's a nice one.

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It's from the late 80s, I believe, in Australia.

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And it was for the Australian Broadcasting Company back in the 80s, I believe.

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And it's a really peppy news song.

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If you've been here for a while, you're here at the top of the wave.

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And if you're joining me for the first time at Gigo and Biological, then you might be

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a skilled TV watcher or what we call a skilled social media user.

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And you might not be staying focused on the biology.

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You might be taking their bait, and you might not love your neighbor.

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And we're going to suggest that those strategies need to be reversed.

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It could be back when ABC was probably more relevant.

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Thanks for spreading the word, ladies and gentlemen.

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Thanks to the supporters.

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Thanks to the people who are spreading these streams.

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This is Gigo and Biological.

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A high-resistance, low-noise information brief brought to you by a biologist.

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And now, since I haven't used that music in a while, this timing was a little off, and

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that thing paused, and who knows why that paused.

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I guess I had to tap that one again.

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I'm just sorry about that.

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There's some old slide combinations here for the beginning.

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I just thought I'd break out something new, old, new, old, new kind of thing.

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Welcome to the show, ladies and gentlemen.

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This is Gigo and Biological.

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The paradigm is shifting right now.

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There are a lot of moving parts.

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It's not something that can be easily understood, but definitely it is responding to our work.

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What we do is having a difference on how people try to push this forward.

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So let's move the ball forward by not participating in their illusion.

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That's really the strategy that I'm advocating for.

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This is, again, Gigo and Biological, a high-resistance, low-noise information brief brought to you

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by a biologist.

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I got my shirt on today.

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Welcome to the show.

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I look, oh, I know what it is.

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These little tiny front lights aren't on, so I look a little better.

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It might be a little better.

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Somehow I look a little, I don't know, greasy or something.

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Anyway, welcome to the show.

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This is Gigo and Biological coming to you live from the back of a garage in Pittsburgh,

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Pennsylvania.

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Thanks for joining me.

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Today, we have a little bit of work to do as far as the study hall goes.

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It's quite a long video, but I'm going to put it at 1.5 speed and we're going to take

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some notes and see what we get from Stanley Prusner's presentation from 2002.

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First, I would like to at least comment on the fact that Jessica Hockett, myself, and

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a friend of Jessica named John, scanning, hold on, that's the wrong male, why did that

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open up, see, I'm starting to get angry at a certain somebody, well, I don't know what

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his name is off the top of my head and I'm not trying to be a creep about it, that's

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the way it is.

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I apologize for that, I should have had that written down, it starts with a K and a very

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clever guy.

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The three of us presented, Jessica presented the longest in the beginning, about a half

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an hour, maybe a little bit longer than that and then John presented it and I presented

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very quickly about five or eight minutes each.

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I think the message was pretty clear.

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The message was a little bit of biology, but just a lot of what the hell happened in

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New York City to explain why we didn't investigate it yet, while there isn't a memorial yet,

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while the names haven't been released yet, why we haven't explained how this all happened

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in such a neat, uniform way and didn't happen in Chicago, even though they had a case a

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year or a month before that and so I felt like it was a decent thing, I thought it went

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pretty well.

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Unfortunately, we were briefing staffers and although I could only see two staffers on

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screen, those two staffers were, my best guess is millennials and one of them told us that

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she had a master's in public health and that any primary literature I could provide to

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back up any of the things that we said would be greatly appreciated and at that stage it

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really dawned on me one of the things that has driven me nuts since the beginning of

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the pandemic but I haven't really been able to express adequately in words but if anybody

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ever says to you to send some primary literature to them which could help them understand something

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unless it's in an extremely specific case of misrepresentation of data or a very specific

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case of a representation of a very specific situation, there is no combination of primary

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literature papers under the number of 5,000 that could be sent to somebody so that they

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could get an adequate idea of how distorted biomedical sciences has become as a result

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of the application of this p-value ritual over the last 20 or 30 years and with intention

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that is.

