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[Music]

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I'm gonna call back a little bit to a

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couple of the sites that Emily mentioned

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before and uh what's exciting for me is

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that I'm in San Diego for the first time

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so I finally might be able to go see one

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of the field sites I'm about to talk

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about and I had no idea that there were

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actually palm trees down here around my

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supposedly extreme field site so that'll

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be a a funny dichotomy there but um so

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yeah we're gonna be following the salt

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uh uh and it's going to be a lot of salt

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so uh uh hang tight there

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so the first field site South Bay

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saltworks which is down the road

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um is a salt Farm which as water is

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taken from the bay it is uh brought in

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and

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um the the people there are are

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basically taking the the hay light out

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and using that to I believe soften the

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water here and then the magnesium

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chloride also gets concentrated and and

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separated and so you get these

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um these really sodium chloride

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concentrated uh regions and magnesium

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concentrated magnesium chloride

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concentrated regions and then you also

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have uh regions which are a lot more

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like seawater and so

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um that's South Bay salt works that's

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one of the sites I'll be talking about a

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lot today and what we're investigating

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there

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um with oceans across space and time uh

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is can bioseignature molecules be

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detected in these sites um with these

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super high salinities and uh then how

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does that input impact our search for

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life elsewhere and

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um then how do these vary with things

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like water activity and ion

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concentration

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and then here's another analog site in

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Western Australia this is Lake Campion

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which is a very interesting site that we

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looked at

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um and uh so these are transient lakes

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and so uh they um they evaporate during

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the drier warmer months and then they

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fill up during the winter months and so

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you get these salt crusts which build up

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on the bottom of them and so uh these

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are really interesting because uh the

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the evaporation process is happening

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very actively so we can get a modern

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look

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um at what's happening there and so in

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general analog sites can give us

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training grounds for robotic exploration

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on other planets they can help us test

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our instruments and uh particularly we

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want to know how salt and acid acidic uh

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um low PH environments affect our uh our

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instruments and then it can give us

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insight into the environmental processes

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and the biology happening there

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and so uh now I'm gonna uh shout out uh

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Luke Fisher's review paper in

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environmental microbiology where he

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talks all about the bioseignature

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preservation uh in Brian's particularly

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in deep hyper saline anoxic basins which

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I won't be talking about today but when

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it comes to brines themselves as opposed

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to evaporates or salt crystals

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um we know that DNA and RNA are well

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preserved while mRNA is not as well

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preserved and that ATP preservation has

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been observed by uh to ovala at all in

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1987 but not much has been done about

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that since then and lipid preservation

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is being studied more and more recently

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and so that's good but but these aren't

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nearly as well explored

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um

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as genetic uh compounds and so uh one

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compound that we're looking at ATP

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adenosine triphosphate it's a

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short-lived molecule and it's used

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um in astrobiology and in ecology as a

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marker of microbial activity and so if

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you have a lot of ATP means that a lot

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of microbial activity is happening if

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you don't have any microbial activity

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then you're probably not going to have

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much ATP but it's very highly evolved so

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as a biosignature it might be less

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agnostic than for example amino acids

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and so we could look at polypeptides or

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we could look at individual amino acids

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and we target biological amino acids in

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this study as well as osmolite amino

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acids which are solute compounds that

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are accumulated by microbes when they're

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under high stress in hyper saline sites

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so and that's well understood in the

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literature that these osmolites exist

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and so first I'll talk about the methods

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that we used in in the field

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um or near the field in the motel room

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we looked for ATP using a luciferase

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assay and so say you have a beautiful

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Crystal

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from the crust of a lake like this from

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Western Australia then you take it to

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your Western Australia motel room and

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you do a uh you set out your samples we

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weigh them out we extract them in hot

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water and then we add our luciferase and

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luciferin and we have a field portable

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luminometer in order to do our analyzes

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and so that's how we uh quantify ATP

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and moving on to what we do at Georgia

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Tech when we bring the samples home so

