Category Archives: D2O Effects on Life
Defense Outline
Just over a week away now…
- Introduction
- What is D2O?
- The history of D2O
- Gilbert Lewis:
- purification
- biological effects
- The hypothesis
- Joseph Katz
- various experiments
- Gilbert Lewis:
- Uses of D2O
- NMR, mass spec
- The need for a D2O adapted organism
- Experiments in DDW
- use for space travel
- cure for cancer?
- The effects on life
- Tobacco Seeds
- The Crumley experiment and repeating the experiment
- Tobacco seed germination rate
- tobacco seed growth rate in low deuterium concentration
- Arabidopsis
- arabidopsis growth rate
- arabidopsis morphology
- E. coli
- growth rates
- adaptation and adapted growth
- morphology
- Yeast
- growth rates
- adaptation – can’t adapt
- morphology
- stall during cell division
- microtubule stabilization in D2O
- Tobacco Seeds
- Molecular effects
- Stabilization of biomacromolecules
- DLS experiments
- Catalase
- Ovalbumin
- YPD longevity
- DLS experiments
- Investigation of HD exchange
- mechanism and exploitation for protein struture studies
- FT-IR analysis
- Cavity ring-down analysis
- low cost measurement of local atmosphere isotopic composition
- Effect on DNA
- The pursuit of shotgun DNA mapping
- optical tweezers
- methods
- overstretching data
- Stabilization of biomacromolecules
- Future Work
- Arabidopsis
- adaptation
- seed growth in low deuterium
- Tobacco growth in low D2O
- Yeast morphology in taxol
- E coli protein expression in D2O and protein structure analysis
- DNA
- overstretching in D2O with intercalators
- Arabidopsis
Well there is my idea of how to present my dissertation. I’m not sure if/where I should put my discussion on open notebook science. Also there are a couple things that I could see going elsewhere. I could describe the yeast and e. coli stuff in parallel instead of one after another. Also the HD exchange stuff could easily go right after the yeast, e. coli, or even the tobacco seed stuff. What to do…
Otherwise I think the story is pretty compelling: history of D2O and the unanswered question by Lewis. Investigations into D2O effects and trying to understand low D2O concentration effects, effects on macromolecules, and the understanding of large volume/long-term HD exchange.
Any feedback you may have would be GREATLY appreciated. I’ll send you a figshare t-shirt, or if you are XL, I’ll send you a hoodie (but I only have one).
Tobacco Seed Growth Rates
I got some awesome new data to show. The first is the compilation of all the Repeating Crumley experiments. And the second is some new data that I’ve been meaning to create and now have with the help of Koch.
The data above is the compilation of all the RC data. Each trial had different water types, but I combined the samples that were the same in every set (DDW, DI water, 33% D2O, 66% D2O, and 99% D2O). Steve adapted his R-code that applies binomial confidence intervals to a data set and used it on this data. If that makes no sense, then just know that the dotted lines are the most probable range of germination rates. For instance, in 66% D2O there is a ~70% likelihood that seeds will germinate at a rate within the dotted yellow lines.
Now it’s time for some brand new data:
Here we went through the pictures from Trial 5 and compared the growth rates of the roots. We calculated the lengths of various seeds in each image and tracked the changes from image to image. We chose DI, DDW, and 1% D2O, because the D2O concentrations are relatively similar and because we wanted to test a hypothesis from a while ago. It’s interesting that the seeds in DDW and D2O grow at the same rate, while seeds in DI water grow at roughly half the speed.
Last Arabidopsis Update
The media is dwindling and the plants will die shortly. I’ll be restarting this experiment in larger environments with more media. Hopefully the plants can do better.
Yesterday Koch noticed that the roots in 10% D2O were much longer than those of the plants in DDW. Something interesting.
Arabidopsis Update (From March 6, 2013)
I took these images several days ago and again forgot to post them. Stupid dissertation…
Anywho, here they are. I’ll try and remember to update tomorrow when I take the images. Tomorrow will be the last day I update this experiment. I’ll be going into full dissertation mode and will be starting a new experiment when I return. I bought some larger test tubes (1in wide), which should give the plants all the room they need and should be large enough to provide more media to keep the plants alive for longer.