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It's not to say that science can't be productive and can't figure things out but what I'm suggesting

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to you is that the distortion, the intentional distortion of certain parts of the general

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scientific understanding and our education surrounding it has resulted in the place that

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we are, allowed us to be led to a place where we can no longer exercise informed consent

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because we simply don't know and trying to hit a home run in that scenario is basically

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where I went wrong, I started this, I'm going to show you the slides that I used and try

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to give the talk as I intended it but since I felt as though John abbreviated his presentation

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greatly in order to facilitate my presentation and I already felt like the staffers were

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looking at their watches and deciding whether they could stay any longer or not or whether

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they needed to listen a couple times, the kind of the head staffer or the one that was speaking

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the most into the microphone said that she's trying to get at our main idea when Jessica

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was talking and it's funny because Jessica started her presentation with the main idea

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which is that we want you to figure out what the hell happened in New York City because what

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happened there was not a natural event, it was like a bomb went off, it was a man-made disaster

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and we want you to investigate it and then she explained all the stuff and so it's weird the

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staffer was still kind of circling back to that stuff so by the time I got the mic John had

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already presented and John had some really nice slides that were actually tacked right on to

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the slides that Jessica had so they could flawlessly change to that and he had one or two slides

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in particular that I want to get him on my stream to talk about which are really slides

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that I've asked actually I believe I've asked Denny Rankor a couple times if he could make

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them which would be to let me see if that's the guy yeah that would be the guy if he could

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make a slide where you did the American data but you took the New York City event out and he had

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that slide and it's actually very striking how intuitively it felt like wow they're really

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using that curve to imply something else because of 24,000 people died in that week and he spread

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them out around America there would still be kind of a signal especially if you started that signal

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from zero right and you know in America 24,000 people have died of COVID so far but the vast majority

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are in New York City asterisk so anyway I started off already with a very good background of course

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so 45 minutes 40 minutes of Jessica's presentation eight minutes of John's statistical you know look

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if you take New York out it's crazy if you look at New York by itself it's so statistically off

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off I mean we're not talking about p-values here you know we're talking about z-score and

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we are we were way way way way and these nobody making these statistics ever thought that

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excuse me ever thought that something like this would be you know a z-score of 200 or something

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like that so my presentation couldn't really go too deep into the biology so I started with the

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idea that informed consent couldn't be exercised by anyone at the start of the pandemic because

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we were misled about the biology and about the whole experience of what was happening

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and that New York City as Jessica and John have pointed out was really central

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to establishing that there was even a pandemic happening and therefore

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I started with again reminding people of this and so we were consciously manipulated into believing

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that a crisis was occurring that it was ongoing that something was spreading in the

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background this kind of thing and this manipulation was on purpose to get us to accept that this

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crisis was occurring but the only numbers we have are the numbers that Jessica just showed you

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are almost assuredly fraudulent that John also backed up with statistics that seem to indicate

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that it's almost assuredly a man made event and I would argue that that man made event

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allowed them to imply the existence of a novel virus that that you and and Senator Ron's staff

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are very much in belief existed and the reason why you're in belief of it I would argue is because

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they fooled you into thinking that the only question was whether it was a bad cave virus or

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whether it was again a function lab league and this mystery that we've been solving over the last

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four years has bamboozled all of us into accepting that what we were told in New York City was the

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start of something very very terrible was actually a mass casualty event that was misconstrued as this

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and the beginning of this the beginning of this whole mystery and I would argue that one of the

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ways that they did it and I went from this to this was that they didn't they didn't genuinely

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describe what all cause mortality was they didn't actually tell you that you know the excess deaths

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where we're experiencing can come from all kinds of things especially if we told doctors with fear

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and uncertainty and doubt that there was a new thing that you had to be afraid of yourself

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and that if this new thing was there you should treat these people entirely different than you

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would have ever treated them had you not been told that this new thing was there

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and all of those new things that we were told to do started killing people and nobody acted because