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uh I was so glad to hear that uh another

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uh person here was using capillary

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electrophoresis so I don't need to

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explain it in its entirety again but um

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it's a separation method that uh in our

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lab we pair with laser-induced

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fluorescence and so laser-induced

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fluorescence mean means that we're

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tagging our amino acids our compounds of

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Interest with a fluorescent dye so we're

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targeting specific compounds

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um and so this is a targeted technique

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which has very low limits of detection

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and very high sensitivity and so

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um we did this at South Bay saltworks on

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one of the samples that has a water

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activity of below 0.4 and magnesium

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concentration of more than four molar

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and uh this is only a one to ten

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dilution so using a method that

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um Marshall Seton and I developed uh we

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got the concentration of amino acids in

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this sample down to tens of nanomolar

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the limits of detection for this method

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General are in the tense of picomolar

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and we did this using a commercial

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instrument

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um

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and again it was only a one to ten

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dilution for this uh hyper saline uh

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sample so we were delighted that uh the

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results came out like this and then for

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an untargeted analysis we can use uh

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microchip capillary electrophoresis

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paired with uh high resolution Mass

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spectrometry so if we take the same

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sample and we look at it using an

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untargeted method where we can get the

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master charge ratio of the compounds

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that we're interested in then uh

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combining the zip chip commercial setup

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with a thermocute exactive orbitrap we

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this is what the chip looks like it's

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very cute uh we get a very interesting

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set of compounds which we didn't

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necessarily Target and so

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uh moving back to the LIF analyzes for

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the results what we first observed was

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that as we increase in magnesium

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concentration and decrease in water

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activity and increase in chaotropicity

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we get a significant increase in the

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dissolved free primary amines that we

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were targeting and so that would be our

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biological amino acids which you've

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identified here and as you go up you

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have higher magnesium sites and we have

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a lot higher Peaks and we also have a

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lot higher values for Quantified amino

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acids and so we attribute this to evapo

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concentration as the sites which have a

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lot of water and a lot of salt and then

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evaporate and leave the salts and the

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Organics behind the concentration is

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higher so that's pretty straightforward

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and simple but it is very useful in the

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case where we're looking at very low

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biomass regions so

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uh next we then put it in micro uh micro

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microchip cems and we looked for these

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same compounds and we found a lot of

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them there and so you can see uh

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histidine Glycine alanine isoleucine and

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leucine are resolved there serine Etc et

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cetera glutamic and aspartic acid

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um and this was only a one to four

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dilution of that same sample so we were

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really delighted by these results as

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well and in the same separation just um

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looking at different traces different M

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over Z's we see a couple polypeptides

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and we see some adenine and guanidine in

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there as well and so these are things

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that we didn't necessarily look for in

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the celif but now that we're combining

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it with an untargeted method we can we

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can get a look at some more compounds

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that we didn't necessarily expect

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and then also osmolites which we did

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expect and did want to look for we found

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those like ornithine sarcosine and

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betaine in our samples with very high

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salt stress being applied to any of the

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microbes that were alive there

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um and and so these are are there in

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significant quantity

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and in Western Australia uh briefly I'll

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just show you some preliminary results

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this is Lake Brown which has a sodium

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concentration of near five molar and a

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water activity of 0.89 so not nearly

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um as low water activity as the South

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Bay saltwork sample I just showed you

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um but it has high relative

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concentrations of a lot of these uh

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compounds including the osmolite

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compound baiting and uh some lower

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concentrations of other compounds

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including a polypeptide

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and then conversely at Lake Gunter which

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has a higher water activity and a lower

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sodium concentration we see that there

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really isn't any easy way to detect

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those organic compounds with the method

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that we're using right now so just

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diluting these samples so perhaps this

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indicates that the uh and of course

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there's only two data points with

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Western Australia but you know looking

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at what we found at South Bay salt Works

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perhaps these saltier sites are actually

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places where it might be easier to

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detect biomolecules because of evapot

