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[Music]

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[Applause]

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I am ready my name is Hannah Dawson and

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I'm a graduate student at University of

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Washington in the School of Oceanography

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and today I want to talk to you about

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using metabolomics as a method to probe

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the physiological adaptations of sea ice

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algae so just to orient us to this

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extreme environment this is a video

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showing you the sea ice extent in the

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Arctic over one season so sea ice is an

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extreme and dynamic environment here on

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earth with four to six percent of the

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global ocean area being covered by sea

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ice depending on the time of the year

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and despite being a relatively harsh

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environment a number of organisms have

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been able to exploit this habitat and

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one of these organisms are sea ice algae

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which are single-celled eukaryotic

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photosynthetic organisms that can live

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within the liquid brine channels formed

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in sea ice surrounded by a freshwater

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ice matrix so here I'm just showing you

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an example of sea ice diatoms inside of

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a brine channel but these organisms

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contend with a number of challenging

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features in this environment and

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temperature is one of the largest

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challenges in this environment where

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throughout the sea ice column

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temperature varies greatly on a spatial

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and seasonal scale so temperature at the

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top of the ice column is much colder and

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in contact with the atmosphere while

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temperatures at the bottom are much

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warmer and closer to sea water

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conditions and like I mentioned there's

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a liquid brine volume left behind when

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seawater freezes and the fat volume also

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scales with temperature and lastly

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salinity varies throughout the ice

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column also with temperature with

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salinities of that Bryan being very high

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at the top of the ice where it's very

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cold and closer to seawater salinities

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towards the bottom of the ice and this

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is also a variable environment on a

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seasonal scale with the melting of sea

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ice in the spring and reformation in the

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autumn and winter bringing along large

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swings and temperature and salinity

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reaching near freshwater conditions in

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melt season and conditions temperatures

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above zero but despite this sea ice

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algae have been able to survive and

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thrive in this environment and here I'm

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showing you a video of what it looks

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like underneath the sea ice in the

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Arctic in Nootka Davich Alaska and here

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stuck a gopro down through a core hole

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in the ice and I think this is a really

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cool video it just gives us a glimpse at

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this otherworldly environment where

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these organisms are you can see a brown

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layer on the bottom of that ice and

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those are all sea ice algae mostly

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dominated by diatoms and in this

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environment they can reach these high

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abundances like we're seeing here high

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rates of primary production and serve as

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an important food source for a number of

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organisms and play largely into

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biogeochemical cycling and climate

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active gas production in this

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environment but we still don't have a

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very good understanding of how they're

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adapted to do this so I'm interested in

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how sea ice algae are physiologically

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adapted to survive and thrive in the

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extreme and variable conditions found in

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sea ice and how we can probe those

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adaptations so one way we can do this is

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by better understanding the metabolic

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reactions going on inside of the cell or

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the suite of biochemical reactions that

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allow a cell to be alive and this is

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just a glimpse at how complicated these

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systems can be these are all the

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metabolic pathways in a and example

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diatoms cell where each line in this

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diagram is a metabolic reaction mediated

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by enzymes and along each of these lines

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there are a lot of precursors

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intermediates and products of these

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reactions which I'll refer to as

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metabolites and one way that we can get

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a better idea of what in tablets are

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inside of a cell how they're regulated

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is by using a method called metabolomics

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so metabolomics we take this entire

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intracellular pool of metabolites inside

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of a cell extract them and then use a

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tandem liquid chromatography mass

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spectrometry approach in order to get

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the entire pool of detectable

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metabolites and in this each compound is

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represented by a peak on the mass spec

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and the area underneath of that peak is

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correlated with the relative abundance

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of that compound and to give you an idea

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of scale of how many compounds we can

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look at simultaneously with this method

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we end up with in my sample type around

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19,000 mass features or Peaks which

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could be potential compounds but to try

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to identify but another approach is to

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look at a pared down list of compounds

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that were interested in going into a

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study and I'm going to

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to those as targeted metabolites and

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again for scale this is around

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eighty-eight compounds in my sample type

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and specifically today I'm going to be

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talking about a group of compounds

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referred to as compatible solutes and

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I'm interested here in how altering

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temperature and salinity conditions

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changed the compatible solute pools and

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sea ice algae and I'm interested in this

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because compatible solutes are these

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small organic molecules that can be

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maintained at very high intracellular

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concentrations serve a number of roles

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inside a wide variety of cells and two

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of the roles that can serve as

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mitigating salinity and temperature

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stress and they can respond very rapidly

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to environmental change so if we start

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with an algal cell out in the

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environment in a marine environment we

