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[Music]

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hello everyone

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my name is jono Abshire I am a second

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year PhD student at Portland State

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University working in the center for

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life in extreme environments more

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specifically out of the extreme virus

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lab and today I'll be giving you a

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little bit of insight into Life In Hell

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understanding the role of toxin

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antitoxin systems in prokaryotic genomes

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and their potential for virus host

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co-evolution tldr extreme organisms the

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viruses that infect them and their

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interactions in these extreme

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environments and our journey kind of

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begins right here so this is a personal

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image of Lassen Volcanic National Park

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it is one of the many areas on our

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beautiful planet that is home to these

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volcanic Hot Springs this is one of the

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main sampling sites of our lab and while

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most people wouldn't believe that there

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are things living in this environment

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there certainly are

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um specifically these microbes from hell

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and I'll get back to these guys in just

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a second but a little bit of background

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about why we why we study these

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particular environments so these

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volcanic Hot Springs are often

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considered and understood to be

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analogues for both for planets both

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outside and inside of our solar system

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referring back to some of the ancient

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Hot Springs found recently on Mars which

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may or may not have looked something

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like this boiling Mud Pot that we find

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in Devil's Kitchen at Lassen Volcanic

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National Park

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getting back to the microbes one such

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microbe is this um micro right here uh

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spherical cell known as sacrolobus so

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factoricus this is a scanning electron

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microscope showing you the topology of

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the cells

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um coined s441 by our lab really thrives

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and uh lives in these hot acidic

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conditions with temperatures from around

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70 to 75 to 80 degrees C and ph's of

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around 3 to as low as one so these

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really extreme environments and when we

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think about these environments and the

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reach ecosystems that uh and habitat

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them we can also think about one of the

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most abundant molecules on the planet so

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that of viruses and yes there are these

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extreme viruses infecting these

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extremophiles uh the one which I work

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with in lab is sopholobus spindle shape

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virus one sacrilebus is a natural host

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of this particular virus this ssv1 this

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is Stanley uses they them pronouns a

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really character by his lemon shape so

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these shapes are really unique to both

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archaeal organisms extremophiles and

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some of these extreme viruses

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and when we think about looking at these

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organisms and the interactions between

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them in these extreme environments we

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can look at some systems currently

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prevalent today in bacterial cells so

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these toxin antitoxin systems usually

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known as addiction modules found on

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plasmids again really prevalent in

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bacterial genomes only recently being uh

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being discovered to have some viral

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encoding

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and what this particular system confers

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is when you have a plasmid that has the

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positive uh or is positive for that

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particular addiction module that cell

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will needs that plasmid in order to

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continue its life in that particular

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environment so here we have the presence

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of the TA should that cell or progeny

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continue with the presence of that

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plasmid you would expect normal growth

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in the environment whereas if it were to

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lose that plasmid it would would have

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been addicted to that particular genome

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it would die off whereas in a negative

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system you would get growth either way

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whether or not there's plasmid loss and

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we can further visualize this particular

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mechanism by looking at what might

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happen to some uncolonized cells some

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sort of event happens in which the

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addiction module is introduced to that

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population

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and you have your addicted Survivor

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going on to make new progeny and then

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conferring that group protection and

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persistence so these systems are really

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understood in bacteria to confer a

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microbial persistence phenotype some

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have been referred to as fate defense

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mechanisms and largely considered uh as

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plasmid stabilizers on the plasm

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um and we can also uh assume that

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another group of uncolonized cells were

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to come in here perhaps that group

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encounter you would still get that toxic

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culling uh from that toxin antitoxin

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system uh one important note they are

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characterized as two genes that are

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typically right next to each other

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um the toxin being just Downstream of

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the antitoxin uh pretty stable toxin

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pretty unstable antitoxin

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and looking at this system with ssv1 a

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previous student did quite a bit of work

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in uh mutagenesis of the ssv1 genome and

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we use this at least these mutants to

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test whether or not some of these uh

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genes or open reading frames are

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essential to the virus and two genes in

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particular this T3 and TX transcript

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these particular two genes we can make

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changes to what I think is the antitoxin

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and we don't see too much

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um difference in terms of viral

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infection viral function whereas if we

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were to delete this uh or insert a

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sequence into this particular open

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reading frame what I think is the toxin

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we do see differences in infection

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mechanisms

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these extreme viruses and just virus

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reproduction overall viruses can

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typically go through two Pathways or a

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combination of both so a lyric cycle in

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which the viral genome or the viral DNA

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will insert itself into the cell

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you have some replication going on

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eventually assembly of those virions and

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the cell will then burst die and those

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viruses will go on to infect other cells

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whereas in a lysogenic cycle the viral

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genome is incorporated into the host

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genome and there's usually a latent

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phase in which it kind of just stays

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there maybe some induction event happens

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in ssv's case it actually just buds from

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the cell without killing the cell and

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one of the one of the two ways that we

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use to test for this viral reproduction

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is of course PCR so amplifying a

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sequence that's specific to viral DNA

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looking for its presence in cell-free

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supernatants another way is through Halo

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assay so very similar to a plaque assay

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in which you look where cells are

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bursting

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we cultivate a lot of cells uninfected

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sacraloba cells like you saw in the

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slide a while back and then we spot cell

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free supernatants which contain these

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mutant viruses

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onto the plate and look for clearings