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there was this illusion of consensus that it was either a novel virus or a lab leak that we were

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responding to and that these people were dying from and the tools that they use to identify it

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the financial incentives that they they use to amplify it all of these things are completely

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ignored by the people that were just in front of you in that senate committee almost none of this

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part of the narrative where hundreds of thousands of Americans were murdered at the beginning of the

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pandemic in 2020 and 2021 that's wholly ignored and completely attributed only to the novel virus

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and all of these people's narrative and this is very dangerous because of course as Jessica just

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showed you New York City is this bump right there that bump right there is New York City and I was

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actually already over here down here and so the mind this bump right here is New York City

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and this could just be people acting really really badly this little rise here there's a

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lot there's a rise there right you can see that right there's not a dip that dip is missing this

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little pattern just completely gets disrupted and now all cause mortality is permanently elevated

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by a combination of confusion and stupidity bad ideas and transfection and the initial casualty

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event is something that Jessica showed you is not a natural phenomenon does not belie the spread of

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a virus but instead suggests a staged event now the reason why I think it's important to understand

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this is because these people who were in front of you in the senate not even a month ago and this is

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exactly what I said in front of your boss excuse me who are also at an event in Romania in November

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of 2023 where they heard Denny Rancor the guy in the square speak about these effects who he

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has actually been speaking since 2020 about the fact that the only deaths that are excess are

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correlative with poverty level household income serious mental illness obesity and the excess

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mortality is a direct correlation with all these things across states irrespective of whether they're

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a blue state or a red state and so he suggests that there's no evidence of spread of a particular

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novel pathogen there's evidence of bad ideas in the form of protocols and how they treat or don't

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treat people and until the vaccines are released the all cause mortality doesn't go up significantly

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with any pattern that would indicate a respiratory virus but all of those people when they were in

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front of you last month none of them said that they all spoke very very generously about the 17

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million people that Denny Rancor said might be or estimated might be having been killed by the

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transfections but they conveniently left out that his data also shows that there's no evidence of

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real novel spreading pathogen at risk additives spreading pathogen in 2020 and 2021 and this is

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very crucial because those same people want us to interpret that what happened in the last

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four years well there was an RNA that caused a pandemic but transfection worked pretty well

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although we rushed it and so they want justice for the RNA that was released and should never have

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been made and they want justice for the people that were injured because we rushed transfection

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before it was ready and this illusion is this this this solution is a lie the reality is is

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that transfection in healthy humans was criminally negligent all along and that every biologist with

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an academic bench in any accredited university in America should have known that transfection is a

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long-standing technology even using a product like lipofectamine is a long-standing technology

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that's been used on the academic bench for acute expression of proteins in order to manipulate

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systems and understand and test hypotheses about different metabolic pathways in any

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any biological system however transfection is never shown proven

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successful in the treatment of anything that's not a deadly disease like a very specialized

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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

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betterment of mankind for the protection of our grandkids from this illusion they're all culpable

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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

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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

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called infectious clones and that's why with regard to ignoring all of these things these

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same people that were in front of you will never really enunciate a hypothesis which has anything

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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

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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

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ready to invert to change pneumonia and influenza what would have normally been classified as

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pneumonia and influenza to a new and novel respiratory pathogen that was a was a national security

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priority because this plan was in existence because not any kind of special technology or

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decree would be necessary for this to occur and because the vast majority if not the entirety of

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the deaths that occurred in 2020 can be explained by the confusion and fear and and

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incorrect behavior that resulted from the financial incentives from the testing and from the the

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messaging on on television there is no way that an RNA molecule can sustain a pandemic however there

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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

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that would support the idea of a circulating novel pathogen and would support the idea of

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rolling out this national security operation en masse would support the idea of co-opting

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anybody that ran to New York City and tried to save the day and found out that it wasn't what

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they said it was on TV and it would justify the use of several different groups of optoratives

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behind the scenes to make sure that this narrative never really got off the rails that nobody ever

