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[Music]

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[Applause]

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I work at the University of Washington

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with a interdisciplinary team and we are

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studying the bio signatures cellular and

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proteomic of extremophiles subject to

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hold and highly saline conditions and

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I'm going to be showing some of our

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first results in this ongoing project so

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first you want to we want to think that

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most of the moons and planets of

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astrobiological interest in the solar

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system have surfaces with temperatures

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well below the limits of life and even

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though it's possible that organisms may

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be living below in deeper warmer

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environments these organisms may be

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transported towers the surface which is

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where we are going to be looking for the

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bio signatures so this raises the

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question of what happens to an organism

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when you take it away from its habitable

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region and remove it in colder and very

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likely South air conditions and to

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answer this question we took a psycho

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philic organism and we incubated it into

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salinity conditions far from its growth

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range so what you are seeing here in

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this temperature salinity graph you have

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the temperature on the vertical axis

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South concentration on the recentl axis

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and this Square this rectangle is

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showing you the limits at which our

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organism can grow so you can see it has

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a nice range of temperatures going down

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to negative 12 but a rather limited

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range in salinity so what we did is we

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incubated it at temperatures in the

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lower range of its temperature growth

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range and saline it is a couple of

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salinities both optimal salinities and

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higher salinities that than what we know

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it can tolerate and after incubating it

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we measured the activity by

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incorporation of a radioactively-labeled

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amino acid we measure the viability that

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is we took the organisms after the

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incubation and we return them to their

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optimal conditions to see estimate the

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number of viable cells that were

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remaining we observe the cells at the

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microscope with that stain and we did

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proteomics with tandem mass spectrometry

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so I'm going to walk you through the

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rationale of what temperature

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salinities did we choose moving into the

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metals and some of the results so you

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see here a simplified diagram of the

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phases of seawater when it's freezing

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you have again temperature and cell

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concentration on the bottom and

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basically this diagram is telling you

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what phases are going to be stable at

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which temperatures and salinity so you

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have liquid water in the warmer areas

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frozen eyes in the cold and salt at

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higher salt concentrations I also want

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to know that sometimes you can get

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solutions out of equilibrium so you can

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get supercool solutions which are common

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for all of us studying in the laboratory

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this type of systems so you have liquid

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water at lower temperatures when it

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should be frozen and an important thing

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in this diagram is the I want to the I

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want to signal you to the brine

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equilibrium line so here what you are

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going to be saying is as this ice is

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freezing the liquid inclusions we seen

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the ice are going to have a salinity

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that is dictated by these lines for

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instance if you are having ice at

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negative 10 the brine inside the ice is

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going to have a salinity or a South

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concentration with an ionic strength of

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3.2 approximately and this is important

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because if you are an organism living

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within the ice this means that as you

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lower the temperature you are

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simultaneously increasing the salinity

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so the organisms it's in the ice

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experiencing are experiencing these two

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stressors at at the same time so here it

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is in the phase diagram you have here

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located the square with the rectangle

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with the growth conditions of our model

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organism which you may have heard in

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other talks coil a secretary at 9:34 H

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and when we so here you can see the

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conditions that we used in the

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experiment so we chose two temperatures

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both in the growth range of Corellia

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negative 5 and negative 10 and we choose

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the following salinities we chose a

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seawater supercooled seawater and we

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chose the brine salinity expected at the

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negative 5 temperature and for negative

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10 we did the same thing

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seawater supercooled and we chose the

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negative 10 Brian salinity so the ionic

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strength of

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those solutions would be 0.74 the

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sea-water 1.8 for the brine equilibrium

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at negative five and three point two for

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the brine et Librium at negative ten and

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I have their concentrations in parts per

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thousand for comparison so let's take a

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look at what we did with the organisms

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themselves we grill them in their

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optimal media at zero degrees and then

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wash the cells transfer them to the high

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salinity media the solutions were

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prepared following the expected

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concentrations for seawater ions at any

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given temperature and then we amended

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the solutions with either a high or a

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low concentration of nutrients we

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distributed these resuspended cells in

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two vials to be into two sets of buyers

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one of them for the activity

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measurements and another one for at the

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same time we took measurements for

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viability cell number and proteomics

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because we wanted to do a study of how

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this evolved through time we took

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triplicate samples for the activity in a

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shorter time frame from 2 hours 12 12 24

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hours and 1 month and we took samples

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for cell numbers and viability at 1

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month and 4th month at after 4 months we

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wanted to see how the organisms had been

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evolving their proteome after being

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subject to these different conditions of

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temperature salinity and nutrients so

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let's take a look at what the organisms

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were doing starting with the activity so

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this graph here you can see the activity

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in the vertical axis and time from 0 to

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24 hours after the incubation the

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vertical lines show you the activity for

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the 42 supercooled solutions in green

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the negative 5 in orange the negative 10

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and you can see that the brine activity

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incorporation of the radioactively label

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amino acid is basically here in the

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bottom you can barely see it above the

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activity of the kill controls so as soon

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as this organism experience a higher

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salinity there is a drastic drop in

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their activity in their metabolic

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activity and after one month if you

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compare the activity

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here in the vertical access with the

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viability of the cells in a logarithmic

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scale in the horizontal axis you can see

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that the populations radically diverge

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so you still have higher activity after

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one month in the supercool samples lower

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activity in the primes and what is

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interesting is the changes in viability

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so for the negative 5 degrees you have a

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higher viability so these cells are

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really happy they are active and they

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are viable when you increase the

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salinity at negative 5 the activity

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drops but the viability is still

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relatively high but for the supercooled

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negative 10 seawater the viability has

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dropped so after amount of incubation

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even though they were picking up the

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amino acid at some point they lost the

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ability to reproduce and the cells that

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were in the negative 10 brine

