You
But you can tell if someone's lying, you know, you can sort of feel it in people
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
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
I'm not sure exactly who he is. His name is JJ Cooey, and I believe he's a consultant for CHD
And I believe he has a PhD in some sort of scientific discipline from what I understand
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.
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. I don't want to be a liar. I don't want to be a liar. I don't want to be a liar.
My friend Denver mixed date.
I wanted to mix it up a little bit, maybe get some other slides out.
We haven't used in a while, I know they're a little more reading.
It's a little more reading.
It's like we got ourselves some readers in the house.
Jimmy Doar made a reference to the reader joke, very, very good Bill Hicks joint, very nice
bit.
Jimmy, Jimmy Doar showed a lot of courage the other night, I'm giving him another shout-out.
It is amazing how many people think they can answer an argument by attributing bad motives
to those who disagree with them using this kind of reasoning you can believe or not believe
anything about anything without having to bother to deal with facts or logic.
It's amazing how many people think they can believe or not believe it or not believe it
or not believe it or not believe it or not believe it or not believe it or not believe
it or not believe it or not believe it or not believe it or not believe it or not believe
it or not believe it or not believe it or not believe it or not believe it or not
believe it or not believe it or not believe it or not believe it or not believe it or not
believe it or not believe it or not believe it or not believe it or not believe it
or not believe it or not believe it Or not believe it or not believe it or not believe it
Oh, that was a bad wolf.
That went wrong.
Sorry about that.
We're just going to go through that one.
That was an old slide that didn't need to go that fast, but that's okay.
The stupor is definitely real.
Brick soup is for lunch.
No soup for you there, Mr. Mark.
You had a really good show yesterday reading some kind of document that I actually never
had any faith would actually exist, but you can still learn that biology thanks to
Mark digging up some of these old descriptions of how early vaccine and let's let's call
them what they were.
They were, they were variolations.
They were variolation preparation.
But this is how they addressed smallpox back then and I think we are just up against
one of the most demonic illusions that has ever been perpetrated on humankind.
Very nicely done here in this graphic.
Don't forget that they danced, don't forget that they kept people who had been married
for 50 years apart in their last days.
Don't forget that they, what they did to us, don't forget, you got to understand what
this is about.
This is a battle, you know, it's a battle between good and evil and yeah, we are trying
to put forth some argument about biology, but even this cartoon might be largely a cartoon
imposed upon us by them.
Most of our understanding is dependent on their, on the very science that they have funded
and put in front of us and this illusion is just, you know, it's something that we've
really got to break through for our children.
There are people who are learning biology.
There are people who knew better and we've got to isolate those people who knew better
and make sure that they, they don't rob us at the last little hope that is available.
And that is in our history.
It's our recent history and our more distant history and the people that are involved in
it, what they did, what they said, what they wrote, what they thought.
Those ideas are still the trap within which we find ourselves and the people that wove
that trap and are involved in maintaining it are the people we need to identify.
I don't think if you watch television or skillfully use social media, you're going
to identify any of the people that matter.
Even when paintings like this exist, you're still not quite seeing, you're seeing still
what they want you to see.
And only in a place where your tongues are completely free are we really free and in
this day and age when a room like this could exist, where Twitter could be an illusion
that is entirely created by rooms like this all around the world.
We've got to keep swinging, ladies and gentlemen.
We've got to keep swinging.
Oh, that definitely was Steven Pinker.
That was definitely Steven Pinker.
Yes, sir.
Rebob, it was.
Yes, sir.
Rebob, it was.
Do you remember this song from way back when what's going on?
Oh, sorry.
Hold on.
Let me pause that for a second.
If you remember this song, I think you must remember this song.
It was used in a while ago.
It's a nice one.
It's from the late 80s, I believe, in Australia.
And it was for the Australian Broadcasting Company back in the 80s, I believe.
And it's a really peppy news song.
If you've been here for a while, you're here at the top of the wave.
And if you're joining me for the first time at Gigo and Biological, then you might be
a skilled TV watcher or what we call a skilled social media user.
And you might not be staying focused on the biology.
You might be taking their bait, and you might not love your neighbor.
And we're going to suggest that those strategies need to be reversed.
It could be back when ABC was probably more relevant.
Thanks for spreading the word, ladies and gentlemen.
Thanks to the supporters.
Thanks to the people who are spreading these streams.
This is Gigo and Biological.
A high-resistance, low-noise information brief brought to you by a biologist.
And now, since I haven't used that music in a while, this timing was a little off, and
that thing paused, and who knows why that paused.
I guess I had to tap that one again.
I'm just sorry about that.
There's some old slide combinations here for the beginning.
I just thought I'd break out something new, old, new, old, new kind of thing.
Welcome to the show, ladies and gentlemen.
This is Gigo and Biological.
The paradigm is shifting right now.
There are a lot of moving parts.
It's not something that can be easily understood, but definitely it is responding to our work.
What we do is having a difference on how people try to push this forward.
So let's move the ball forward by not participating in their illusion.
That's really the strategy that I'm advocating for.
This is, again, Gigo and Biological, a high-resistance, low-noise information brief brought to you
by a biologist.
I got my shirt on today.
Welcome to the show.
I look, oh, I know what it is.
These little tiny front lights aren't on, so I look a little better.
It might be a little better.
Somehow I look a little, I don't know, greasy or something.
Anyway, welcome to the show.
This is Gigo and Biological coming to you live from the back of a garage in Pittsburgh,
Pennsylvania.
Thanks for joining me.
Today, we have a little bit of work to do as far as the study hall goes.
It's quite a long video, but I'm going to put it at 1.5 speed and we're going to take
some notes and see what we get from Stanley Prusner's presentation from 2002.
First, I would like to at least comment on the fact that Jessica Hockett, myself, and
a friend of Jessica named John, scanning, hold on, that's the wrong male, why did that
open up, see, I'm starting to get angry at a certain somebody, well, I don't know what
his name is off the top of my head and I'm not trying to be a creep about it, that's
the way it is.
I apologize for that, I should have had that written down, it starts with a K and a very
clever guy.
The three of us presented, Jessica presented the longest in the beginning, about a half
an hour, maybe a little bit longer than that and then John presented it and I presented
very quickly about five or eight minutes each.
I think the message was pretty clear.
The message was a little bit of biology, but just a lot of what the hell happened in
New York City to explain why we didn't investigate it yet, while there isn't a memorial yet,
while the names haven't been released yet, why we haven't explained how this all happened
in such a neat, uniform way and didn't happen in Chicago, even though they had a case a
year or a month before that and so I felt like it was a decent thing, I thought it went
pretty well.
Unfortunately, we were briefing staffers and although I could only see two staffers on
screen, those two staffers were, my best guess is millennials and one of them told us that
she had a master's in public health and that any primary literature I could provide to
back up any of the things that we said would be greatly appreciated and at that stage it
really dawned on me one of the things that has driven me nuts since the beginning of
the pandemic but I haven't really been able to express adequately in words but if anybody
ever says to you to send some primary literature to them which could help them understand something
unless it's in an extremely specific case of misrepresentation of data or a very specific
case of a representation of a very specific situation, there is no combination of primary
literature papers under the number of 5,000 that could be sent to somebody so that they
could get an adequate idea of how distorted biomedical sciences has become as a result
of the application of this p-value ritual over the last 20 or 30 years and with intention
that is.
It's not to say that science can't be productive and can't figure things out but what I'm suggesting
to you is that the distortion, the intentional distortion of certain parts of the general
scientific understanding and our education surrounding it has resulted in the place that
we are, allowed us to be led to a place where we can no longer exercise informed consent
because we simply don't know and trying to hit a home run in that scenario is basically
where I went wrong, I started this, I'm going to show you the slides that I used and try
to give the talk as I intended it but since I felt as though John abbreviated his presentation
greatly in order to facilitate my presentation and I already felt like the staffers were
looking at their watches and deciding whether they could stay any longer or not or whether
they needed to listen a couple times, the kind of the head staffer or the one that was speaking
the most into the microphone said that she's trying to get at our main idea when Jessica
was talking and it's funny because Jessica started her presentation with the main idea
which is that we want you to figure out what the hell happened in New York City because what
happened there was not a natural event, it was like a bomb went off, it was a man-made disaster
and we want you to investigate it and then she explained all the stuff and so it's weird the
staffer was still kind of circling back to that stuff so by the time I got the mic John had
already presented and John had some really nice slides that were actually tacked right on to
the slides that Jessica had so they could flawlessly change to that and he had one or two slides
in particular that I want to get him on my stream to talk about which are really slides
that I've asked actually I believe I've asked Denny Rankor a couple times if he could make
them which would be to let me see if that's the guy yeah that would be the guy if he could
make a slide where you did the American data but you took the New York City event out and he had
that slide and it's actually very striking how intuitively it felt like wow they're really
using that curve to imply something else because of 24,000 people died in that week and he spread
them out around America there would still be kind of a signal especially if you started that signal
from zero right and you know in America 24,000 people have died of COVID so far but the vast majority
are in New York City asterisk so anyway I started off already with a very good background of course
so 45 minutes 40 minutes of Jessica's presentation eight minutes of John's statistical you know look
if you take New York out it's crazy if you look at New York by itself it's so statistically off
off I mean we're not talking about p-values here you know we're talking about z-score and
we are we were way way way way and these nobody making these statistics ever thought that
excuse me ever thought that something like this would be you know a z-score of 200 or something
like that so my presentation couldn't really go too deep into the biology so I started with the
idea that informed consent couldn't be exercised by anyone at the start of the pandemic because
we were misled about the biology and about the whole experience of what was happening
and that New York City as Jessica and John have pointed out was really central
to establishing that there was even a pandemic happening and therefore
I started with again reminding people of this and so we were consciously manipulated into believing
that a crisis was occurring that it was ongoing that something was spreading in the
background this kind of thing and this manipulation was on purpose to get us to accept that this
crisis was occurring but the only numbers we have are the numbers that Jessica just showed you
are almost assuredly fraudulent that John also backed up with statistics that seem to indicate
that it's almost assuredly a man made event and I would argue that that man made event
allowed them to imply the existence of a novel virus that that you and and Senator Ron's staff
are very much in belief existed and the reason why you're in belief of it I would argue is because
they fooled you into thinking that the only question was whether it was a bad cave virus or
whether it was again a function lab league and this mystery that we've been solving over the last
four years has bamboozled all of us into accepting that what we were told in New York City was the
start of something very very terrible was actually a mass casualty event that was misconstrued as this
and the beginning of this the beginning of this whole mystery and I would argue that one of the
ways that they did it and I went from this to this was that they didn't they didn't genuinely
describe what all cause mortality was they didn't actually tell you that you know the excess deaths
where we're experiencing can come from all kinds of things especially if we told doctors with fear
and uncertainty and doubt that there was a new thing that you had to be afraid of yourself
and that if this new thing was there you should treat these people entirely different than you
would have ever treated them had you not been told that this new thing was there
and all of those new things that we were told to do started killing people and nobody acted because
there was this illusion of consensus that it was either a novel virus or a lab leak that we were
responding to and that these people were dying from and the tools that they use to identify it
the financial incentives that they they use to amplify it all of these things are completely
ignored by the people that were just in front of you in that senate committee almost none of this
part of the narrative where hundreds of thousands of Americans were murdered at the beginning of the
pandemic in 2020 and 2021 that's wholly ignored and completely attributed only to the novel virus
and all of these people's narrative and this is very dangerous because of course as Jessica just
showed you New York City is this bump right there that bump right there is New York City and I was
actually already over here down here and so the mind this bump right here is New York City
and this could just be people acting really really badly this little rise here there's a
lot there's a rise there right you can see that right there's not a dip that dip is missing this
little pattern just completely gets disrupted and now all cause mortality is permanently elevated
by a combination of confusion and stupidity bad ideas and transfection and the initial casualty
event is something that Jessica showed you is not a natural phenomenon does not belie the spread of