Arabidopsis update
This will probably be the last update. I’m going to (1) need to take a break until after I defend, and (2) need to start a new experiment because the plants are running low on media. I did just buy these awesome 1in diameter test tubes which should give the plants all the media and water they could need for a longer period of time. Anyways let’s go to the pictures:
Arabidopsis Update
Here is the update for my arabidopsis growth. It still seems to me that the plants in 10% D2O are doing better than the plants in DDW. I’ll have to make a huge purchase of D2O and DDW (still don’t understand why DDW is more expensive than D2O since one is a by product of the other) and try lower concentrations of deuterium (like 0-10% amounts).
Notice that the seeds in 99% D2O germinated (mostly likely due to D-exchange), but haven’t grown in 3 weeks.
I wanted to take images of the root growth, but my camera couldn’t see clearly enough in the agar to take decent pictures.
YPD aggregation study
Two summers ago Kenji Doering (an REU student that summer) and I did some protein aggregation studies comparing D2O and DI water. The results were pretty consistent in that proteins don’t aggregate under the same conditions in both samples. What that meant was the D2O is almost certainly better for longevity of chemicals.
A few months ago I noticed that my ypd stocks go noticeably bad after a couple of weeks. But they seemed to last longer in D2O, I just never quantified that. Well now I’m going to.
I have two cuvettes sitting on my bench. One is filled with 1ml of DI YPD and the other is 1ml of D2O YPD. They are sitting next to the benchtop cooler to supply a bit of heat to speed up the aggregation reaction. I will take daily pictures of the solutions to compare the aggregation times of each media. Here is the day=0 time point (on the left is the DI YPD sample, and the right is the 99% D2O sample):
Arabidopsis Update: From Feb 13, 2013 (6 days ago)
These pictures are a bit late, but better late than never. They are an update of the growth progress of arabidopsis in varying amounts of D2O. The group shot is inconsistent with the others because the sample captured for 0% D2O (DDW, deuterium depleted water) is different than the sample used in the individual 0% D2O image.
With that said, there are a couple morphological things I would like to point out. In my opinion the plants are growing better (bigger, faster, etc) in 10% D2O than they are in DDW. They also appear to be slightly more green. And finally there are little hairs on the leaves that seem to be more prominent than in the other samples.
Meanwhile, in 60% D2O the plants are very yellow in appearance. It is interesting that more plants sprouted in that sample, but they are significantly behind in development.
I’ll update tomorrow for the weekly update. And this time I’ll upload the images in real-time. Promise!
Looking into hydrogen-deuterium exchange
H-D exchange (or D-exchange as I’ve sometimes referred to it) has been a problem I’ve dealt with in the lab for some time. It is essentially something that I need to minimize but can never actually stop. It is also a process that I know almost nothing about other than it happens, it occurs somewhat instantaneously, but may be mediated by evaporation rates (when dealing with DDW and 99% D2O). Now I’m perusing the internet looking for some information. Come take a walk with me:
- At ScienceOnline 2013, I met a person who pointed me in the direction of a Dr. Richard Zare. She told me he was very knowledgable in the field and so I looked up his papers. He had five papers relevant to exchange reactions, all of which are way above my head. So I’ll start on Wikipedia and work my way up.
- For those unaware, hydrogen-deuterium exchange is a reaction where a covalently bonded hydrogen is replaced with a deuterium atom. In the case of my experiments this happen with water. If I have deuterium depleted water and I keep it in contact with the environment (which has deuterium in it at around 16mM), eventually it will reach equilibrium and the deuterium level (of the DDW) will rise. The mechanism that causes this, I presume, is D-exchange.
- Apparently the reaction is pH dependent. It can be quenched around pH 2.6, but seems to work best at pH 7.0-8.0. That’s really interesting to me. The pH levels that I’m normally working around are optimal for these reactions. Unfortunately there is nothing I can do about that.
- The reaction may also be quite slow. According to Wikipedia, exchange is slow/unlikely intramolecularly, but is quite rapid via exposed surface hydrogen bonds. For the purposes of NMR, in vivo deuterium incorporation would be valuable and is hard to attain by dissolving proteins in D2O.
- The first person to measure H-D exchange was Kaj Ulrik Linderstrøm-Lang, and I would read some of his stuff right now but they are locked down. The mechanism he used to study D-exchange was pretty interesting and involved a density gradient tube. If I read this correctly, KULL filled a long tube with oils of different desities. He could place a drop of water, with a small amount of proteins in the drop, into the oils to determine the density. As reactions occur, the density would change and the drop would move accordingly.
Ok I’ve reached the limit of what I can do tonight without using the UNM network for access to papers. I’ll try to look for more information this weekend. The quest for a basic understanding of H-D exchange continues…