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would expect them to have a baseline

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concentration of compatible solutes in

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order to mitigate osmotic stress between

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the interior of the cell and their

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saline environment surrounding them but

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if they're in colder or saltier

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conditions like in sea ice brines we

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would expect them to have higher

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concentrations of these compatible

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solutes to prevent water loss our

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freezing and if they're introduced into

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warmer and fresher conditions algal

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cells can rapidly dump these compatible

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solutes out into the surrounding

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environment and once they're in the

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environment they can be taken up and

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used as compatible solutes again by

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other organisms used as a carbon

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nitrogen or an energy source by

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heterotrophic bacteria in the sea ice or

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serve as climate active gas precursors

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and here are just three examples of

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compatible solutes that have been found

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to be abundant in sea ice algae in

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particular including DM SP the climate

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active gas precursor glycine betaine and

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prolene so to get at this question of

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how CSL G used compatible solutes in

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response to changes in temperature and

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salinity

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we used a lab study of the Antarctic sea

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ice diatom mitchell Laconte grown at two

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different temperatures and two different

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salinities minus 1 and plus 4 degrees

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Celsius and solemnities of 32 and 41 and

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then compared this to environmental

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samples collected in the Arctic in new

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geographic in a mixed sea ice community

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dominated by a niches species and this

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was at similar conditions around minus 1

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degrees Celsius and a salinity

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32 and first off we could see from

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looking at the general physiology of the

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cells growing these different treatments

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there were relatively small changes in

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something like growth rates so we saw

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less than 10% changes in growth rate

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which is in stark contrast to a lot of

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studies of compatible solutes that use

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large shock treatments with bigger

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temperature and salinity changes where

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growth often is reduced or stopped so

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we're assuming that these cells are

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generally well adapted to the conditions

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we grew them under but we still saw

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large changes in metabolite abundances

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including those compatible solutes we're

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interested in so just to give you a

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broad idea of the power of this

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metabolomics method we wanted to first

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look at a suite of potential compatible

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solutes that we've seen evidence in

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other organisms that they could be

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compatible solutes and here this is

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again just this matrix of cold vs warm

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and salty versus fresh and when we

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looked at this potential broad suite we

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saw a number of reactions we would

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expect compatible solutes to fall mainly

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up here higher in abundance in the cold

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and salty treatment but we saw some

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respond that way and some respond only

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in higher abundance in just the cold

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treatment or just the salty treatment

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another group that responded either

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oppositely or had mixed reactions to

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what we'd expect for compatible solute

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action and another group that were not

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significantly changed in response to any

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of the temperature or salinity

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treatments we used here so we're seeing

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a really broad reaction to these

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conditions and today I'm just going to

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talk a little bit more about a few of

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these compounds in particular so those 3

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compatible solutes I mentioned that are

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common in ice algae in particular here

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I'm showing you prolene

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DM SP and attained for prolene we saw

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that even though we were getting those

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less than 10 percent growth rate changes

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really large magnitude changes in the

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concentration of prolene both in

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response to cold temperature and in

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response to higher salinity with an

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additive effect up to a four-fold

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increase in pearling concentrations and

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reaching high concentrations around 50

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milli molar and here I have this as

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normalized peak area which is like

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relative abundance but we do have

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estimates for absolute concentration

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well which is that fifty mili molar and

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then if we look at DMS P again we're

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looking at the normalized peak area

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against the salinities and the two

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different temperature treatments minus

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one and plus for DMS P increased in

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response to cold temperature and higher

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salinity around twofold but did not show

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the additive effect we saw four prolene

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and we don't have absolute

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concentrations for DMS P using this

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method but we can assume from previous

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work they're similarly high and for

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glycine betaine there are at pretty high

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concentration still between 27 and 60 1

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millimolar but showed a more complicated

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response than what we're seeing for

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these other candidates with a general

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increase in response to higher salinity

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but a more complicated temperature story

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and one of the cool things that this

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method can do is not only look at those

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compatible solutes we already know

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exists and important in ice algal cells

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but to find new potential candidates

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that is compatible solutes and one of

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these that came out of the study was d

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HPS which is dihydroxy propane sulfonate

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and this is a relatively newly studied

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metabolite in the oceans in general and

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we don't have a great idea what it's

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doing in any ocean environment let alone

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in sea ice but it has shown potential as

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a compatible solute and we are seeing

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similar results here where a thps was

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increased around twofold in response to

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cold temperature and to higher salinity

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but again not the additive effect we saw

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with prolene but reaching really high

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intracellular concentrations up to 91

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milli molar and just to give you an