a virus but instead suggests a staged event now the reason why I think it's important to understand
this is because these people who were in front of you in the senate not even a month ago and this is
exactly what I said in front of your boss excuse me who are also at an event in Romania in November
of 2023 where they heard Denny Rancor the guy in the square speak about these effects who he
has actually been speaking since 2020 about the fact that the only deaths that are excess are
correlative with poverty level household income serious mental illness obesity and the excess
mortality is a direct correlation with all these things across states irrespective of whether they're
a blue state or a red state and so he suggests that there's no evidence of spread of a particular
novel pathogen there's evidence of bad ideas in the form of protocols and how they treat or don't
treat people and until the vaccines are released the all cause mortality doesn't go up significantly
with any pattern that would indicate a respiratory virus but all of those people when they were in
front of you last month none of them said that they all spoke very very generously about the 17
million people that Denny Rancor said might be or estimated might be having been killed by the
transfections but they conveniently left out that his data also shows that there's no evidence of
real novel spreading pathogen at risk additives spreading pathogen in 2020 and 2021 and this is
very crucial because those same people want us to interpret that what happened in the last
four years well there was an RNA that caused a pandemic but transfection worked pretty well
although we rushed it and so they want justice for the RNA that was released and should never have
been made and they want justice for the people that were injured because we rushed transfection
before it was ready and this illusion is this this this solution is a lie the reality is is
that transfection in healthy humans was criminally negligent all along and that every biologist with
an academic bench in any accredited university in America should have known that transfection is a
long-standing technology even using a product like lipofectamine is a long-standing technology
that's been used on the academic bench for acute expression of proteins in order to manipulate
systems and understand and test hypotheses about different metabolic pathways in any
any biological system however transfection is never shown proven
successful in the treatment of anything that's not a deadly disease like a very specialized
cancer and the fact that they either didn't speak up because they didn't know or didn't speak up
because they were unwilling they did not have the principles to dictate that they should for the
betterment of mankind for the protection of our grandkids from this illusion they're all culpable
they're criminally negligent I lost my job at the University of Pittsburgh School of Medicine
because I spoke out about that specific thing and since then in the four years that I've been trying
to understand what is and what isn't well understood RNA virology I've come to the conclusion that RNA
cannot pandemic and they've always known it in fact it's a it's a central sort of
it's a foundational part of the understanding we have or don't have about RNA viruses that they are
almost intractable in culture because of the nature of how RNA viruses and especially RNA viruses like
coronaviruses are are purported to replicate and those shortcomings are overcome by a technology
called infectious clones and that's why with regard to ignoring all of these things these
same people that were in front of you will never really enunciate a hypothesis which has anything
to do with that that core technology of RNA virology which is infectious clones using DNA to make the
RNA that is infectious and so our hypothesis my hypothesis is that the who declared a particular
a dangerous pandemic of a particular sign and that's not obviously I said it better than that
the the who declared a pandemic of a dangerous novel virus and it was a background signal that
they that they misconstrued as spread they used an event in New York City to to create the illusion
that that an impending crisis was coming and at the same time they already had a plan where they were
ready to invert to change pneumonia and influenza what would have normally been classified as
pneumonia and influenza to a new and novel respiratory pathogen that was a was a national security
priority because this plan was in existence because not any kind of special technology or
decree would be necessary for this to occur and because the vast majority if not the entirety of
the deaths that occurred in 2020 can be explained by the confusion and fear and and
incorrect behavior that resulted from the financial incentives from the testing and from the the
messaging on on television there is no way that an RNA molecule can sustain a pandemic however there
is a way for that sequence to be found in many places around the world and that again is an
infectious clone and that could have been used if that was part of the national security
operative to make operation to make absolutely sure that there was a seamless body of evidence
that would support the idea of a circulating novel pathogen and would support the idea of
rolling out this national security operation en masse would support the idea of co-opting
anybody that ran to New York City and tried to save the day and found out that it wasn't what
they said it was on TV and it would justify the use of several different groups of optoratives
behind the scenes to make sure that this narrative never really got off the rails that nobody ever
questioned a novel virus so that even when we got four years into the pandemic and people were in
front of the senate and speaking about it that the novel virus would never be questioned that
how many people died from it would never be questioned that we would already have moved on to whether
or not the transfection worked or didn't work and how many people were hurt by it
and this theater is actually an orchestrated attack on the united states from the from within
orchestrated by people outside of the united states in cooperation with people inside of the
u.s. government and that's what i think we need to understand most is that this is not an this
is a national security issue for americans and what's happening in canada is separate from us
and their emergency what happens in australia is their emergency and even the people that were in
front of ron johnson in the senate are very very particular in how they express the emergency it's
all of us it's everyone it's the who the globe has threatened no
the only handles of power that we have the only way that we can act is through our government within
our borders within our legislative system that's all we've got once we start trying to organize
with australians or organize with canadians or or or then we're actually doing their work
for them we're we're globalizing in a different way
we need to decentralize that means that we don't need the help of people in other countries they
have their own country to save and i think that's part of the reason why you should be very suspicious
of having a senate hearing where half of the people are from different countries
where half of the people have been on stage with the other half of the people in other countries
multiple times why were they invited them none of this stuff was said to the staff i stopped a
long time ago already but these were the things that i used i had this slide in there but i didn't
really read it because i didn't i just went back after i what after this slide popped up i said i
don't think i should use that slide should leave it here let you read this and then i'll stop for
any questions and there weren't any questions of course but that was already an hour into the
meeting and i think the out the meeting was supposed to be a half an hour and so they were ready to
leave as soon as i quit so some things went really well other things weren't ideal i don't think my
presentation was ideal i was a bit riled up i was going too fast i had already become frustrated
with one of the guys one of the people on the staff because of an earlier comment
i'll just i'll just comment on it now at one point in time when jeff was talking
uh he was saying that um that there is evidence from previous winters and flu seasons that you know
we've had nastier flu seasons in the past that we've not really made any fuss about and so what if
these are actually coronaviruses in the background this background signal is always there and now
they just misconstrued it as something else um of course i'm parsing his words incorrectly but
she said um oh that's really interesting because of course there's lots of evidence or some people
are saying that it was circulating in the background before 2020 and uh we didn't have any PCR testing
and so that's what explains all this and they kind of went on but didn't go on because i think jeff
was just trying to hurry up and he kind of acknowledged it um tried to sort of say
something but it was the way that she trailed off her her comment that made it kind of just kind
of stumbled through and ended up the presentation moving on and then i unmuted really quick and i
was like whoa whoa whoa whoa i just want to go back one step because what um she just said we really
need to point out a little better how ridiculously contradictory that statement would be how holding
those two thoughts in your head shouldn't be possible if you listen to Jessica's presentation
and you heard that New York City is this you know highly isolated event with you know orders of magnitude
more dead bodies than in any other weeks that have ever come before after that and it's a very
strange anomaly that occurs with at-home deaths and also nursing home deaths and all these things
are at the same time um and so it's very strange um to believe that that represents a the spread
of a particular dangerous pathogen those deaths but it was spreading before that and we didn't have
tests and 20 000 people for a week weren't dying because we weren't locked down we weren't aware
we were all just going to restaurants and drinking and and nobody was wearing masks and
and so at some point you know like there was this oh yeah but it's a variant and all it's
this and what about the lockdowns and you know so there was a lot of discussion but the end result
always with Jessica's data is that but you can't explain this with that hand waving because it
essentially death starts out very normal at 4 000 people per week and then goes
or 4 000 people per April and then it goes and there's no biological explanation for that
there would be EMTs that are traumatized from the sheer number of bodies they had to handle
there would be nurses that are traumatized from the sheer number of people
there would be memorials because it's like eight nine eleven events
and so if if it was circulating before then but we didn't know how to track it and we were not
doing anything to close down the borders then how in the hell didn't more bombs go off in other
cities before this and she got very defensive and was like I don't think that's what I said I
don't I don't I don't remember exactly what I said but I don't want to repeat it because I'm
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
deadly novel pathogen was circulating but it didn't kill anybody before we started testing for it
or we didn't kill anybody before Tony Fauci made the 15 days to stop the spread announcement
so that's where we were um you know that we're still right here right intramuscular injection of
any combination of substances with the intent of augmenting the immune system is dumb
transfection in healthy humans is criminally negligent in RNA cannot pandemic we're also
beyond our own hypothesis here we're also trying um yeah I forgot I wanted to say I'm
wearing Jimmy Dorr's shirt and I saw Jimmy Dorr the other day and I think although he doesn't have
everything right he still thinks there was a novel virus it's probably gained a function and they
lied about it I do think that his courage comes from a place without expertise right so all he
knows for sure is that he was injured by the vaccine and people didn't take him seriously and that
pissed him off and he realized that people were lying to him and now he's people he's realizing
that a lot of people are liars and a lot of people can hold contradictory positions in their head
and so he's as frustrated as I am that at some point in time it seemed like Robert F Kennedy
Jr. was a really legit possibility as a normal everyday well-read smart guy with a lot of
you know forward-looking ideas and tell you to hear him go on about climate change or Israel
and so you know we're all we're all a bit frustrated with the world right now
and so I think it's always good to in those times to to take a chance just kind of listen
and take notes and and learn or maybe not learn from people who like it or not are having a very
big impact on today going forward I have argued that we are probably should be expecting to see
more crowds spelled yachob's disease more more protein misfolding disease is simply because we
are starting to roll out mRNA transfection in old people all around the world and especially in
the United States and in Europe and that rollout as it continues with the flu and RSV and the
pneumonia and whatever else they decide to roll out on people maybe even the shingles shot
you're going to see more and more of these what have already been well-seeded in in the mainstream
narrative as as ramifications of a gain of function virus and potentially a designer spike protein
but actually it's from transfection and they knew this already for a long time they knew
as they moved forward that the vaccine schedule as it stood was probably going to eventually fall
apart that it wasn't going to stand this the the scrutiny of of the citizenry forever as they
ramped it up that's part of the reason why I still find it so amazing that the average young parent
isn't even isn't even curious about how many shots and when they give them in other countries but
if you do that math if you do that research you're going to find out that the American
vaccine schedule is just an exclusively early and dense and so even if some of these purported
intramuscular augmentations of the immune system were somewhat effective the way that they use
them and deploy them in such early lifetime is absolutely absurd and also contradictory to what
we understand about everything to do with neuronal development and and immunological development
in fact it's funny because you can get a you can see a nice talk by Michael Warby from before
the pandemic when he's talking about vaccinating people for flu and given the fact that they have
lifelong immunity to the flu that they're exposed to as a kid we probably have to start vaccinating
people before birth if we really wanted to get ahead of this but of course that's actually the
plan right that's why they want you to think that vaccinating pregnant women's no big deal
yes you shouldn't eat sushi or uh unpasteurized cheese but vaccination is fine intramuscular
injection of any combination of substances is fine for a for a pregnant woman
let's look at Stanley Prusiner and Prions because again I think that this narrative has been
seeded was given a Nobel Prize for goodness sakes I mean let's just talk intellectually a
little bit about a Nobel Prize that I was very very very very very very very very very very
cursorily connected to the Nobel Prize in 2014 was awarded for place cells and grid cells and
spatial coding in in the brain of rodents to John O'Keefe and Edward and Maybert Moser
I was lucky enough to have worked with and studied with and trained with Edward and Maybert Moser and
uh probably more importantly for me Edward uh Menowitter in in Norway um for almost five years
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