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concentration

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and we also want to look at the

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normalized abundance of certain amino

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acids just to see what sort of patterns

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we could find and if we have water

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activity increasing from left to right

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here

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that certain amino acids are upright

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regulated in this magnesium heavy site

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and others are down regulated

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and with that information we decided

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maybe we could classify our sites based

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on the amino acid distribution so if you

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take the distribution in you put in a

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classifier either logistic regression or

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random Forest you could get out one of

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these three different site types and

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this is a small data set and more of a

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proof of concept than anything but it's

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interesting to see that we have a very

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good area under the curve of you know 1

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or 0.83 which uh and and we also can see

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which compounds are driving that the

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most and so that's an interesting thing

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to take note of that might be used in

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the future if we have much larger data

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sets

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and so the last thing I wanted to talk

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about ATP if you remember so uh on the

279
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x-axis here's magnesium concentration

280
00:10:42,170 --> 00:10:40,500
and then we have three y axes because

281
00:10:44,210 --> 00:10:42,180
why not and

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00:10:46,490 --> 00:10:44,220
um uh first of all I want to mention the

283
00:10:49,310 --> 00:10:46,500
cell counts by microscopy which is uh

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00:10:51,710 --> 00:10:49,320
from Ben klempe uh showed that the

285
00:10:53,269 --> 00:10:51,720
number of active cells in these sites go

286
00:10:55,430 --> 00:10:53,279
down as you get to the super high

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magnesium concentration regions which is

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00:10:58,550 --> 00:10:56,339
also

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00:11:02,090 --> 00:10:58,560
um what Emily showed in her talk and so

290
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uh what we then can look at is the ATP

291
00:11:08,210 --> 00:11:04,560
concentration which strangely goes up

292
00:11:10,250 --> 00:11:08,220
even as the concentration of active

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00:11:12,110 --> 00:11:10,260
cells goes down and the expected

294
00:11:15,050 --> 00:11:12,120
activity goes down and so we wondered

295
00:11:17,449 --> 00:11:15,060
why is that happening because

296
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um as we know the amino acids are

297
00:11:20,509 --> 00:11:18,899
concentrating but amino acids don't

298
00:11:22,790 --> 00:11:20,519
Decay on really short time scales

299
00:11:26,329 --> 00:11:22,800
they're not broken up by hydrolysis or

300
00:11:28,670 --> 00:11:26,339
by atpase enzymes but

301
00:11:30,470 --> 00:11:28,680
we think is that something must be

302
00:11:34,970 --> 00:11:30,480
driving this and if it's not evapo

303
00:11:36,949 --> 00:11:34,980
concentration uh well uh we're wondering

304
00:11:41,030 --> 00:11:36,959
you know since the ATP hydrolysis rate

305
00:11:43,790 --> 00:11:41,040
is uh much quicker than the evapo

306
00:11:45,350 --> 00:11:43,800
concentration rate uh why would the ATP

307
00:11:47,210 --> 00:11:45,360
accumulate doesn't really make much

308
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sense but perhaps

309
00:11:51,829 --> 00:11:49,860
it's being preserved so this to ovala at

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all paper from 1987 that I mentioned way

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earlier uh they found that ATP was

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preserved in low water activity sites

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and so that could be preventing the

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enzymatic uh and natural hydrolysis of

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ATP leaving it to uh persist in the

316
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solution and additionally it could also

317
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be used as an osmolite compound as well

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and be accumulated by those

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microorganisms before they end up in a

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super high magnesium site and not very

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active themselves

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so in conclusion we can detect

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biosignatures at micromolar

324
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concentrations in near saturation brines

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using celaf and microce Ms

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and amino acid distribution ratios can

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be well classified based on their brine

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type

329
00:12:41,389 --> 00:12:39,600
and osmolites are present and detectable

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in some but not all of the South Bay

331
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saltworks in Western Australia Brines

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and we found preservation of ATP at

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00:12:51,290 --> 00:12:48,560
Southbay SolidWorks and so

