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mitochondrial uncoupling

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#1 caliban

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Posted 14 August 2010 - 07:12 PM


Barry Halliwell is Tan Chin Tuan Centennial Professor at the National University of Singapore.

Dr. Jan Gruber is a senior postdoc in Professor Haliwell's group and scientist who will steer this research project. Find attached a recent CV. Jan heads a group of young researchers specialising in aging research.

Attached File  image012.jpg   31.5KB   6 downloads, Attached File  Bimage001 (4).jpg   15.11KB   6 downloads

You are invited to post questions to Dr. Gruber below. First and foremost, we want Jan and his colleagues to get on with their important research, so please understand if it may take some time to address your questions.

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#2 caliban

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Posted 16 August 2010 - 09:12 PM

Alright, let me kick things off:

Hi Jan. Welcome to ImmInst.

The study uses protonophore 2,4-dinitrophenol (DNP) to 'treat' the worms. As DNP has already been shown to decrease ROS production, and decrease DNA damage, resulting in increased lifespan of mice - what is the reason for repeating these studies in nematodes?

#3 AgeVivo

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Posted 16 August 2010 - 09:55 PM

Hi Jan, concerning mitochondrial uncoupling extending mouse lifespan,
In mid 2008 Michael Rae posted a message that was not much in support of DNP. Do you think things have evolved since? :
http://mfoundation.o...post.php?p=3028

this is just 'the usual nonsense': ALL the animals were short-lived, and those that got DNP lived a little longer, but still not nearly as long as a normal, healthy, well-husbanded, non-genetically-messed-up mouse (which will on av'g live ~900 days) (2). In this study, "Median longevity in the control group was 722 days, versus 771 days in the DNP group, while mean lifespan was 718.8 days in the control and 769.7 days in the treated group. Although the change in life span promoted by DNP was not large, it is statistically significant (p = 0.038). " . And there was no extension of maximum LS at all, even compared to the short-lived controls; extension of max LS is the very essence of the CR life extension effect, which is exactly what marks it as the sole genuine anti-aging intervention validated in mammals to date)

The human analogy would be a study in which you took a bunch of people , put half of them on a dangerous drug for their entire lives, and at the end of the study the average control lived just 67 years (rather than 82, you'd expect from a random sample of the USA) -- but look! People taking the drug lived to be 70! It's a miracle anti-aging drug!!

What happened? I note that "Food intake was also equal in both groups (Fig. 1c), while DNP-treated animals presented significantly lower body mass (Fig. 1d)"; I'm going to GUESS that they just let the mice eat themselves into obesity, and DNP partially corrected for the ensuing metabolic mayhem.

The conclusion is not that DNP is an anti-aging drug; the conclusion is tht DNP can help some sick mice, and these folks need to learn to take better care of them.

Thank you.

#4 Jan Gruber

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Posted 17 August 2010 - 10:13 AM

Hi AgeVivo, Caliban,

Thanks for pointing me to this post – I had not seen it.

No, I do not think anything has changed much since that post in 2008 and I do agree with much of what Mr. Rae has said – although not with all of it (but when do scientists ever agree totally, see below).

Some points regarding the assessment of the 2008 paper by da Silva et al.:

1) I am not sure that there is strong evidence that the mice used in the paper are unreasonably and worryingly short lived.

The citation given in support of the notion that this is the case clearly illustrates that different mouse strains have very different expected longevities.

It does not, however, give data specifically on the female Swiss Webster outbred albino mice used by da Silva et al. So the question is, what is the “normal” mean and maximum lifespan of this specific type of WT mice.

I do not work in mouse lifespan studies myself and I do not know the normal lifespan for the specific strain used in the paper (if anybody has hard data on this issue, it would be useful indeed). However, the paper by da Silva cites one other publication [1], which seems to support their assertion that the lifespan observed is pretty normal for females of this type of outbred mice.

Furthermore, the longest surviving mice in the study lived to about 140 weeks (980 days or almost 2.7 years). Compared to other published studies on mouse lifespan, 140 weeks does not strike me as suspiciously short lived – unless somebody has a reference regarding this particular type of mice that says otherwise.

The method section of the da Silva paper also indicates careful animal care protocols including specific pathogen free conditions.

2) Maximum lifespan is commonly measured as the average of the longest surviving 10% of a cohort. By this measure, there clearly is at last a trend to increased maxLSP with DNP treatment (Figure 4 in da Silva et al.). Whether this is statistically significant is not clear but this is probably at least partially a question of n-numbers (30 animals in each group – so the top 10% are only 3 individuals). Mouse lifespan studies are expensive and n-numbers are often regrettably small (one of the reasons to consider working with simple model organisms, IMHO).

I guess my point here is that the judgment that this work is in some way “nonsense” is maybe not well supported by evidence in the original post. In fact, I think it is quite a good paper on this approach, although I agree that they may be overstating the CR case a little in the discussion.