in my life um Norway was a great place to live um and I met a lot of great people there um I
actually got I actually got an email from the universe sorry from the Society for Neuroscience
inviting me to register for the meeting and uh at the bottom was the you know the SFN
meeting committee chair signature is Laura Colgan somebody who I worked with and met in the Moser
lab um in the first couple years I was there a really nice um smart girl I shouldn't say girl
smart woman um who's now I still believe working in New Orleans but I'm I apologize Laura for not
knowing where you are now but being the chairperson for uh for the chairman for the Society for Neuroscience
meeting a meeting of over 35 000 um neuroscientists every year um it's a pretty big step um I guess
that's on the way to being president of Society for Neuroscience um she was uh always gonna be
you know head of her department at some point so I'm really happy I was really happy to get that
letter and just um so anyway what was I saying Norway is a place where Edward and Maybers
Maybers Moser uh work and they in troll time and they actually won the Nobel Prize in 2014
um it was a year so after I had left I moved to Norway the Netherlands in Rotterdam to try and
get tenure there and try to find a permanent job there we had intended to raise our kids um
in in Holland but that didn't work out um and so in 2016 we moved to Pittsburgh um but in 2014
they won that Nobel Prize and so you would have thought that at least that should help a little
bit with my grant applications and with my work but I was doing something in Rotterdam that was so
methodologically close to impossible that I was so obsessed with um that I basically dropped the
ball on getting funding and you know getting any big publications myself and so I found myself
where I was at that time because of you know choices that I had made in mountains that I decided
to climb so I'm not blaming anybody for those years um they were very taxing on our what on
our marriage because I was so obsessed with with making things work at work um the reason why I'm
talking about this is because um the year that they won the Nobel Prize they also won the Nobel Prize
with John O'Keefe who whose first discovery of play cells was actually in the early 70s you might
say 1971 or 1972 is when this happened and then his students actually um two students from Norway
who came to his lab to learn the physical technique of making the electrodes that they recorded these
play cells in the hippocampus from as the animal freely navigated in a small box or maze and uh
they were they were keen on learning those and doing experiments with him and then they recorded
from uh different uh upstream brain region anatomically um giving a lot of input to the
hippocampus as suggested by meno litter and there is where they found their discovery the grid cells
the point is is that that Nobel Prize was awarded to a total of three people essentially a teacher
and his students who had changed the field both several different methodological ways in the
sense of enabling the recording of these these neurons and and and show and developing the
anatomical techniques to follow where you recorded from and and use them for leisure all kinds of
things that essentially change the way that this kind of neuroscience was done these experiments
were done these these methodology spread all around the world and people were putting these
electrodes in all different heads and recording you know uh play cells and then trying to study
how play cells encoded memory and and and how they express themselves in different behavioral
paradigms though I mean they actually changed or even created a field that before them didn't
really exist the hippocampus was originally really exciting because it was this one place where
bliss and lomo were able to create this long-term potentiation that everybody thought was memory
if you could change the electrical signal from a weak signal to a strong signal that was sort of
like this is like the first sort of in vitro preparation of memory but the hippocampus itself
and its functional sort of a model of its functional understanding really comes from this
decades of work that started with the play cells in in in London in John O'Keefe's lab in the 70s
and a and a and a Nobel Prize in medicine wasn't awarded until 2014 Stanley Prouzner got a Nobel
Prize for for prions and the idea of prions before everybody in the field even agreed that prions
were a thing I believe seven years after he coined the term
it should as as Peter Thiel says it should make you suspicious because the potential for people to
lie and exaggerate has been greatly increased and so your default should be yeah your default
assumption should be that there is lie and exaggeration here so let's take a look at this and see what
we can get out of it shall we
you
people who have dementia are demented
and so I think it's okay to talk about demented people what I'm going to tell you about is a
very special journey that happened here at UCSF that begins in 1972 and really is continuing up
to the present so we'll start with this slide and I'm assured the lights will go down so I'm going
to tell you about a saga that represents the triumph of scientific investigation over prejudice
it's really a journey from heresy to orthodoxy it's about prions really a new principle of infection
and disease now the diseases we're going to talk about our crew of New Guinea Natives
Croitesville the Ocob disease called CJD. Curseman stories are shanker disease and fatal insomnia
then scraping of sheep mad cow disease or BSE of cattle and chronic wasting disease of mule deer
and elf now it was 1972 as I mentioned to you a moment ago that I became interested in these
diseases and I had a patient from Marin County a 60 year old woman who was dying of Croitesville
the Ocob disease I was a resident in neurology here and as I began to learn about the animal
disorders scrappy and about the foray people in New Guinea who suffered of crew I thought
this could be really a fascinating area to pursue and really the fascination came from the chemical
point of view because there were a few tidbits of chemistry that suggested that the particles
causing these diseases must be much different than anything that was known this is a slide
by my colleague Steve D. Arman showing you what the brain looks like of a patient who typically dies
of Croitesville the Ocob disease it's a rare disease about one person in a million or one
every 10,000 deaths this is a much less common form with these huge vacuoles that you see
you have to excuse my voice uh that's this is a virus causing it not prions
now for many years scientists thought that scrappy
sorry I got brought brought lunch so I'm eating in the background so first of all let's go back
a little bit here to see that um he doesn't really tell you what this is is his brain or what
um I presume it's brain but it's interesting how quickly you went through it not to tell
you what to look for what to see here there's no not normal form I mean it's again are we
are we here to objectively inform people or are you implanting an idea that you they must accept
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
but I think you'll get used to it really quick yes this is a much less common form with these
huge vacuoles that you see you have to excuse my voice uh that's this is a virus causing it not
prions now for many years scientists thought that scrappy was caused by a virus because the
disease is transmissible the agent is small there's a rise in scrappy agent tighter that
precedes disease so that's the concentration or amount of scrappy agent and there are different
strains of scrappy agent that produce different patterns of disease so there was a lot known
when we got into this and what I thought in 1972 was that we really needed to do was to develop
a method to isolate the infectious particles so the infectious particles in this cartoon are
represented by these little red squares and everything else is junk and we needed to separate
these so we could under this is gonna be hard to do while I'm uh I'm gonna try and eat lunch
here and get done so this list of assumptions was gone through way too fast for this to be so
scrappy agent is small and filterable that sounds like you know 1920s virology a rise in scrappy
agent tighter precedes the disease yeah I wonder what that's really based on I wonder what
does that mean that the more you inject in the brain of an animal the quicker it gets sick
you see these assumptions are already sketchy to me the strains of scrappy agent produce
different patterns of disease I'm willing to bet also is based on a very small number of observations
meant to be used to generalize to this extent and to go through this slide this quickly
um well anyway you'll see and we got into this now what I thought in 1972 was that we really
needed to do was to develop a method to isolate the infectious particles so the infectious
particles in this cartoon are represented by these little red squares and everything else is junk
and we needed to separate these so we could understand what they were made of well the problem
became that the particles did not behave very well so instead of having a very steep curve here
the curve was very uh very gradual I won't go into the details of some of the more difficult
pieces of science but this shows it a little more easily so the ideal behavior of a particle
whether it's a virus or a protein or a piece of the classic acid whatever you might want to isolate
it would when we fractionate it had a bit one piece of whatever you might want to get a bite
instead of having a very steep curve that you could understand what they were made of
and so now what we're looking here
log scraping infectivity and so this is a cartoon that's sort of
supposed to show you that somehow or another they've tried different ways to purify
the scraping agent and it doesn't behave the way an agent would be expected to behave
and that for them should be a problem it should indicate that maybe there isn't one
agent but instead they try to stick very hard in fact he will stick to the assumption till the
end of the talk that there is a single causative agent that they just have to develop better
techniques in order to study well the problem became that the particles did not behave very
well so instead of having a very steep curve here the curve was very uh very gradual I won't go
into the details of some of the more difficult pieces of science but this shows it a little more
easily so the ideal behavior of a particle whether it's a virus or a protein or a piece of the
plaque acid whatever you might want to isolate it would when we fractionate it have a bit one peak
it looks like that but instead what we found was that the scrapey agent was spread throughout
the entire uh gradient
so again that's a problem right because it's not one thing
now the argument I believe they're going to use for a little while at least is that the
aggregation of a repetitive protein might produce this kind of smear
however I think that that's you're going to very quickly see that in the end they're going to get
back to it being a single thing and uh so just keep this in mind he's just telling us a story
right now and so we're just listening to his story what we found was that the scrapey agent
was spread throughout the entire uh gradient
now the problem was compounded by the fact that when I started we needed to use 60 mice
and we needed one year to carry out what was called an endpoint titration
and all the red ones are positives and all the white ones are negative so what we would use
are 60 mice as I said uh for a single sample so at each tenfold illusion we would have six mice
and this assay took a year now we were able in the late 1970s to develop a new assay and this was
based on the work of Richard Martian Wisconsin and Richard Kimberlin who was working with Richard
Marsh but he was from England and we turned to the hamsters we followed their lead and we were
able to reduce the number of animals from 60 down to 4 and the time from about 130 days for high
titered samples to 70 days and we did this by constructing a standardized curve and then just
simply reading the dose off of the curve and since we were interested in making more and more
enriched preparations this worked to our advantage because we didn't have to wait to get this endpoint
where we had the negatives and the positives after a whole year we could just simply read off the
titer or the concentration from the standard curve this was a huge step it allowed us to do
more experiments in a period of three years and have been done in the entire field over previous
century now as I said a few moments ago isolating the scraping agent was a nightmare because it
behaved I will admit that I don't really get that I'm gonna play that one more time and see if I
can catch it better a more rapid and economical assay for the scraping agent it seems like to me
what he's saying is if you want to find the scraping agent you have to inoculate 60 animals
and then some of the animals will develop it and then from those animals you can isolate the scraping
agent and with hamsters it's faster or something I don't let's listen the number of animals from
but it was from new assay assay took a year now we were able in the late 1970s to develop a new
assay and this was based on the work of Richard Martian Wisconsin and Richard Kimberlin who was
working with Richard Marsh but who was from England and we turned to the hamsters we followed their
lead and we were able to reduce the number of animals from 60 down to four and the time from
about 130 days for high-tired samples to 70 days and we did this by constructing a standardized
curve and then just simply reading the dose off of the curve and since we were interested in making
more and more enriched preparations this worked to our advantage because we didn't have to wait
to get this endpoint where we had the negatives and the positives after a whole year we could
simply read off the piter or the concentration from the standard curve this was a huge step it
allowed us to do more experiments in a period of three years and have been done in the entire field
over a previous century now as I said to you a moment ago isolating the scraping agent was a
nightmare because it behaved very oddly and also because the method of measurement when we entered
the field was extremely difficult one year and 60 animals so having now moved away from mice
into hamsters able to accelerate our studies by nearly 100 fold so what we could do in one year
would have taken us a century to do and there are few investigators that live a century much
less have a productive lifetime of the century so now what we could do is develop what's called
a purification scheme so we could use detergents and enzymes and back to the centrifuge and these
gradients and now we could follow up the lead that was present in the literature from a woman
named Tig Valper who was a radiobiologist working in England and she had argued that because the
scraping agent was so resistant to UV and ionizing radiation that it did not contain RNA or DNA
nucleic acid and what we found was that all the procedures we use that alter nucleic acids did
not diminish the infectivity and the procedures that modified proteins led to an activation of
the scraping agent but we could only see this after we removed more than 99 percent of the junk
the unwanted molecules and having done that and that's me by the way with brown hair at the time
I thought that by 1982 actually the spring of 81 that we had enough information to say that
this particle was not a virus or a tiny nucleic acid called a viroid discovered by Ted Deener in
the late 1960s and that we needed to give it a new name and so I had the audacity to call these
particles prions that's the long arm of the scientific community pushing on me and people