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basically my argument is then why don't

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00:12:54,829 --> 00:12:53,100
we follow the salt and see if we can

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find biosignatures there on other worlds

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foreign

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we have time for two questions

339
00:13:21,769 --> 00:13:19,430
Chad thank you for the talk um that was

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00:13:23,569 --> 00:13:21,779
a beautiful separation that you showed

341
00:13:26,449 --> 00:13:23,579
uh showed earlier

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00:13:30,170 --> 00:13:26,459
um so I'm I'm curious uh you did a

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00:13:33,050 --> 00:13:30,180
connotation of amino acids using celf

344
00:13:38,110 --> 00:13:33,060
method and it looks like you've done

345
00:13:42,530 --> 00:13:38,120
some work using cems have you tried uh

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00:13:45,769 --> 00:13:42,540
quantifying the amino acid content in

347
00:13:47,629 --> 00:13:45,779
the same sites using microchip cems and

348
00:13:49,550 --> 00:13:47,639
seeing what those look like to try and

349
00:13:50,930 --> 00:13:49,560
like cross validate both methods and the

350
00:13:53,030 --> 00:13:50,940
using the same sample yet or have you

351
00:13:55,730 --> 00:13:53,040
not got to that yet nope so we just

352
00:13:58,310 --> 00:13:55,740
started doing the micro CMS work over

353
00:14:00,410 --> 00:13:58,320
the past month and so we're really happy

354
00:14:02,509 --> 00:14:00,420
how it's been doing with qualitative

355
00:14:04,430 --> 00:14:02,519
untargeted work but we haven't gone into

356
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quantitative because the ion suppression