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they have no recognisable activity or

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viability after one month so this is

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really stressful for them and you can

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see that the salt is driving the

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difference in the activity and the

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temperature the difference in the

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viability of the organism so you have

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here in this area growing cells here

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cells that are with low activity but

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still viable cells that are active but

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not viable and here in this condition

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which is when most of them are non

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viable and non active so let's take at

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cell numbers here you have the cells and

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the viability and I'm going to be

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showing two treatments the light colors

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are going to be without nutrients and

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the dark colors with nutrients and these

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triangles he'll show you the initial

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conditions so these were the cells how

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the state of the cells when we started

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incubation and after one month in the

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negative 5 supercooled and seawater most

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cells are similar to how they started

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except that if they don't have very many

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nutrients the number of cells decreases

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and this outlier here is interesting

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because this sample was frozen so the

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organisms here were actually

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experiencing not the seawater salinity

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but the brine salinity at the negative 5

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temperature and that had an effort in

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the viability so let's take a look at

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the brine samples how do they do the

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brain samples effectively with the brine

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the viability decreased and it was more

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drastic effect when there were no

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nutrients available for the organism at

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negative 10 there was a bigger loss

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in the in the viability and the effect

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of the nutrients was ever ha even higher

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so cells really need the presence of

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nutrients to be able to survive

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incubations

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in these more extreme conditions here

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there were we had a lot of frozen

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samples which we think were driving even

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further down the viability of the

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organisms because these samples were

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experiencing the brine conditions at

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negative 10 and those were really

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extreme for the organism at negative 10

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divided will be had dropped to 0 but

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what is interesting for us is that the

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number of cells was preserved so these

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cells that were suddenly exposed to a

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very high salinity they couldn't

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maintain their activity near their

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viability but the cells remained and we

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think that this may be important when

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you think in the context of finding bio

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signatures somewhere else so what were

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these cells doing for that we're going

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to be looking at the proteome of these

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cells I'm going to be showing two plots

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the first one is anime and in MDS plot

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in which each one of the dots is

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comparing the proteome of that sample

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with a proteome of all of the other

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samples the second type of plots I will

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be showing our string networks in which

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each one of the dots is a protein and

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what you are going to be seeing is how

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the proteins are related to other

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proteins where they are and how are they

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related related in the metabolic

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pathways so when you look at the

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proteome is nice because the treatments

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are separating we have here the negative

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5 with and without nutrients the

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super-cold this is the brain here up you

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have the supercool negative 10 and here

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are all the negative 10 brain cells that

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were not doing very well so let's see

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what do these cells do when you increase

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the salinity when you go from

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supercooled sea water to the increased

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salinity the supercooled sea water cells

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are doing the normal metabolic paths of

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the cell but when you increase the

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salinity they start pressing all of

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their stress response they become motile

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they start assembling clusters of iron

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which we think can help with the stress

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of the oxidative stress and they start

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repairing their DNA so they are stressed

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and they are responding to it these two

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treatments had nutrients in the absence

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of nutrients what are these

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doing so they are looking for metals we

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think a specifically iron and they start

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expressing an interesting protein which

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is called a connotates and we think is

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interesting because this protein has a

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remarkable stability at high salinities

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according to the literature and also can

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take a dual role depending on the

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presence or absence of iron so we see

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that the cells are looking for iron and

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that they are over expressing

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approaching that has something to do

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with or that uses iron and can have

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different roles depending on the

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presence of iron now let's see how the

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metabolic networks looked so here you

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have the negative 5 super cool brains

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and you will see how they compare with

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when the salinity is increased so here

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is super cool negative 5 and here is the

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increase in salinity as soon as the

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Cellini's increase a cell that initially

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had a lot of metabolic networks acting

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active and working simplifies its

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proteomic Network to just focus on

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finding metals and and become mobile

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when you do not have nutrients the

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networks are simpler and they are again

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focusing on achieving of acquiring iron

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and expressing their qualities protein

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when we compare these negative 5 brains

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with more extreme negative 10 brains we

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see that the proteome has simplified

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into making proteins that are going to

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sterilize the DNA under extreme

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conditions so these cells are really

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stressed just looking to maintain their

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DNA so to conclude what do we see when

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we expose a cycler file to extreme cell

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conditions they decrease the activity

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they reduce the metabolic pathways focus

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on iron acquisition and motility in the

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negative 5 brine the nutrients help it

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be viable and in the negative 10 brine

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they lose all the viability and the

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proteome just simplifies to take care of

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the DNA and make it more stable from the

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if you think about the bio signatures we

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think that the Econo taste may be a good

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candidate because it's all regulated in

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these cells Express exposed to the

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brines

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and it's also very stable at high

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salinity and the other aspect that we

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found interesting is that the cells are

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preserving their their shape

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DNA even though they are exposed to very

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high salinity conditions and this is

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even after four months of incubation so

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I want to thank everybody in the team

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including me none damage that is just

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join our team two weeks ago to help us

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process all the proteomic data and our

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funding agencies thank you I do we have

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any very quick question Justin Lawrence

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00:14:08,030 --> 00:14:06,570
just super interesting talk I'll say the

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second question which is much longer

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the first question one as you mentioned

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super cool brands how do you hold us a

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seawater salinity sample at negative

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five for a month how do we hold it

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liquid absolutely that's very common

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it's the opposite state is how do we

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make them freeze so because these are

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eppendorf tubes and we are used very

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clean water to make our samples the

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water juice is in supercooled state

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because it doesn't have anything that

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nucleates the ice so for us the problem

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is actually freezing the samples Kolia

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doesn't seem to nucleate the ice we saw

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00:14:44,120 --> 00:14:42,330
it could help but it doesn't it doesn't

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have an effect the only thing we have

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found is scratching the tubes that does