say well what do those people look like and here they are at the table that's my best slide you can
go home now and it became very very clear that prions eventually became very clear
our infectious proteins and I'm going to take you through a lot more evidence that builds the case
that prions our infectious proteins and then so basically I guess he won the Nobel prize for that
hamster work where the incubation time bioassay right is where they take scraping agent
maybe it's prepared no better than what Marx describes on his stream for the other day
is then injected directly into the brains of mice and it used to take a year
but eventually they were able to accelerate that in hamsters I don't know if they altered the
scraping or if they had to change the PRP protein in a certain way to get that to happen we will
look at that in the coming journal clubs but in this summary and I apologize for eating lunch
I'm so hungry the problem with this is though is that I think that we went so quickly to assuming
that this is the real deal and that more importantly that this recapitulates it
right that's the problem here is the the animal model of a gain of function virus is one where
we go into a bat and we swab it we PCR test it we amplify all of the RNA that we see there
whatever genes are missing we replace by something that we find in a catalog and we rebuild
the coronavirus that was in that bat based on the fact that well if you assume the spike
protein is a new spike protein then I guess that's a new virus we can build it we can't grow it
we can't grow it and make a lot of it we can't culture it and make a lot of it
but we can use computer algorithms and and nonspecific PCR primers to amplify signals in the
background that may have already been there that may have nothing to do with the health of the
animal that may be a a bacteriophage signal for all we know because we do nothing to differentiate
between them this is very similar to that if we're going to have an animal model
that has nothing to do with it if we start with humanized mice and infectious clones
that are purported to be the same as the stuff they found in the bat and now we have this model
of coronavirus disease where if we squirt this RNA or or the the cell culture products that are
produced after we transfect a cell culture with this RNA if we put that material into a mouse
that we call that a model of ARDS or SARS then then then we have a whole industry that can evolve
from that that has nothing to do with whatever the original natural phenomenon was
and so it's it's at least worth extreme inspection to find out whether this animal model that's just
been sort of just presented to us as accepted and and proven and well understood
is actually an adequate bridge model to come from what happens in scrapie or what happens in
crowds filled yachab or what happens in kuru and notice that this entire lecture and all the
lectures about protein misfolding have absolutely never ever mentioned the immune system responding
to misfolded proteins do you see how significant it is that if you study protein misfolding you
don't worry about the immune system at all the immune system is not involved this is a completely
different thing that has to do with aggregation and cell death and you know whatever
so now he's going to purify the protein and use using sucrose gradient which he showed earlier
didn't work and so now we're at the development of the prion concept procedures altering nucleic
acids don't diminish the infectivity so chemicals that are that are thought or assumed to get rid
of all nucleic act nucleic acid still the infectivity of these preparations is apparently still preserved
when you do what when you inject them into the brain of an animal or when you use some other
probably not very adequate experimental correlate for what you what you would think happens in
these natural forms i'm not suggesting that the natural form of kuru doesn't exist
crowds felt yachab as a result of using an adenavirus encoding a viral spike protein fusion protein
could potentially have caused crowds felt crowds felt yachab disease in 28 people like
luke montanier's paper said i'm suggesting to you that genetic technologies like transformation
and transfection we're already known a long time ago to interfere with and maybe even induce
maladaptive states in healthy animals and so what this means is is that we need to be very careful
about how many of these very recently seeded biological phenomenon biological ideas were
actually seeded by people who have intention of confusing us and about this sounds very paranoid
but this is the same year that sars came on the world 2002 when he's given this talk is the year
that sars is going to be released or the year after i guess it's 2003 really when it happened
so you can almost imagine in fact a scenario over the last couple decades where this
this potential bio hazards from mother nature these mythologies were already these were
mythologies were seeded and while we're being told that these phenomenon are natural we we
have no way of knowing we have no way of knowing we do have pretty good reason to believe that
they've been messing around with all sorts of biotechnologies for the last 30 years in attempt to
figure stuff out and so there's no question that one of the things that could have resulted from
are these kinds of diseases there's also no question in my mind that that kuru and eating
your dead relatives might be a bad idea that would involve your immune system going a little
going a little haywire can imagine that i can
but i do think that an infectious protein is much more interesting from a bioweapon perspective
than it is from a biological perspective not diminishing the infectivity and the procedures
that modified proteins led to an activation of the scrapey agent but we could only see this
after we removed more than 99 percent of the junk the unwanted molecules and having done that
and that's me by the way with brown hair at the time i thought that by 1982 actually the spring
of 81 that we had enough information to say that this particle was not a virus or a tiny nucleic
acid called a viroid discovered by Ted Deener in the late 1960s and that we needed to give it a
new name and so i had the audacity to call these particles prions that's the long arm of the
scientific community pushing on me and people say well what do those people look like and here
they are at the table that's my best slide you can go home now and it became very very clear
that eventually became very clear our infectious proteins and i'm going to take you through a
lot more evidence that builds the case that prions are infectious proteins and then as we move through
this we'll get to some very new data and we'll talk about bse and we'll talk about other prions
diseases of humans and then at the end we'll talk about therapies and there'll be a lot of chemistry
at the end but it won't be complicated because i put the chemistry in the beginning you'll all
leave so here we have there is there is something to be said about too many jokes
have this purification scheme where we remove 99 percent of the unwanted particles we then knew
that a protein was important and we went on to identify and find this protein and then the next
thing we did was to try to determine whether we could separate this protein from the infectivity
and so we used a lot of approaches so we're now measuring the biological activity enhancers
and we're looking at the protein by physical methods and we tried to separate it and what we
found is that every time we destroyed the protein we destroyed the infectivity or every time we
destroyed the infectivity the protein was removed or destroyed and when we eventually figured out
what was going on we found that there was a normal form of the protein in all of us now this is a
gel and the proteins migrate and the smaller they are the faster they migrate through this gel this
porous material sort of like jello and then we can stain them with antibodies and so what we found
was that the protein was a protein in all of us which is a normal form of the prion protein
and when we treat with enzymes that destroy proteins the protein was completely destroyed
but in the brains of these hamsters that became ill with scrapi what we found was that there was
both a normal form of the protein and another form of the protein that when we treated with
enzymes that destroy proteins there was a residual piece and here they're higher-ordered multiverse
they're larger forms that are aggregated of the scrapi causing protein and eventually
the protein that I just showed you in the gel after we treated with proteases what we did was to use
a lot of very complicated equipment in collaboration with a man named Leroy Hood who was at Caltech at
the time and Darlene Groff who's been working with me for 25 years went to Caltech over a period
of a year about seven different times and we finally were able to work out what's called the
amino acid sequence the N terminal 15 amino acids so right in this region now this was more
complicated because it was ragged because we'd thrown in these proteases but what we had found
of course is that if we threw the proteases into the normal preparations there was nothing well
now we had the possibility of doing what's called reverse genetic engineering and we went backwards
and with Charles Weisman and Zurich we found the gene that codes for the prion protein and we found
that all of us have this gene and it became very clear that all of us have the normal form of the
prion protein and then by a process that we still don't understand in great detail it could be
converted into a protein which is infectious and which can eventually kill us as human beings or
kill animals like the cattle in Europe so now we knew that there was a gene for the protein of the
prion and the protein of the prion we called PRP scrapey and it's and that this gene encodes
the precursor protein PRPC there's a mistake here it's not found it's only found in six mammals
but the gene is found in all mammals so now we had two proteins the precursor protein PRPC that
we all have a normal protein and the protein found in okay so just notice the wording that
he's using here a precursor protein we have to assume that it does something normally
and that's the normal protein and that the the prion scrapey protein has to be the abnormal
protein so it's really weird that he doesn't say it in the way that his disease model implies it
should be said um as if the protein only exists to cause the disease in its precursor form it's
fine in its functional form it causes disease that's really not the way we should be thinking of this
especially if
we have to work from the assumption that the that the that the prion protein does something
otherwise what is it there for on the membrane a protein that's on the memory that doesn't do
anything sounds like a very bad idea to me had two proteins the precursor protein PRPC that we
all have a normal protein and the protein found in the animals that were ill with prion disease
we did another set of experiments and we asked what about the messenger RNA so the messenger
RNA is the intermediate between the gene and the protein and we said well the messenger RNA ought
to rise as the as these animals go out toward the these are now mice that get sick at 130 days
as incubation time gets longer and longer and so the n-terminal amino acid sequence of of
prion protein 27 to 30 is determined isocoding DNAs used to retrieve PRPC DNA clones
so my guess is is that they used
DNAs that would encode for the amino acid sequence of the n-terminal then they use those DNA probes
and they hybridized against the DNA of a human or a mouse or something like my human I guess
and found a genomic DNA to which that n-terminal DNA hybridized to
expected the prion protein mRNA levels were similar in uninfected controls with animals with
scraping so when they look for the mRNA for the for the protein they find it and two forms are
identified cellular and pathogenic and is derived from pr by n-terminal truncation let's see how
he explains this hider the prions goes up the messenger RNA ought to rise but in fact the messenger
RNA state exactly the same it never changed and this worried us a little bit where we're looking
at the right protein but we got lucky because using that same technology genetic engineering
we now looked at black mice or brown mice and we looked at these white mice the white mice had
long incubation times about 225 days the short ones about 130 days and what we found was that this
gene the prion protein gene that i've been talking about determined whether the mice had a short
incubation time or a long incubation time and the mice with long incubation times had two amino
acids out of 250 that were different and so this began to cement the idea that this protein PRP
that we discovered was key in the disease process wow so that's a really key thing to realize because
what is this established this is we need to find this paper so i'm going to write this one down
because this is a really important one it's 86 through 87 and this is brown and white mice huh
the reason why this is important is because this whole thing would have been established
by a p-value test right if the p-value between the incubation time and the brown mice and the
incubation time and the white mice in the experiment was significant as determined by whatever probability
test they use that ritual would be enough to establish this as fact and then he would go on to
say that these two amino acid differences between those mice and those mice are the reason why the
incubation time is different and then that whole field moves on based on the idea that well i mean
obviously think about how that that could potentially set up years and years of being wrong
that's not definitive proof that this protein's involved
how can that be even if there is a p-value difference there how can that be assumed
is that the only difference between the brown mice and the white mice
wow isn't that special
isn't that special the lady from mit told us that all those heat shock proteins are really
responsible for protein folding and misfolding and that they're like chaperone proteins and
it's so cool because yeasts are eukaryotes and were eukaryotes and we have the same proteins
that chaperone are protein folding and prevent them from misfolding
and so it doesn't matter that the brown mice might have different heat shock protein variants
than the white mice do it doesn't matter at all i think the lady from mit who's dead now would
probably disagree but i don't know what's going on here that's how p-values are used to create
the distortion of information and knowledge from noise
it's a single experiment
where the difference between incubation times to injection of material in their brain
has nothing to do with looking at what was injected or where it was injected or how much
exactly was injected it has to do with those two amino acids in that one protein that has to
be i mean look i mean it has to be look at the other experiments we did there was like a gel
and stuff
out of 250 that were different and so this began to cement the idea that this protein
PRP that we discovered was key in the disease process
so what i've told you is that not only did the pre-on protein track with scraping
infectivity so every time we tried to alter the protein we altered the infectivity and vice versa
but also amino acid substitutions changed the incubation times so these findings link the phenotype
which is called the disease characteristics in this case to the genotype that changes
their mutations in the DNA okay so to a certain extent this is interesting because what it now
implies of course is that function is related to structure which is one of the real overarching
principles of reductionist biology one of the things that we all agree on as we move through
the world trying to understand what's going on if you want to understand how a tree works
