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[Music]

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hi everyone I'm Emily and I'm a PhD

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candidate in the DECA slab at Stanford

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University and I study the limits of

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life in hyper saline environments

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so as we all probably know Brian's are

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one of the targets for a life detection

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mission to other ocean worlds including

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Europa and Enceladus and even past ocean

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worlds like Mars and so by understanding

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the limits of salty life on Earth we can

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understand how and where to look for

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life on these other places

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one of the ways we can assess a brine

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environment is through this measurement

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called water activity and water activity

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depends on vapor pressure but you can

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think of it about as how many water

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molecules are available to a cell in

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solution so on the left is an image of

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what pure water would be this is where

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all the water molecules are free to

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become Vapor move about the cabin that

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would be a water activity of one

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now if there's ions in solution water

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would be attracted to those molecules

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and a water activity would decrease so

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the lower the water activity the more

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uninhabitable an environment is

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and since water is required for all Life

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as We Know It water activity can be used

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to assess assess habitability so some

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common items in their water activities

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are listed on the left including sea

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water which has a pretty high water

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activity and the salinity of seawater is

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about 3.5 percent

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and then honey has a water activity of

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0.6 and some of Mars special regions or

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areas where we might detect life on Mars

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are in no lower than 0.5

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and there's been a lot of really great

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pure culture work on the limits of life

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and cell division at different water

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activities and to sum it up the

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predicted or theoretical water activity

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limit for all life on Earth including

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eukaryotes and prokaryotes is about 0.6

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and this is based on pure culture so

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like our speaker last night mentioned

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not all the things in the world are in

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pure culture yet so that leaves out

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about 90 or more of the biodiversity on

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Earth and our ability to assess what the

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limit of life for each of those strains

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so brine environments are really

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widespread on Earth they're everywhere

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um and a lot of great work has gone into

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assessing metagenomics like the

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taxonomic diversity the potential

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metabolic metabolic potential of

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microbes in these environments

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metatranscriptomes and even some bulk

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activity measurements but metabolic

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activity is rarely measured at the

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Single Cell level

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and this can be really important because

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when a cell is stressed out they might

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have really low levels of activity that

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are not detected with both techniques

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so today I'll be talking a little bit

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about my work in solar cell turns and

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then transition into the my current work

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in Western Australia aesthetic brands

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so like I mentioned when life is

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reaching its limit a metabolic activity

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can be really low and so single cell

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analysis can help us detect the slow

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level of activity by lowering detection

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limits so a cell needs to acquire 50 new

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biomass to be able to divide into two

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new daughter cells however with Nano

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Sims for example we only need to detect

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about five percent maximum we can even

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get as low as less than one percent of

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new biomass to detect active life in a

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sample it also provides quantitative

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results so we can do a lot with this

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data on a quantitative level and it also

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reveals trends that are often obscured

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by bulk analysis

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and when I'm talking about metabolic

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activity in this talk I'm talking

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specifically about anabolic activity so

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there's catabolic activity that produces

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energy for a cell and then there's

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anabolic activity where a cell can take

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substrates from the environment like

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amino acids and glucose and take that

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carbon and nitrogen and then produce new

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cell biomass which includes proteins DNA

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so first the work that I've done at this

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um solar Salter and it's called South

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Bay saltworks it's in here in San Diego

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and basically they bring sea water in

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from San Diego Bay and it goes through a

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series of evapo concentration ponds and

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as the water evaporates out of the sea

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water different salts are left behind

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producing a series of ponds that range

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in water activity from 0.98 that of

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seawater to that well below the known

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limit of Life at 0.4

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and so we measured anabolic activity of

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over 6 000 individual cells from a

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series of five of these brines with five

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different isotopic substrates from amino

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acids ammonium and bicarbonate glucose

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and nitrate and that sort of thing

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we measured their anabolic activity with

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nano SIM so on the left this is what

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this looks like cells that are circled

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in red are considered enriched and this

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is for carbon from amino acids and then

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the cells that are circled in white

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would be considered less active or less

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enriched in the sample

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and when you plot this with water

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activity you see that microbial activity

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decreases exponentially with water

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activity however there's certain areas

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so you can

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see certain subsets of cells are more

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active than most of the cells in