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and this Halo assay is made possible

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because of the fact that ssv1 buds from

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its cell without killing the cell

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um and the onion the infected cells grow

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quite slower so it really converts this

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growth stunting phenotype in the

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sacrolobus cell

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and looking at the toxin protein and

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large a large majority of my work has

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been looking at mutants that we've made

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in this toxin protein and several uh

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machine learning softwares have

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indicated that there's a quite a high

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probability of a cleavage site over here

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behind this very hydrophobic

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uh Helix you right here and so I sought

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to characterize at least this particular

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mechanism in ssv1 through using some of

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these mutants that we already have

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um and while largely these over here are

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our wild type so really shouldn't expect

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any kind of change in viral function you

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can see some really nice Halos and we

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can confer that you know the cells are

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not dying but they're sick

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and in our mutants where we have

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insertions in in that specific toxin uh

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open reading frame we don't see any Halo

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formation

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um likewise I sought to substitute those

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two uh residues at the cleavage site

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since it's largely important for protein

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maturation protein function and we still

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don't see a Halo just switching those

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two residues but the main takeaway from

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this is that we still get virus

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replication

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um we still get quite a bit of at least

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high levels of viral replication from

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some of these mutants and one of these

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mutants particular we didn't see

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anything so maybe that one's detrimental

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to that particular open reading frame

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but we do see that there is virus

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replication happening in the cell

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supernatant when we screen for DNA

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um and it's important to kind of note

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that this this protein isn't just unique

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to ssv1

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um in in looking at these systems in

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these extreme organisms uh this one in

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particular up here is another SSV so

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another spindle shaped virus in which we

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see really low sequence similarity

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across these particular genes but quite

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a hot quite a high bit of um structural

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similarity between these genes and a lot

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of these are

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genomes of extremophiles while some are

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virally encoded

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and uh one of the main takeaways really

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just wrapping uh wrapping it all back

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around looking for these particular

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mechanisms and how they confer

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persistence with both their

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extremophilic organism and their viruses

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would be a really nice first start in

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looking for these biosignatures perhaps

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um and ways that we can look into how

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the microbes might be interacting

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in these particular extreme environments

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this is another sampling video

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um just of Lassen Volcanic Park pretty

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pretty close to where s441 was isolated

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from

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uh looks pretty similar to maybe

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something we'd see on another planet

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that we could sample at

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and then a Shameless plug but looking

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for viruses in space I think is the

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obvious next step we know that they're

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the most abundant molecule on the planet

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and just understanding that these

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mechanisms and that these systems are

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out there allowing these cells to thrive

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in this particular environment would be

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a great uh in starter conversation for

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looking for these small biomolecules in

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space

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and with that I just want to thank my

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lab

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um my group over here at Boiling Springs

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Lake when we collected samples and my Pi

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[Music]

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thank you General we have a time for

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hey my name is Pia and I'm really

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interested in like Christopher Cross

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Community of microbes

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um have you looked at all into the

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interactions of other phage defense

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systems

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um yeah so at least in terms of uh this

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toxin antitoxin system they're pretty

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understood in bacterial cells to like

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incorporate themselves into the genomic

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crispr cas9 there are some proteins that

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um that look pretty similar to this a291

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and some of these other virally encoded

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genes that do have some kind of crispr

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casts editing mechanism so yeah I think

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they it might be leaning towards that

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way but they're pretty well

283
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characterized in terms of like how they

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work with proteins and mRNA so um yeah

285
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not too far there yet but

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um definitely definitely somewhere in

287
00:11:49,790 --> 00:11:46,310
uh thank you for the talk um Marshall

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Seton JPL and I I was curious so um

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you're talking about looking for viruses

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and extraterrestrial environments um do

291
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you think there would be or I'm curious

292
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I'm completely ignoring yeah so no no I

293
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dropped out of biotube so for this

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um for viruses specifically like it

295
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would small molecule biosignature

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classes vary from viruses to like what

297
00:12:12,769 --> 00:12:09,660
you'd be looking for to cells yeah yeah

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I I for sure and that kind of brings me

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at least back to a point like of looking

300
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for fossils and stuff like that so

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viruses and sediment

302
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um and at least our lab there's been

303
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some work in like silica and coding

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viruses and how you know the stability

305
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of them so maybe there's a pretty silica

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Rich environment in which we might be

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able to look for any kind of these

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signatures so like proteins

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um or or yeah just specific things that

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might might have been preserved in those

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particular environments

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um considering that these viruses live

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and thrive in in pretty extremes

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um you know they're they're they got

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they got to be out there at least in my

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mind so you mentioned um looking at like

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morphology and fossils and things like

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that I know that at least for

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um looking at cells and things they've

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been shown to to mimic abiotic systems

321
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uh pretty well because humans are very

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good pattern recognition and so um has

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anything been seen like that for viruses

324
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yeah not the not that I'm familiar with

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um I know it's at least a brand new

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conversation of starting to look for

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like viruses in space Astro virology

328
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things like that so uh yeah we're just

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kind of getting up and running oh yeah

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yeah no sorry I don't mean to like play

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20 Questions oh no I just thought it was

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00:13:37,610 --> 00:13:35,870
yeah there's a there's a little cure lab

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that like goes on right now that I teach

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so I'm obviously not there but they're

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any other questions