it's probably related to its structure it's its structure in the macro sense and also structure
at the cellular level and the molecular level but structure begets function is something that holds
at the protein level from everyone's perspective the the amino acid sequence determines its structure
the the RNA sequence that encoded the amino acid is to a large extent synonymous
but we know that synonymous mutations that don't change the amino acid for which they code
can still be highly correlated with genetic disease
and so clearly the cartoon about yes we have DNA we translated it to RNA the RNA translates
to amino acids and the amino acids arrange in a certain way because there are three-dimensional
electrostatic shape inside of a three-dimensional electrostatic solvent called water and so they
form a particular shape to try and best optimize their energy distribution in a stable way
but that's a variable process a process that isn't understood a process that
occurred to the dead lady from MIT actually goes wrong an awful lot and we waste a lot of energy
misfolding proteins all the time and that prion protein now is a very special protein that has
the propensity apparently as we move forward we're going to hear how it can can cause other
proteins with a similar sequence to misfold as well so prion protein misfolds other prion protein
even though we don't know what prion protein really does in the in the healthy animal it's
there it's mRNA is there all the time and in in some animals like hamsters it develops faster
than other animals like mice and in some brown mice it develops longer faster than in white mice
now we come to a whole different approach and we spent six years here at UCSF
trying to identify the difference between PRPC the normal form of the protein that all of us have
and the scrapey form of the protein that's found only in the disease and we looked and looked so
far the only evidence that they have of a different protein is that gel keep that in mind right do
we need to go back to that a little bit if I just go back to the gel
this is the gel right this this is the gel that he explained is the evidence that he has for
prion protein being different this is the normal protein this is the lane with the normal protein
after they digested it with proteases this is the protein apparently from the the scrapey infected
animal i don't know what all this other crap is that didn't show up in this line which is also
very frustrating how am i supposed to compare this experiment if this lane is supposed to be
equivalent to that lane except this lane has four more bars in it than this one does
what kind of shit experiment is this and how is he able to just get through the all
gel it moves uh the smaller fragments move faster than the the heavy fragments and uh so uh yeah
so what are these fragments well anyway his argument is is that this is the protease
digested side and here they still have a protein whereas here it's all gone and so in that in the
in the normal functioning prion protein format it can be completely digested by protease and here
it can't that is the only evidence that they have for an alternate form of a protein that's it
because the sequence is the same
as best they can tell it's just one gene
these are not as related complementary experiments as he's implying and without careful
and maybe even several months of hard studious work you can't claim to be an expert on prions
because nobody explains it well enough to be this is not a good enough explanation of prions
for me to say oh yeah i guess i believe it this is hand waving
this is how they explain to you how pandemics work and why masks work and why lockdowns if done
correctly work and why zero virus after the end of a pandemic is a useful goal
it's that kind of it's that kind of explanation on protein track with scraping
infectivity so every time we try to alter the protein we alter the infectivity and vice versa
but also amino acid substitutions change the incubation times so these findings link the phenotype
which is called the disease characteristics in this case to the genotype the changes are
mutations in the DNA thank you solar flare i'm clicking a whole different approach and we spent
six years here at UCSF trying to identify the difference i'll take it between prpc the normal
form of the protein that all of us have and the scraping form of the protein that's found only
the disease and we looked and looked for a chemical difference because if we could have found a
chemical difference then it would be very easy to make that chemical change on prpc
and convert it into prpc but we never found it this was a work of a young postdoc Neil Stahl
who was working with Mike Baldwin who's still here and who's a professor in the school of pharmacy
and mass spectrometry so then Fred Cohn became involved and we began to think about models
and the first thing that Fred did he said i wonder if this isn't really the case that
the normal form of the prion protein is full of these helical structures called alpha helices
and that some of these are reduced and that there's a lot of beta sheet these beta strands
these blue ribbons and at that point what we did was we knew that there was a lot of beta structure
from other studies from our won't work and from that of other people we went it off and purified
out of brains of animals the normal form of the prion protein and we found that Fred in fact was
correct that there was very little beta structure and then more recently Tom James and his colleagues
here with the help of peter right now i thought it was really hard to purify the prion proteins
from animals with the disease and now he just says we're going to go do it and we found out
that there's more beta sheets i don't know pain dyson scripts uh from protein that was made by
genetic engineering and bacteria and determined its structure so here we're sort of halfway
through the protein around residue 90 and then you see this long unstructured region then one the
first alpha helix then more of this unstructured region with a small amount of beta structure
than the second helix and the third helix and what's called the c-terminus now what we
hypothesized was that the first helix and in fact we thought the first helix was made up of two
this whole region was made up of two helices and we were wrong and Kurt Rudrich and Zurich showed us
that we were wrong much to our dislike and unhappiness and so this was the model that we had for a number
of years so what i've just told you is that when prpc is converted into prp scraping the protein
undergoes a profound change in shape or as scientists call it confirmation prpc is rich in alpha helix
whereas prp scraping is rich in beta structure now if you believe alpha fold and you believe
people who study proteins alpha helix is something that you can predict from the sequence
and a beta pleated sheet is something you can predict from the sequence and it's not very clear
to me that those two are are not mutually exclusive i i was i was taught that those are kind of
mutually exclusive in the sense of we don't really understand how proteins fold but we do
understand that they have two general motifs that of an alpha helix and that of a beta pleated sheet
and these two general motifs of course have very specific expression but these general motifs are
used to construct or or or or build out some of these protein machines
so the idea that one part of a protein could be both an alpha helix and a beta pleated sheet
is already a very novel idea in my humble opinion and that i believe is what he's implying here
and we went on to do some studies in collaboration with Dennis Burton at Scripps
and this is the work of David Parrots who's still here at UCSF and what we did was to make
antibodies that reacted to different regions of the prion protein so this is the three helix
protein now what's really incredible about this is that antibodies are reacting to a three-dimensional
static shape they don't necessarily react to only one and antibodies are very
non-specific so it's very very would be very interesting to go back
and see how these antibodies were raised they're raised to different epitopes as you can see here
they are raised to epitopes that are about 22 amino acids this is 23 right this is no that's 30
30 28 i don't know i can't do that math it's not very many
so they're small epitopes presumably then what injected into a rabbit or something and then
they take the plasma out and they get those antibodies and those antibodies although they
were raised to that epitope aren't necessarily all going to be the same antibodies with the same
specificity so unless they used a monoclonal antibody as they were made in the 90s that would be one
thing to look at here and what they're doing is trying to identify using antibody binding whether
or not particular structures are present or not present
so i i just wanted to be sure you knew what they were doing with these antibodies
let's see what he says hey this is helix a helix b and helix c that i showed you before
and this is residue 90 and in this first region the the prion protein binds antibodies and the
native form of the prion protein binds them quite well but the binding region or epitope disappears
when prp scrape is formed so that was an important discovery and we use that information and we
contrasted that with the green where but remember that when prion protein scrape is formed they can't
separate it from the native prion so again these pluses and minuses are binding strength and whenever
you have one key tip i would say that whenever you have a paper that wants to basically
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
one and that's a you know you should take it with a grain of salt here both the native form of PRPC
and the native form of PRPC mine antibodies that's down here
so these epitopes are regions so that's the second basic prince proof that the scrappy protein in
its in its infectious form is a different shape than this one but in this preparation we're not
sequencing a protein we're not starting with a pure protein in my i i don't think i don't know
what we're doing here we got to we got to find that out we got to know we hope we're doing a
pure protein here but is it a synthetically pure made protein is it a so that's again we have we
have a lot of work to do here to find out where p values are essentially holding up what is a what
is a what is a house that shouldn't be holding up we already have seen a couple examples of this
like the brown and white mice thing with two amino acids difference and this protein means that
the difference that we saw in the experiment had to do with those two amino acids that's the
kind of assumptions that we're always talking about here these numbers plus two plus three is that
really is he really saying that the native and the denatured pre-on scrappy protein are different
by a factor of one out of three that's their scale i think that's down here
so these epitopes are regions of the alpha helix rich precursor protein p rpc of
prions are exposed but become buried when the protein is refolded into an infectious form
p rpc scraping so why didn't they make um anybody see this is the problem i have with that
they have the the protein here native pre-on protein scrappy so why don't they make antibodies
to that why don't they make antibodies to the pre-on protein scrappy part and then show me
that those antibodies bind pre-on scrappy but they don't bind this one why don't they do the
reverse experiment apparently they have a pure form of this right otherwise you couldn't do this
this assay so they raised antibodies to these epitopes but they don't raise antibodies to the
epitopes of the pre-on scrappy because it's so hard to isolate or something i don't know
they can't get it to form badly in a in a dish or using the right ribosome or what
you see the problem here here
so these epitopes are regions of the alpha helix rich precursor protein p rpc of prions are
exposed but become buried when the protein is refolded into an infectious form p rpc scraping
now when we treat with proteases and we cleave off the n terminal region and we form the ragged
n terminus that i showed you that we sequenced and we call this prp 2730 this protein and this
is the one that i showed you before where we've treated with protease so it's a little bit shorter
that protein polymerizes into these rod shape structures that you see here
so after they treat it with an enzyme it forms rod shape structures there's nothing to do with
the physiology of it at all right this is an in vitro preparation with an in vitro result
and yet somehow or another we got here by him saying something about the refolding
now when we treat with proteases and we cleave off the n terminal region and we form the ragged
n terminus that i showed you that we sequenced and we call this prp 2730 this protein and this
is the one that i showed you before where we've treated with protease so it's a little bit shorter
that protein polymerizes into these rod shape structures that you see here and there was a very
famous man at Berkeley who died about 15 years ago roblie Williams was really the father of the
electron microscopy of viruses and he was working with us at the time because i really thought we
were going to find an interesting virus not a protein and we would see these rod shape structures
and he called them rods some people would call them fibers and one day i guess we'd been working
together for almost a year and a half i was looking at a book on the electron microscopy of proteins
and all this looking like amyloid proteins now amyloid is a mammalian protein these are usually
cathologically laid down in big fibers and there are many many diseases like alzheimer's disease
where there are amyloid plaques of many of these proteins but so the first connection here so far
seems to be that one microscopist called it a fibro and now they the people that study alzheimer's
disease have also called the proteins involved in alzheimer's disease fibers and so now that's
okay now they must be the same thing it turned out that this broke that what we were seeing was that
the protein of the prion was forming amyloid and that's what these rods represent and then it formed
plaques in the brain and i'll show you some of those plaques a little later now we also found
and this is the work of holder willy who's here at UCSF along with the help of david agar and
many others uh hana serven uh darlene groth that the protein forms these
rods that the plaques a little later amyloid and that's what these were this broke that
what we were seeing was that the protein of the prion was forming amyloid and that's what
these rods represent the protein of the prion was forming amyloid so what i hear here is that prion
protein is the sub component of amyloid is that what i'm hearing is that right
hmm that's interesting that's that i didn't think that they were the same i didn't think
they were the same protein i thought amyloid was a different protein um i didn't know that it
was prion protein that was folded differently without an n-terminus because that's what he just
said these are prion rods that are formed by the prion protein after you cleave its n-terminal
off with proteases and these form under the microscope and now he seems to be indicating
in 2002 that the amyloid plaques are actually prion protein did i not hear that did i not hear that
on the electron microscopy of proteins and all this look like amyloid proteins now amyloid
was is a mammalian protein these are usually depend logically uh laid down
in big fibrils and there are many many diseases like Alzheimer's disease where there are amyloid
many many diseases like Alzheimer's disease where amyloid plaques are laid down i don't
don't know many many many diseases where amyloid plaques all this look like amyloid proteins
now amyloid was is a mammalian protein these are usually Happyl logically uh laid down in
is like Alzheimer's disease, where there are amyloid plaques,
so many of these proteins.