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example to ground that in a temperate

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diatom species celesia cyrus sudan ana

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DHS has only found around seven

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millimolar this is potentially a really

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potent compatible solute in the sea ice

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environment in particular and the next

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thing so CH the CI sarà Tom Mitchell

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Laconte maintains and regulates a

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diverse suite of compatible solutes with

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variable sensitivities to temperature

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and salinity even when growth is not

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large virtually change but the next

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thing we wanted to look at is if the

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metabolize diatom culture was similar to

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that of a mixed he ate sea ice community

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and what's actually going on in the

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environment so here I'm just showing you

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the overall number of detected targeted

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metabolites from that

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smaller list I mentioned earlier and

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this is for the field samples which were

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a mixed community but dominated by a

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niche Agenor as well nature for Geetha

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and comparing that to our niche Allah

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can take culture and we conceded the

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majority of metabolites in this targeted

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pool were shared but there were unique

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compounds found in both sample types

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with more unique compounds found in the

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field and the next thing I wanted to

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look at is how the concentrations of the

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compatible solutes in particular compare

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between these sample types and here I'm

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just showing you for a select number of

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compatible solutes the absolute

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concentrations for both the field and

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the culture and the first thing I want

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to point out is the difference in y axis

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where the right axis in green is the

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millimoles of metabolites per mole

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carbon in the culture and the right in

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blue is in the field and you might

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notice that there is about a tenfold

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difference in concentration between

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these two sample types with

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concentrations being higher in the

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culture which we would expect and

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comparing a pure exponentially growing

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culture to a mixed field community where

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all the organic matter might not

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necessarily be living and growing but

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overall we saw that DHBs was the most

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abundant of the compatible solutes we

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quantified in both sample types with

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beteen and prolene scaling down in a

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similar pattern from that but there were

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also a number of compatible solute

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candidates that were in relatively high

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concentrations in the field like home

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marine and ECT onic acid that were not

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found in high levels within our culture

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samples and another group of compatible

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solute candidates that were strictly

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unique to the field not detected at all

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in our culture's but at relatively low

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concentrations hinting at some

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interesting possible species specific

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differences in compatible solute use and

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just to give you an idea of the scale of

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the concentrations of these compounds

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out in the environment and their

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potential to impact the environment I

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want to remind you that these are highly

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mobile compounds that can be rapidly

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dumped from cells such as during the

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spring melt when they're introduced into

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those warmer and fresher conditions and

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they can dump up to 80% of their

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compatible sawyou

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inventory in less than an hour so if we

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just take the nitrogen containing

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compounds I have quantified here and

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pull them together this is equivalent to

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around

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point two micro molar concentration of

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organic nitrogen that's capable of

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rapidly flux into the surrounding

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environment and this is on a comparable

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scale to the inorganic nature

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concentrations in Batam sea ice during

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the spring which are around 0.5 to 10

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micromolar nitrate so this is a

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potential large source of nitrogen for

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this environment so to wrap up I want to

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conclude that metabolomic is a powerful

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tool that can detect a wide range of

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intracellular metabolites simultaneously

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in microbial organisms and quantify

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their response to varying environmental

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conditions here we saw that CAS algae

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maintained and tightly regulated complex

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suite of compatible solutes with

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variable sensitivities to temperature

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and salinity and to follow up on this we

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want to do continued work on

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environmental metabolomics of sea ice

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and compatible solute use in sea ice and

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we're doing that with an NSF funded

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project studying the spring melt season

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in Antarctica and seeing how that can be

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the sea ice community response to melt

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conditions so I'd like to thank everyone

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who is involved in this project everyone

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who helped generate the data it's

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particularly the young angles and Deming

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labs at u-dub and I'll take any

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[Music]

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thank you very much Hannah we have time

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for one question so there's a microphone

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up front and center please identify

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yourself when you ask your question

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state your name and institution anybody

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00:15:11,100 --> 00:15:08,490
have any questions for Hannah thank you

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hi my name is joy I'm at Carnegie

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00:15:15,420 --> 00:15:13,209
Institute in Washington DC really great

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thank you for sharing your work um so

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you talked about the eat flux of these

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metabolites containing nitrogen is there

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a way for you to understand the

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partitioning of extracellular versus

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intracellular metabolites with this

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00:15:30,480 --> 00:15:28,000
method currently it's a little difficult

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to look at the dissolved pool of

419
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compatible solutes but that is something

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that the Ingalls lab is currently

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working on adjusting and that's

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something that hopefully in the future

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we'll be able to quantify a little bit

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better but currently we're just looking

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at the