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seawater at a higher water activity with

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even just low decreases in water

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activity

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um we also detected cell biomass through

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dappy and nanosims in the slow water

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activity magnesium chloride brine

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however we didn't detect any activity

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here and the incubation time was two

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days long so there's several reasons for

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this

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estimate a new predicted lower limit of

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Life at 0.54 so that's a little

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significantly biologically significantly

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lower than the 0.6 previous limit

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detected sorry predicted and this data

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I'd love to talk more about it but it's

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been submitted and I just got word that

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it's now in review as of this morning so

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it's really exciting and if you'd like

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to hear more about that please talk to

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me later

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so now to kind of get into the topic of

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this talk which is I'm talking about the

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limits of life in acidic brine

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environments there's a lot of things

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that happen in brines so you can have

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salinity as a limiting factor you can

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have high metal concentrations you can

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have high ionic strength and

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chaotropicity but one thing that really

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stood out to me about this sample set

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was that most of the brines that had

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high levels of activity actually had a

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pH about seven or eight so like

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relatively neutral to slightly basic pH

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however this magnesium chloride brine

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which had no detectable activity had a

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pH of about 5.4

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and so my question was how does pH

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affect microbial activity in brine

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environments and is this an important

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factor

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in the limits of life

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so last summer our team went to Western

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Australia to sample a bunch of acidic

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brines and these brines are considered

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Mars analogs because of the geochemistry

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and weathering systems that created them

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over millions of years

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um just in a short oxidation of sulfides

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acidified the water and then evaporation

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concentrated ions and other salts over

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time creating these really acidic Briny

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environments

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there's hundreds of these lakes and we

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were really lucky to sample a really

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broad spectrum of them so on the x-axis

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is water activity and on the y-axis is

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pH and I was really thrilled to get a

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nice group of lakes to sample for

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activity analysis especially these ones

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that are boxed these are were at a water

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activity at 0.7 and they range in PH so

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it's a really good opportunity to kind

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of look at how pH affects metabolism at

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low water activity

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so at this this study I analyzed

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metabolic activity specifically anabolic

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activity by incubating these brines with

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an analog amino acid called hpg

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cells that are active in the sample will

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take up hpg and incorporate them into

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their new proteins that are produced and

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then the hpg has a alkyne group on the

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end that later on in lab you can perform

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click chemistry on and click a 404 to it

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so all the cells that are active in the

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sample turn out green under the

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microscope or in flow cytometry and then

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that can be counter-stained or compared

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to cells that are stained blue via DNA

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stain of doping and so with this data

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you can calculate the percent of active

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cells across these Lakes

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so this is just an example of what this

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data set looks like these are just four

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of the 13 or 14 lakes that we were able

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to do this with on the left the lakes

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are ordered and decreasing pH and I

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thought it was also kind of interesting

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the color changes so you can admire

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those I don't know why they do that but

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that's the way it is and then in the

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middle are the dappy stain cells under

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the microscope and then on the right are

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the active cells that are tagged with

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the green fluorophore

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and you can also notice like the

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different cell morphologies and

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abundances that change with ph which is

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really fascinating and also that pH

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didn't affect the assay itself we do see

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some cells that are active at these low

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PHS and sometimes you have brines where

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so when you calculate the percent of

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active cells and you put this all in a

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plot I'll just Orient you so on the

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x-axis here we have water activity

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decreasing and on the y-axis is pH

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decreasing in this direction so you

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would expect the most extreme acidic

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brines to be kind of in this region

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this dotted line is that newly predicted

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water activity limit of life that I

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talked about earlier and then this

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dotted line represents the percent of

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active cells dropping below 50 percent

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at this pH so this color block each of

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the dots is one Lake and the color bar

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was colored on a green gradient if the

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percent of active cells is above 50

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percent and on a gradient towards black

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if it was below 50 so you can kind of

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just visually see that all of the green

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like high percentage act cells cluster

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up here and all of the low percent of

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active cells clustered down here and

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when you look at it statistically there

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is a moderately stronger correlation

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with ph and activity compared to water

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activity and activity so so many times

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you can count how many times I say

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activity in this talk

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um and so some thoughts on this

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a pH of six is really high for

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environmental samples and as we've heard

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from a lot of people and as we know