357
00:14:08,210 --> 00:14:05,480
effects

358
00:14:09,710 --> 00:14:08,220
might be a bit of a challenge to

359
00:14:11,329 --> 00:14:09,720
overcome so that'll probably be a little

360
00:14:12,949 --> 00:14:11,339
bit of a longer project what we have

361
00:14:16,069 --> 00:14:12,959
done to try to corroborate the amino

362
00:14:18,410 --> 00:14:16,079
acid concentrations is look at the

363
00:14:20,629 --> 00:14:18,420
expression of um or not the expression

364
00:14:21,730 --> 00:14:20,639
rather but the presence of certain

365
00:14:25,910 --> 00:14:21,740
proteins

366
00:14:28,190 --> 00:14:25,920
in the genetic code of the microbes that

367
00:14:32,150 --> 00:14:28,200
are were found to be present here by

368
00:14:32,750 --> 00:14:32,160
other uh host collaborators and

369
00:14:35,090 --> 00:14:32,760
um

370
00:14:36,769 --> 00:14:35,100
we find a moderate correlation between

371
00:14:39,290 --> 00:14:36,779
the amino acids we see and the amino

372
00:14:40,730 --> 00:14:39,300
acids that they see but since we don't

373
00:14:42,170 --> 00:14:40,740
have transcriptomes from all of these

374
00:14:44,210 --> 00:14:42,180
sites we don't know exactly what

375
00:14:46,550 --> 00:14:44,220
proteins are being made and so I think

376
00:14:48,650 --> 00:14:46,560
that's the next step is is to connect

377
00:14:50,509 --> 00:14:48,660
those from metabolomics to

378
00:14:53,750 --> 00:14:50,519
transcriptomics to genomics just as the

379
00:14:59,269 --> 00:14:53,760
talk last night uh was uh was discussing

380
00:14:59,279 --> 00:15:10,250
any last question for Chad

381
00:15:14,150 --> 00:15:12,050
um I have a two question actually oh

382
00:15:17,269 --> 00:15:14,160
maybe I didn't really catch you what's a

383
00:15:19,129 --> 00:15:17,279
pH of the site that you show the

384
00:15:21,230 --> 00:15:19,139
Magnesium concentration is more than

385
00:15:22,550 --> 00:15:21,240
four molar that's a great question I

386
00:15:23,810 --> 00:15:22,560
don't know off the top of my head but I

387
00:15:25,189 --> 00:15:23,820
can get back to you in a moment yeah

388
00:15:27,710 --> 00:15:25,199
okay

389
00:15:29,470 --> 00:15:27,720
um and also how would you determine your

390
00:15:32,150 --> 00:15:29,480
atp's

391
00:15:34,730 --> 00:15:32,160
accumulated or protected under high

392
00:15:37,670 --> 00:15:34,740
selling water or like a low water

393
00:15:40,610 --> 00:15:37,680
activity how did I sorry say again how

394
00:15:44,210 --> 00:15:40,620
did I how do you know that your ATP is

395
00:15:47,210 --> 00:15:44,220
not hydrolysis so yeah I don't know that

396
00:15:49,550 --> 00:15:47,220
it's not hydrolyzed necessarily but I am

397
00:15:50,870 --> 00:15:49,560
predicting that so the the typical

398
00:15:53,329 --> 00:15:50,880
destruction

399
00:15:54,590 --> 00:15:53,339
um method of ATP uh either in the cell

400
00:15:57,290 --> 00:15:54,600
or in the environment is through

401
00:15:59,230 --> 00:15:57,300
hydrolysis either performed by proteins

402
00:16:01,850 --> 00:15:59,240
or just

403
00:16:04,430 --> 00:16:01,860
happening naturally in the environment

404
00:16:07,550 --> 00:16:04,440
with water and so those are the those

405
00:16:10,129 --> 00:16:07,560
are the things that must uh from what

406
00:16:13,490 --> 00:16:10,139
I'm observing have been stopped

407
00:16:16,009 --> 00:16:13,500
if the ATP is going to persist so long

408
00:16:17,389 --> 00:16:16,019
for it to be evapo concentrated to those

409
00:16:19,790 --> 00:16:17,399
high of levels

410
00:16:22,069 --> 00:16:19,800
um the huge Spike there likely wouldn't

411
00:16:25,129 --> 00:16:22,079
have been possible if uh of Apple

412
00:16:28,250 --> 00:16:25,139
concentration weren't a component and so

413
00:16:31,250 --> 00:16:28,260
um you know unless uh the uh the

414
00:16:33,410 --> 00:16:31,260
osmolite effect is is uh so much more

415
00:16:35,210 --> 00:16:33,420
significant than I had thought

416
00:16:37,970 --> 00:16:35,220
um that could be another reason but I

417
00:16:41,269 --> 00:16:37,980
still think that uh the the preservation

418
00:16:44,629 --> 00:16:41,279
of ATP is yeah is linked to the the lack

419
00:16:47,749 --> 00:16:44,639
of a um a rapid breakdown mechanism uh

420
00:16:48,670 --> 00:16:47,759
have you ever considered that ATP with

421
00:16:51,530 --> 00:16:48,680
um

422
00:16:53,629 --> 00:16:51,540
form some nanostructures to make the

423
00:16:56,030 --> 00:16:53,639
things happen yeah so whether it

424
00:16:59,150 --> 00:16:56,040
stabilizes it in some way the yeah so

425
00:17:00,710 --> 00:16:59,160
I've I don't honestly know much about

426
00:17:02,389 --> 00:17:00,720
how it would investigate that I've

427
00:17:04,549 --> 00:17:02,399
spoken to someone recently about um

428
00:17:08,329 --> 00:17:04,559
potentially modeling it uh using

429
00:17:10,669 --> 00:17:08,339
molecular Dynamics but uh I haven't you

430
00:17:12,350 --> 00:17:10,679
know waded into the um you know the

431
00:17:14,569 --> 00:17:12,360
actual uh

432
00:17:15,770 --> 00:17:14,579
the structural chemistry of whether of

433
00:17:17,809 --> 00:17:15,780
whether that would happen but if you

434
00:17:20,210 --> 00:17:17,819
have any ideas I'd love to hear so