But it turned out that this broke that what we were seeing
was that the protein of the prion was forming amyloid,
and that's what these rods represent.
And then it formed plaques in the brain,
and I'll show you some of those plaques a little later.
Now, we also found, and this is the work of Holger-Willy,
who's here at UCSF, along with the help of David Agard
and many others, Hana Sermon, Darlene Groth,
that the protein forms these rods,
and then at one end, we sometimes
see these two-dimensional crystals.
And we don't know whether the crystals form first,
and from that, the rods form, or the rods
begin to dissolve, and we form the crystals on the surface.
But we were able to use those images of the crystals
and then do what's called correlation averaging.
Now, keep in mind, this is what Robert Malone
purports to have simulatedly done at the beginning
of the pandemic for the three CL protease of the coronavirus.
He made a virtual X-ray crystallography model of that enzyme
and then used that three-dimensional X-ray crystallography
model to scan all known pharmaceuticals and nutraceuticals
in the FDA catalog in less than three weeks
to identify ivermectin, fluvoxamine, silicoxib,
and remdesivir has four potential repurposed drugs
to respond to the pandemic.
He spun up his volunteer team and developed an X-ray crystallography
model, computer-generated, similar to what
is being described here, but just a computer-generated version
of it, of the three CL protease and then
scanned all known drugs and pharmaceuticals
using the domain program, which was a DITRA program run
by his friend, David Hone, and came up
with four candidate drugs, of which at least one of them
was tested by Steve Kirsch, through his early treatment
fund, something something before he started his VRSF fund.
I mean, that's neither here nor there with regard
to what's happening here.
This is just to say that when you can crystallize a protein,
then you can take high-resolution pictures of it
and get some idea of the structure of the protein
because the crystal is a ever-repeating lattice of a pure protein.
So this is a ever-repeating lattice of the prion protein,
not the prionscrapi protein, which they still
can't produce separately from the prion protein, which
is interesting because the prion protein is not
an infectious protein.
And then we can average some more.
So these are all imaging techniques.
And we can develop these power spectra
and get very, very detailed images.
And so we went through a lot of this.
And eventually, we're able to show
that a piece of the prion protein in the middle of the protein
is found in the center in these black regions
and that the sugar chains are found around the edges
of the black regions.
And that gave rise to two different models.
What we call a trimer of dimers, so there
are six here, or simply a trimer, as shown here.
And this is the right-handed model.
This is the left-handed model.
And here, you simply see one of these now.
It's a much different-looking structure
for the model of prp-scrapi.
It has what are called beta helices.
So this beta structure is now in a helix.
This is an alpha helix.
This is a beta helix.
This is PRPC, as I talked about earlier, with helix A,
helix B, and helix C. Here, you see part of helix B
preserved, and all of helix C preserved.
And the rest of this now is changed into beta helix.
This is a current model.
This is what I was thinking about this.
It may or may not be right.
In the way we obtained these images
was using what we call it.
Now remember, he doesn't have the crystal of this.
So I don't really know what their molecular basis for assuming
there's beta.
That's a beta-pleated sheet.
He called it a beta helix.
I don't know.
I thought you called that a beta-pleated sheet.
I could be wrong.
There could be also a beta helix.
I mean, I'm not a protein guy.
This is the current model.
This is what I was thinking about this.
It may or may not be right.
In the way we obtained these images
was using what we call the old electron microscope, which
means it's no longer in service.
But I'd simply show you this.
And now what we're doing is working
with a new microscope, which was purchased
with the help of the Fairchild Foundation.
This is a $2 million instrument.
And you can maybe understand all this new detail that
wasn't there in the last slide.
So you can see that it's really essentially blank.
And now we begin to see all kinds of information.
So we're very optimistic that this will yield much more
structure in the near future.
So prions are composed of prion proteins
that are rich in beta structure.
And we don't know whether this is beta helix or beta sheet.
But all the evidence so far argues
for this new model of beta helix.
So there are beta helix and beta sheet, according to that,
which is good to know.
I still don't think he's provided a lot of evidence
that prion in an alternate form can cause other proteins
to form that way yet, right?
We haven't even been able to establish
that prion scrappy exists as a separate protein.
He's only shown as a crystal of prion protein
in its natural form.
Keep that in mind.
When the prion protein PRPC is synthesized,
it's made deep in the cell in what
is called the endoplasmic reticulum.
Here we go.
It travels through what is called the Golgi apparatus
out to the surface.
And on the surface in these what are called
rafts or cholesterol rich microdomains,
where there's a lot of cholesterol on the surface
in these little regions, the normal form of the prion
protein PRPC is converted into PRP scrappy.
And then it travels deep into the cell again
and all the way down into what are called bicosomes.
So does this look like anything that you've seen before,
another cartoon of some intracellular biological process
is just to be assumed to work exactly like it's drawn
because there's a guy with a laser pointer shining on it?
Because nothing that we've talked about so far
in this discussion has led us to believe
that this part of this thing is like real.
It's an assumption.
The cartoon is an assumption.
And they're not going to do very many experiments
to test any predictions that this thing might make.
They're going to test to see if they can find data
which fits this cartoon.
See, it's converted into PRP scrappy.
And then it travels deep into the cell again
and all the way down into what are called bicosomes.
And of course, this is a part of the biology
that they want you to ignore that there
are these lipid rafts that are in our membrane
which are cholesterol-rich regions
where most of the intracellular extracellular communication
and trafficking occurs.
And so it's one of the reasons why,
besides the fact that myelin in your brain
is made up of a lot of cholesterol,
it's one of the wonderful reasons why statins are a terrible
and that reducing cholesterol is just a terrible health motive
that could have only been put out there
by people who are idiots or people who are malevolent.
And this is really feeling like covering up
for people that are malevolent more than anything else.
This doesn't feel like a legitimate biological story
but it feels like mythology to me.
Where proteins are normally degraded
but PRP scrappy persists.
But it doesn't persist forever as I'll show you
a little bit later and it's changed the way
we think about this.
So the precursor of the prion
is an alpha helix-rich protein PRPC
that's synthesized in what's called the ER
and a plasma-criticulum of the cell.
The infectious form of the prion protein PRP scrappy
of the prion is rich in beta structure
and it is formed from PRPC on the surface of the cell
in these cholesterol-rich micro domains.
So this, all of this right here,
besides the cholesterol-rich micro domains,
all of this here is based on very, very little evidence,
sometimes only one paper, just keep that in mind.
Now recently we discovered that the prion protein
was around a long, long time ago
and that it has a cousin, actually a sister or a brother.
It was an ancient gene duplication
before men and mice diverged.
And with the help of Lee Hood and David Westaway
who was here at the time
and later Richard Moore who came from Edinburgh
and Patrick Tremblay who came from Montreal,
we were able to unravel this mystery.
So here's the prion protein gene
and 16,000 bases downstream,
we predicted there was a new gene
which would be for the doppel protein.
And when we began to look at this gene, it became...
And so what are they doing?
They're looking for gene homology, right?
And so this could be a duplication,
this could be any number of things
that happens in our genome.
But they're starting to make an argument
that some of these motifs may or may not be other proteins
in the genome with potential to be infectious.
And clear that there were many common features
and that helix A existed in both,
helix B existed in both,
and helix C existed in both.
But there were differences.
When we looked at individual amino acids one by one,
there were only 25% that shared sequence homology
that were identical.
But when we looked at the structure,
and this is with the work of Jane Dyson and Peter Wright
and they're post-cycled.
It's one existed in both and helix C existed in both.
But there were differences.
So helix C and helix B existed in both.
Listen to him say it.
And when we began to look at this gene
it became clear that there were many common features
and that helix A existed in both,
helix B existed in both,
and helix C existed in both.
Okay, they exist in both.
So both of these proteins apparently have the same sequence
because otherwise that alpha helix,
you wouldn't call it alpha helix A
if it's not the same sequence of amino acids
because it would be a different alpha helix, right?
But there were differences.
When we looked at individual amino acids one by one,
there were only 25% that shared sequence homology.
So if there are only 25 amino acids
that share sequence homology,
then how can you call it sequence the same sequence?
It's not alpha helix A.
It's not alpha helix B.
It's not alpha helix C.
Because we know that proteins use these motifs,
alpha helix, beta pleated sheet, beta helix,
as standard motifs.
They're not so different these 20 amino acids
that they don't fold similarly
depending on their main attribute,
which is hydrophobicity or hydrophilic.
They either like to be near the polar solvents of water
or they wanna fold away from it.
And so again, those amino acids
make a huge difference to the propensity
by which that alpha helix curls up tightly away from water
and bends to the left or the right.
These differences are huge.
And he says there's only 25% homology between these,
but I mean, we'll still call them alpha helix A, B and C.
Holy cow, this goes against the actual principle,
the very foundational understanding that we have
that the amino acid sequence has something directly to do
with the potential structure and function of the protein.
We're identical.
But when we looked at the structure,
and this is with the work of Jane Dyson and Peter Wright
and their postdoc, Wapping Mo, at Scripps,
comparing this to the structure done in Zurich
by Kurt Vootrick that I mentioned earlier.