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questioned a novel virus so that even when we got four years into the pandemic and people were in

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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

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or not the transfection worked or didn't work and how many people were hurt by it

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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

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and their emergency what happens in australia is their emergency and even the people that were in

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front of ron johnson in the senate are very very particular in how they express the emergency it's

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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

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our borders within our legislative system that's all we've got once we start trying to organize

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with australians or organize with canadians or or or then we're actually doing their work

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for them we're we're globalizing in a different way

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we need to decentralize that means that we don't need the help of people in other countries they

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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

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any questions and there weren't any questions of course but that was already an hour into the

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meeting and i think the out the meeting was supposed to be a half an hour and so they were ready to

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leave as soon as i quit so some things went really well other things weren't ideal i don't think my

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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

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i'll just i'll just comment on it now at one point in time when jeff was talking

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uh he was saying that um that there is evidence from previous winters and flu seasons that you know

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we've had nastier flu seasons in the past that we've not really made any fuss about and so what if

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these are actually coronaviruses in the background this background signal is always there and now

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they just misconstrued it as something else um of course i'm parsing his words incorrectly but

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she said um oh that's really interesting because of course there's lots of evidence or some people

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are saying that it was circulating in the background before 2020 and uh we didn't have any PCR testing

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and so that's what explains all this and they kind of went on but didn't go on because i think jeff

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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

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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

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more dead bodies than in any other weeks that have ever come before after that and it's a very

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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

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of a particular dangerous pathogen those deaths but it was spreading before that and we didn't have

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tests and 20 000 people for a week weren't dying because we weren't locked down we weren't aware

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we were all just going to restaurants and drinking and and nobody was wearing masks and

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and so at some point you know like there was this oh yeah but it's a variant and all it's

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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

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and so if if it was circulating before then but we didn't know how to track it and we were not

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doing anything to close down the borders then how in the hell didn't more bombs go off in other

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cities before this and she got very defensive and was like I don't think that's what I said I

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don't I don't I don't remember exactly what I said but I don't want to repeat it because I'm

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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

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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

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any combination of substances with the intent of augmenting the immune system is dumb

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transfection in healthy humans is criminally negligent in RNA cannot pandemic we're also

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beyond our own hypothesis here we're also trying um yeah I forgot I wanted to say I'm

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wearing Jimmy Dorr's shirt and I saw Jimmy Dorr the other day and I think although he doesn't have

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everything right he still thinks there was a novel virus it's probably gained a function and they

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lied about it I do think that his courage comes from a place without expertise right so all he

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knows for sure is that he was injured by the vaccine and people didn't take him seriously and that

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pissed him off and he realized that people were lying to him and now he's people he's realizing

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that a lot of people are liars and a lot of people can hold contradictory positions in their head

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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

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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

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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

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more crowds spelled yachob's disease more more protein misfolding disease is simply because we

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are starting to roll out mRNA transfection in old people all around the world and especially in

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the United States and in Europe and that rollout as it continues with the flu and RSV and the

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pneumonia and whatever else they decide to roll out on people maybe even the shingles shot

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you're going to see more and more of these what have already been well-seeded in in the mainstream

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narrative as as ramifications of a gain of function virus and potentially a designer spike protein

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but actually it's from transfection and they knew this already for a long time they knew

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as they moved forward that the vaccine schedule as it stood was probably going to eventually fall

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apart that it wasn't going to stand this the the scrutiny of of the citizenry forever as they

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ramped it up that's part of the reason why I still find it so amazing that the average young parent

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isn't even isn't even curious about how many shots and when they give them in other countries but

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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

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them and deploy them in such early lifetime is absolutely absurd and also contradictory to what

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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

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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

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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

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uh probably more importantly for me Edward uh Menowitter in in Norway um for almost five years

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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

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in my life um Norway was a great place to live um and I met a lot of great people there um I

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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

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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

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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.

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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.

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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

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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.

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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

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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.