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there's a lot of microbes that live in

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way more acidic environments so what's

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actually happening here I don't have an

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answer to that yet and I'm hoping to

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figure that out with future analyzes

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that include nanosims work on these

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Lakes

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um and then if this is a true signal and

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this is what what is going on in the

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environment then microbial activity may

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be detected at lower water activities

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than we know of now as long as the pH is

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within the seven to eight maybe like not

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so acidic range

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um so figuring out what that PH range is

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and how that interacts with water

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activity limits of life

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um is something of ongoing investigation

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so with that I'd like to acknowledge my

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lab the deaca slab pictured up in the

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right all of our funding sources the

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oceans across space and time team which

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is a nasa-funded project you'll hear

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about uh you'll hear from a lot of us

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throughout this conference and all of

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our collaborators in Western Australia

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including Molly the dingo who of course

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this work would not have been possible

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without

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all right and then I'll take any

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questions and I'll leave this up so you

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can stare at the dots for a little bit

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longer

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[Music]

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[Applause]

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hi uh that was really cool talk thanks

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so much

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um I was just wondering if he could

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expand a little bit on why you

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concentrate onion anabolic

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um activity as opposed to catabolic

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activity yeah so

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let's see scientifically the reason why

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I would focus on anabolic activity I

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mean both catabolic and anabolic

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activity are essential for life right

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life has to make energy to assimilate

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substrates and ground divides so both

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processes are really important

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um and they are processes that I'll be

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combining or looking at in future field

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sites

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um with anabolic activity the

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interesting thing here is that you could

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imagine that when a cell gets really

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stressed out it would stop dividing

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potentially and still accumulate

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substrates to repair DNA repair cell

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walls do all that stuff until conditions

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get better and so the idea was that or

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my original hypothesis was that by

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um that anabolic activity would extend

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beyond the known cell division limit of

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life because even as cells are um cells

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would be able to survive just by taking

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up materials and so that taking up

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process is the anabolic Activity Part

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sweet talk

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I have a question so um you mentioned

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that you guys have essentially like uh

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extrapolated uh new

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like limit for life like in terms of

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water activity it's kind of a power move

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so we're not the first ones to do this

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okay but okay but uh could you explain

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how you extrapolated that because I

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think maybe I missed that or I didn't

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understand yeah um okay we're gonna get

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into the weeds here but let me go back

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to that slide so this this idea or this

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like extrapolation is based on

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um

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these this study here so they did the

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same thing and this is how they got this

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theoretical limit of life that has now

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been cited almost 200 times from food

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preservation fields to life detection

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Fields so

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um

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hasn't been published yet

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um so the reason so they used a

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different technique and they based it on

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cell division and so we're

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um

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so hang on let me answer your first

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question so

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um the way that we did this is single

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cell analysis allows you to separate the

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cells that are active from those that

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are inactive and in a brine environment

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this is really important because brines

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are really salty and cells can fall in

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from anywhere bird poop wind whatever

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and get preserved and those can be

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inactive so those can contribute to that

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bulk analysis and not necessarily even

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be important in that environment so with

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single cell analysis we took out the

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active cells average their anabolic

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activities so we're just focusing on

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who's contributing to metabolism in that

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environment and then use detection

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limits and and math to extrapolate and

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predict or propose this new predicted

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limit of detectable life and so this is

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not the limit of life this is what our

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measurements can detect and if we found

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an environment at 0.54 Water activity

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with similar conditions to this

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environment that we extrapolated it from

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we would technically be able to detect

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life in that environment

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and this is also a really conservative

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limit so

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um we even upped the number of standard

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deviations that we would normally do for

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our like detection limit so if we were

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to do like our data processing in the

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normal way it might even be lower that

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hey George eibel from Montana State I'm

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just curious uh you're looking at the

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Active cells were you did you look at

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who they were at all whether that was by

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fish or sequencing so in this study we

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did not look at cell identity and we

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focused um on metabolism

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in Western Australia I don't have plans

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to look at the actual like I don't know

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I could look at the Active fraction of

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cells from the bond cat analysis I

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haven't decided if I want to go down

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that road yet but in the next field site

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we are planning on on doing that so

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um

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yeah not in this not in a city

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all right thank you very much Emily yeah