So this is mouse doppel and mouse PRP.
So what you see is that over time,
the structures were preserved, here's helix A, helix A,
helix B, helix C, helix B, and helix C.
And yet, the sequence is diverged enormously.
75% of the residues are different,
but the overall structures are extremely similar.
They're not arguing that those overall structures
would suggest that their function is identical, but they are.
Don't you see they are?
Even though they know so little,
they are already making that argument.
We don't know what either of these proteins does.
We don't know the function of either protein.
Ha, ha, we don't know the function
of either of these proteins in 2002.
Now let's turn to the human preon diseases for a moment.
The sporadic form of these diseases
is called Croites Valley Aqua disease.
And this accounts for 85% of all preon disease.
The inherited forms account for 10% to 15%
and are called Gerson stories are shanker disease,
familial CJD, and fatal familial insomnia.
These are autosomal dominant diseases,
so half of the family members are afflicted
if they live long enough.
And the infectious forms of these diseases are very rare.
And they include kuru among new-getting natives.
It was transmitted by ritualistic cannibalism.
Iatrogenic Croites Valley Aqua disease caused by growth hormone
derived from human to doitaries.
And I'll talk much more about new variant CJD in Britain.
See, so they gave growth hormone to people
and then they got CJD.
So is that an immune reaction or is that a protein
that you can only have one of?
I don't know, I'm still skeptical
of these things all being one phenomenon
that is protein misfolding out of control.
Hormone derived from human to doitaries.
And I'll talk much more about new variant CJD in Britain.
Sporadic CJD is a disease of older people.
So the peak is around 70 years of age.
This is Larry Schoenberger's data from the CDC.
It's a 20-year experience in the United States.
So very few young people develop sporadic CJD.
Now, the genetic forms of this disease
have a fascinating history because the first reports
of Croites Valley Aqua disease were in 1921.
And 10 years later, the first pedigree
was brought of familial CJD.
And in 1973, Ray Rus was currently
chair in neurology at the University of Chicago,
working with Carlton Gajosecond Joe Gibbs
at the NIH, who did all the early work on Kuru.
And all the transmission work on Kuru and CJD into apes and monkeys.
They three of them wrote a paper.
And they reported the first familial cases
transmitted into non-human primates.
They offered three explanations.
First, that the CJD virus, as they thought it was in 1973.
They thought it was a virus.
Was transmitted among family members living in close proximity.
Second, there was a genetic predisposition
to a ubiquitous CJD virus that was everywhere, like the ether.
And third, like in AIDS, there was
vertical transmission of the CJD virus
from parent to offspring.
So it turned out that all three explanations were wrong.
And in 1989, Karen Chow, who had just
finished her neurology residency here
and came to work with me as a postdoctoral fellow,
identified first gene mutation in the PRPG
in a patient who had died of GSS at UCLA.
And from that, it became very clear
that this mutant form of PRPC refolds into PRP scraping.
It became readily apparent that it
misfolds into PRP scraping.
That's a very bold statement.
And we're going to have to find that paper.
It's a 1989 paper.
That this mutant form of PRPC refolds into PRP scraping.
Now, subsequent to this, this became an industry.
And there are more than 30 gene mutations
that have been identified, all of them above the line here,
that cause these inherited forms of the disease.
We've also identified what are called polymorphisms.
And those are shown below the line.
This one right here, and this one right here in sheep,
protect people from CJD or from scraping.
And we've been doing a lot of drug development
with Fred Cohn and one of his postdoctoral fellows,
Marty May.
So he's suggesting that some of these single point mutations
here can protect people from the misfolding disease, which
is a pretty remarkable statement that I
can't necessarily dispute.
And he's suggesting that these are all associated
with familial disease.
I'm still at least skeptical.
And we've been doing a lot of drug development
with Fred Cohn and one of his postdoctoral fellows, Marty May.
Now, let me turn to Prian's and Mad Cows
and the transmission to people, which
has really created a public health crisis that threatens
the food supply as well as the blood supply worldwide.
Just yesterday, Poland joined the food and blood supplies
an interesting statement to make.
It's definitely relevant for public health, then, isn't it?
Countries of the BSE club, the first case.
Now, we believe that BSE arose through industrial cannibalism
in the late 1970s.
And there's an argument whether it arose spontaneously
in a cow, perhaps a mutation, or it came from sheep
where scrapies endemic, but nevertheless,
it recycled through cows and then into humans.
Here's a typical newspaper clipping.
And it's not only a British problem.
This is also a problem throughout the con.
And while there are over 100 cases,
a new variant CJD in young people in Britain,
there are now four cases in France.
This is the BSE epidemic that begins in 1986
with the discovery of the disease by Gerald Wells.
It takes around 19 years.
So cannibalism would imply something
other than protein misfolding, right?
Because if you feed healthy cattle,
the remains of healthy cattle, that
doesn't mean that you're giving them an infectious disease
unless they already had it.
So for me, this implies a autoimmune reaction.
It implies autoimmunity caused by eating stuff
that's too closely related to you
and having that go through your payer's patches
and cause a four alarm fire.
That seems a lot more understandable
and more likely than the idea that,
again, we're dealing with something
where you get the wrong amino acids in the wrong order
and it's just one of those is all it's needed
and you're eventually gonna be dead.
That's an amazing mythology that I don't think
any of this past talk has done anything to substantiate.
92 with almost 40,000 cases
and then the number of cases keep declining.
But we keep finding more and more cases
because more and more sensitive assays are being used
or measurement techniques to detect the prions.
All of these animals were clinically ill
than at least 36 months of age.
This represents, and these little balls or circles
are represent one case, whereas each square
represents a thousand cases on the y-axis.
So what we're seeing is that there is this continuous
increase in the number of cases per year,
this is the yearly numbers of new variants, CJD,
except in 2001, the numbers down here.
What it'll be in 2002, we don't know.
So we see that this disease appears about a decade
after the beginning of BSE,
so we think the incubation time exceeds one decade
and in from crew studies, we know it can be up to four decades.
Wow, so we could be waiting right now
for it to happen to all of us is what is impre-
what is implication is.
That's pretty cool because if it happens
from a transfection that's 20 years in the future,
he will have already predicted it's gonna occur.
Boy, that's creating a pretty wide envelope
for naturally occurring prion disease
to just show up and mass across the population, isn't it?
Oh man, we shouldn't have been eating those cows 20 years ago.
Right, we shouldn't have been eating all that beef 20 years ago.
Wow, I can't believe it.
We better stop eating beef all together.
Maybe we should just eat fake beef
because I mean, prions take 40 years to incubate.
Obviously, the reason why everybody's dying of prions now
is because we've been eating meat for 40 years.
Just listen to this talk.
This is an MRI scan and it shows,
if we didn't have that light on over there,
you could see this much better,
this hyper-density is what it's called ate
and all the way down into the pulvinar,
which is this dorsal nucleus of the thalamus
and this is called a hockey stick sign.
And that's very typical of patients with new variant CJD.
Patients with sporadic CJD have all these hyper-intensities
that are seen bilaterally,
but they don't have the lighting up of the pulvinar.
I said I would show you some of these analog plaques
and in patients with new variant CJD
and this is the work of Steve D'Armond again
with slides sent to, I should say,
sessions sent to us by Bob Will and Jim Ironside
and Edinburgh.
So these are the plaques around by this halo
of sponge-formed generation.
They stain with antibodies to the prion protein very intensely.
So which antibody, how do you raise an antibody
to the prion protein if it's in that form?
You reuse the one that binds to the parts
that are still there.
You don't have to be specific about that at all right now.
Given the fact that just 20 minutes ago
you were using antibodies as an indicator
of whether it was prion protein in a natural form
or in the infectious form,
don't you think you could have used that same technique
to show what fraction of that protein was in
in a badly folded form
or in a dangerous folded form?
Or do you just think that he wasn't clever enough
to ask that question with the tools that he already had?
BUZZER.
This is why they hate me to be in the audience of these talks,
why they hated me in the audience of these talks.
Oh, very intensely.
I've already said this seven times.
Now, one wanted to try to figure out
where did all of this start?
Did it really start in sheep?
Did it start in cows?
And did it come from the cows to the humans?
And if we were looking for a virus,
we could do this very easily.
The virus would be simple to track
because the virus would have a nucleic acid,
a piece of DNA or RNA,
and we could easily track it into the sheep and see.
Viruses are easy to track according to Stanley Prusen
or easy, I mean, come on, that's easy work.
Easy work.
BUZZER.
And it had nothing to do with the sheep,
but that same piece of DNA or RNA
appeared in the cow and then appeared in the human.
We can't do that.
So what we had to do was really begin
to look at what are called prion strains.
And what's happening is that PRP scrape
is signified by a square.
It's stimulating the conversion of bovine PRPC.
This is from the sheep into PRP scrapey of the cow.
And the same thing, we can reiterate this into the human.
Now, there's a lot of data, but I'm
only going to show you very little.
And what we did was to use transgenic mice
to investigate the origin of BSE
and the transmission of prions to humans.
Oh, so we're going to use an animal model
to understand the cartoon that he says definitely happens,
that the sheep protein causes the refolding of the cow protein
that causes the refolding of the human protein.
There's three assumptions there on top
of three different cartoons.
And yet somehow or another, we're
going to move right on to a transgenic animal model.
Let's hear it.
Now, there's a lot of data, but I'm
only going to show you very little.
And what we did was to use transgenic mice
to investigate the origin of BSE and the transmission
of prions to humans.
There's now compelling evidence that indicates
that prions and beef products were ingested by humans, who
later developed variant CJD.
What we began with, and this is Jim Mastrioni's work,
Glenn Telling's work, was what we call a humanized mouse.
So now this mouse behaves toward prions like a human being.
And when we take sporadic CJD or familial CJD
and transmit into the mice brain extracts
from people who have died of these diseases,
all of the mice get sick in about 200 days.
But when we took new variant CJD from young people in England,
people in their early 20s or late teens,
we found that about 25%, this is wrong, it's not 60%,
but about 25% of the mice became ill by 500 days.
And when we took brain extracts from cows with BSE,
none of the mice developed disease by 600 days.
Now, Mike Scott did an experiment
where he created bovinized mice.
So these are mice that behave like cows
with respected prions.
And when he took mad cows, brain, 240 days,
and all the mice were sick.
With new variant CJD, what he found
was that 100% of the mice developed disease
at 260 days after inoculation.
So this was quite a shock.
And that when we took sheep's scrapie from the United States,
the mice were even more susceptible, 210 days, 100% were sick.
Now, we would have to look at what this mouse model is.
I don't know if they have overexpression of the bovine protein
in their brain or the human protein in their brain.
That's my guess.
My guess is that this time, I'm sure
I'm right.
Okay, I would bet my, I bet everything on it.
In 2002, and when this was done,
it would have been before 2002,
there would have been no way for them to knock out
the native gene and replace it with the bovine gene.
So what they're most likely working with here
is a mouse that also expresses the bovine
prion protein driven by some genetic technology.
And then when they inject that animal
in the brain, it gets sick.
And so we would have to, again, look into these papers
and figure it out.
But my guess is that this isn't gonna be as clean a result
as what he's suggesting it is here,
simply because the experimental model is not as clean
as he wants you to believe it is.
It's a bovine, it responds like a cow with respect to prion,
it responds like a human with respect to prion.
You can hear him say it again if you want to.
Into the mice, brain extracts from people
who've died of these diseases.
All of the mice get sick in about 200 days.
But when we took new variants, CJD from young people in England,
people in the early 20s, late teens,
we found that about 25%, this is wrong, it's not 60%,
but about 25% of the mice became ill by 500 days.
And when we took brain extracts from cows with BSE,
none of the mice developed disease by 600 days.
Now Mike Scott did an experiment
where he created bovineized mice.
So these are mice that behave like cows
with respect to prions.
See bovineized mice and then again,
they're injecting into the brain.
Again, how does that equivalent to eating your dead relative?
How does that equivalent to eating another cow?
It's just all not the experimental model that you want it to be
if you want to understand what he was showing
in the previous slides.
And when he took mad cows, brain, 240 days
and all the mice were sick.
With new variants, CJD, what he found was that 100%
of the mice developed disease at 260 days
after inoculation.
So this was quite a shock.
And that when we took sheep's scrapie from the United States,
the mice were even more susceptible, 210 days, 100% were sick.
And we looked at the neuropathology
against Stevie Arman's work.
What Steve found was that if we began with sheep's scrapie,
there was very little of the prion protein deposited
in what's called the corpus callosum,
the structure that connects both sides of your brain.
And some people with intractable epilepsy,
this structure is cut.
When we looked, if we began with BSE in these bovineized mice,
we saw large amounts of the prion protein
deposited in these big plaques.
And if we took new variant CJD,
the image was indistinguishable.
So when we put all this together,
the indistinguishable neuropathology,
the very similar incubation times,
it became very clear that there was little doubt
that the people had become sick from the cows.
And that's in addition to all the human epidemiology
showing the disease has confined largely to Great Britain
with four cases in France, as I said,
one in Italy and one in Ireland.
And now there's actually one in the United States,
but this is a woman who, 22 years old,
22 years old, living in Florida,
who lived in Great Britain for nearly 10 years.
And it's a British citizen.
So all of this put together,
Mike Scott began to think about this
in a slightly different way.
And he's begun to wonder if, in fact,
all sheep with screpy prions, the blue ones,
also have a few BSE prions that multiply more slowly.
And that what happened in the late 1970s
was that when they reduced the temperature
and also the process, they did less severe,
what they call the rendering process,
the screpy prions were still destroyed,
but the BSE prions survived.
And now they multiplied in cattle
where they became pathogenic for humans.
And there were no more screpy prions,
the blue ones, the whole down the amount
of the red ones that could be made,
because whether that's true or not, I don't know.
So this is a hypothesis.
But there's no way for that to happen
unless you're talking about prions moving separately
as animals breed over time and building up separately
in the population, even though they're not passed on
through birth.
That's incredible.
Or maybe they are passed, I mean, wow.
That's a pretty funny model.
It makes a lot of predictions that I bet
they won't bother testing.
We're trying to pursue this.
So variant CJD is caused by prions
in beef products from mad cows.
Over 85% of all human prion diseases
are sporadic 10 to 15% are inherited.
So I just want to point out that while there are about 5000
cases of CJD across the planet each year,
we're talking about 25 cases of variant CJD so far.
So just to give you a little perspective on the numbers.
Let's now turn to therapeutic approaches to prion diseases.
So there are a large number of approaches
that we've taken here at UCSF.
And I won't talk about rational drug design based
upon what is called dominant negative inhibition
or gene therapy.
And I'm also not going to talk about what
is called enhanced clearance.
But I come back to talk a little bit about clearance of prions.
I am going to talk about antibodies
that were used to inhibit prion replication.
And then I'm going to talk about a drug called quinacrine
that we're giving to patients in a study with Bruce Miller
and Michael Geshwin and many, many other people here at UCSF.
So this is David Parris' work using these antibodies produced
by Dennis Burton and Anthony Williamson at Scripps.
And again, the same blue antibodies react out here,
more toward the N-terminus, the green ones
at the C-terminus, and the red ones in the middle region.
That's Helix A, Helix B, Helix C.
All this looks a little complicated.
But let's just look over here at this graph.
So we're adding increasing amounts of antibody
to cultured cells.
And we're seeing the decrease in the amount of PRP
scrapey, the protein of the prion.
And the red antibodies work better than the blue antibodies
but only a little bit.
But both red and blue work much better than the green antibodies.
And if we look here, now instead of having
the concentration of antibody, it's the duration of antibodies.
Are you at all surprised that one
of the therapies he wanted to talk about
was using monoclonal antibodies to fight prion disease?
Are you at all surprised?
This is at a time when monoclonal antibodies
are right at the hottest biologic there is to make.
And the IP law is ripe for the writing.
Antibody treatment.
So we picked one concentration of antibody.
And now what we've done, and you see that the red ones
work quite rapidly compared to the blue ones.
And what this allows us to do is to look at when
we see a half maximal decrease, when we've gone down 50%.
And that's about at this point at about 30 hours right in here.
It was actually IMM, UNITY.
That's what I got, bodies anti.
He spelled immunity not by antibodies.
And in 1990, Bruce Cheesebrow and Byron Coe
working at the Rocky Mountain Laboratory
did a lot of work on the synthesis of PRPC
and defined what is called the Half-Life for formation.
Very rapid.
And David Borchelt working here in San Francisco confirmed
that and then showed that the Half-Life time for degradation
is about six hours.
And that PRPC was made even more slowly
with a half-time of formation of about three to 10.
OK, so here they're transfecting these two.
And now PRPC grapey actually must have a different sequence
because otherwise you're expressing the same protein.
It gets folded in two different ways.
So either you're trans, how can you
transfect neuroblastoma cells with this protein
unless it has a different sequence?
What's going on here?
What is going on here?
David Borchelt working here in San Francisco confirmed that
and then showed that the Half-Life time for degradation
is about six hours.
And that PRPC grapey was made even more slowly
with a half-time of formation of about three to 10 hours.
Now, I had thought that PRPC grapey was complete granite
and that it was never degraded.
But in the last slide that I showed you,
I don't get that at all.
This is really represents the degradation of PRPC
the loss of prions from the culture.
They couldn't separate the two
because they're the same weight.
They can't separate the two
because they exist together in their cartoons.
But then they shouldn't be able to exist together
because the one causes the other to become it.
And yet they have the same amino acid sequence.
So how would you express one or the other in a cell culture?
You'd have to use the same RNA.
I don't get it.
And so we were able to calculate a number,
as I mentioned before,
where 50% of the PRPC grapey disappears of about 30 hours.
This has important implications
for thinking about all the purposes.
I'm just an idiot then.
I got more reading to do.
That's all.
See, this is the reason why I haven't started teaching it yet
because I've got more read.
I don't understand that.
And that contradiction seems pretty big.
The whole talk has been explaining
about how difficult it is to sort this out
because it is a pre-...
It is a protein that has alternate forms
that alternate forms are hard to study.
And yet now we can express them
in different models when we want them.
Tells us that cells are capable
of degrading both PRPC and PRPC grapey.
And it raises the question whether PRPC grapey
is normally found at very low levels in normal cells
and has a physiological function.
And that is a very appealing way of thinking about all this
because it makes much more sense
than thinking that PRPC grapey is something totally apparent.
It raises the question of whether or not
we really have an issue.
It's a kinetic race between the formation of PRPC grapey
and the cell's ability to clear PRPC grapey.
And that when the cell can keep up with the formation
and clear it like it does with all other proteins in fact,
when that happens everything is functioning fine.
But when the cell gets out of balance,
it can no longer clear PRPC grapey at the rate that it's formed.
I'm talking about very, very low levels
that we can't detect even by these animal assays.
Then something goes awry and we begin to accumulate
more and more PRPC grapey and eventually the animal gets sick
and goes on to die or the human being.
Now, I promised you a little chemistry at the end.
And if you just look down here at chloropromacy
and this is thoracine, this is one of the first
antipsychotic drugs and this is the structure of it
and it has these three rings.
And when Karsten Korth added one micromolar, this amount,
he still saw these protease resistant bands.
So the three bands are the ones with no sugars,
one sugar chain and two sugar chains,
they're shed a little better here.
So two sugar chains, one and none.
I'm gonna go back.
Or even up in here.
It's a very appealing way of thinking about all this
because it makes much more sense than thinking
that PRPC grapey is something totally apparent.
It raises the question of whether or not
we really have an issue, it's a kinetic race
between the formation of PRPC grapey
and the cells of the clear PRPC grapey.
And that when the cell can keep up with the formation
and clear it like it does with all other proteins, in fact,
when that happens, everything is functioning fine.
But when the cell gets out of balance,
it can no longer clear PRPC grapey at the rate that it's formed.
And we're talking about very, very low levels
that we can't detect even by these animal assays.
So very, very low levels, kind of like asymptomatic.
You're asymptomatic for CAD, all your life.
And if you lose the race, then you become,
so it's actually not what a lot of these worst case scenario
people said, which is you just need one molecule.
You have one molecule all the time by this hypothesis.
And it's just a question of whether you fall behind
with degradation or whether you inherited too much
to be ever catch up in the beginning or something like that.
It's extraordinary what is being said here.
Then something goes awry, and we began to accumulate
more and more PRPC grapey, and eventually the animal gets sick.
And it goes on to die.
And still we're talking about a very specific protein
with a very specific sequence in a very specific structure.
And then sometimes we're not.
Sometimes we're talking about a very specific protein
with a very specific sequence in a very specific structure
that has two structures.
And those two structures, apparently, one of them
is more stable than the other, so much so that it can cause
the good one to form the bad one.
But that part of this cartoon is almost wholly missing
from the explanation.
And instead we have incubation times
and p-value differences between brown mice and white mice
with two amino acids difference in this protein.
And then that being a sign that the protein is involved
in whatever happens.
Okay, it is 335.
I need to go to basketball.
I'm going to play the funny opening that I started with again
as a way out in case you didn't see it because it's just funny.
I got a new toy, which is actually a new toy for
it is a new toy, which I am planning to use on the wheel.
And also maybe on my motorcycle if we get a little better weather.
And that toy, of course, was a little camera
that allowed me to make this video.
But anyway, thank you very much for joining me.
This has been Giga Ohm Biological, a high-resistance,
low-noise, oh, that's not the right song.
You funny man, that's not the right song.
This is the right song.
That's it.
This has been a production of Giga Ohm Biological.
And this was, of course, not meant to make you take me seriously
as a basketball player, but at least to let you know that I do
actually go to the gym with my kids.
And I'm going to try and figure out a way to make neurobiology
shorts and immunology shorts while I'm shooting around.
And I thought this camera would kind of help me.
It actually works surprisingly well.
It also works surprisingly well on the one wheel.
So I think I'm going to be able to make some shorts
that I haven't been able to make before because I just don't like
putting three GoPros on something and having to edit for hours afterwards.
And that thing seems to have solved that problem a little bit.
I can give a GoPros to my wife for her yoga.
Anyway, thanks very much for joining me.
And when Giga Ohm Biological, we are trying to distribute the knowledge
that can dispel this big E enchantment.
Thanks very much, and we'll see you again soon.
Thank you very much.