3) I also fully agree that there is little clear evidence that DNP has slowed ageing or mimicked CR in these animals. Showing effect on ageing would require not just clear extension of maximum lifespan but ideally evidence for a higher mortality doubling times (lower ageing rate). Assessing mortality patterns is very difficult (expensive) to do in mice because of the large number of animals that would be required. This is, however, one thing that is fairly easy to do in nematodes where 100s or even 1000s of animals can be used relatively easily (and cheaply).

4) I do further agree that there are many open questions regarding “what happened”, in particular regarding the mechanism of action of DNP in these mice. A reduction in body fat certainly is one possibility here. Also, it is interesting to note that DNP affects the brain in particular and that it is here that the markers of oxidative damage are most changed. This is interesting but also represents a challenge, as different organs are affected differently.

5) Most of all, I emphatically agree that DNP is a dangerous drug and should not be viewed as lifespan extension medication for humans.



Does this mean that there is no reason to carry out the work we have proposed ?

Clearly, I do not think so - our proposal is very explicitly a basic science proposal and we consider DNP an interesting proof-of-principle compound (laboratory reagent) for testing a specific “metabolic tuning” approach in the context of the mitochondrial free radical theory of ageing (mFRTA). This work is also intended to test aspects of the mFRTA itself (which has been much debated and challenged recently).

The fact that DNP has already been shown to extend lifespan in mice just makes it even more interesting to figure out how it might (or might not) work.

Unlike da Silva et al. we have the luxury of doing in vivo ROS dose-response curves first, in order to get an ideal dose to work with. We can also carry out studies big enough to evaluate maximum lifespan and maybe even mortality changes. We routinely observe parameters of mitochondrial function and damage and we will have to worry less about complex pharmacokinetics effect.

If we can use DNP to significantly reduce oxidative damage to mitochondria (without other obvious detrimental changes) and this does NOT lead to maximum lifespan extension / slower ageing rate – that, in my view, would be a serious challenge to the free radical theory of ageing. If on the other hand chemical uncoupling can be used to reduce damage and preserve function (in this simple model organism), it would lend support to this avenues of research and might ultimately lead to compounds much safer than DNP. Again, the fact that there is already evidence that this approach has at least some beneficial effects, even in higher organisms, is actually very encouraging.

To summarize, I think the paper by da Silva et al. has illustrated that mild chemical uncoupling might be an interesting strategy for modulating lifespan. I do not think that there are problems with their work so severe to suggest that there is nothing further to explore. However, I fully agree that many important questions remain unanswered, which is why I wrote the proposal in the first place 8)

I hope this goes some way towards answering both AgeVivo’s and Caliban’s concerns / comments – let me know if it does not ;-)


[1] Guayerbas N, De La Fuente M (2003) An impairment of phagocytic function is linked to a shorter life span in two strains of prematurely aging mice. Dev. Comp. Immunol. 27, 339–350.

Edited by Jan Gruber, 17 August 2010 - 05:17 PM.

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#5 caliban

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Posted 22 August 2010 - 08:50 PM

Mind has agreed to schedule a show with Dr. Gruber.
This should a a 15-20 minute broadcast. Audio unless we need to use slides.

Stay tuned for details.
If the show is live, people can ring in with questions, or you are invited to post any questions for Mind to put to Jan here.

#6 brokenportal

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Posted 25 August 2010 - 04:44 AM

The youtube has been granted the non profit status now. One of the additional functions this gives us is a tool for overlaying promotional things on our videos. We could advertise this fund, but since we are so close to the goal already we could probably plug this ones success, and the next fundraiser.

#7 AgeVivo

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Posted 25 August 2010 - 05:50 AM

show with Dr. Gruber

great!! when and at which url for example?

#8 Jan Gruber

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Posted 30 October 2010 - 02:27 AM

Hi - I was asked to give a quick update on the Project. Nominally, we are two month into it. Well, plans do not usually survive much beyond the point where they make contact with reality. We have had some of that...

It turns out that it takes more than 4 weeks for a US check to clear here. Since it took a little while to receive it in the first place, the short update is that the check has not yet cleared and we have not been able to officially hire the student (Mr. Fong Sheng) yet.

This does of course not mean that we have not started work, only that we had to be a little creative. Fong Sheng is currently volunteering and we made a "visitors" account to get him into the building and give him access to the library ect. The first thing he did was to update and extend the literature review from what we did back when we first played with this idea.

One thing we have found is a, hopefully, better method to directly measure membrane potential in vivo in nematode mitochondria. Membrane potential is what we aim to modulate using DNP, and we will try out this method to see if we can use it for our titration experiments. Luckily, Fong Sheng has found somebody who had previously used the same dye (meaning that we were able to borrow some and did not need to purchase any). He is now trying to make this assay work with some wild type, mutant and uncoupled worms. We will see....

More when we get the results from this !

Edited by Jan Gruber, 30 October 2010 - 02:29 AM.

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#9 Jan Gruber

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Posted 14 December 2010 - 04:56 AM

OK - time for another update:

First of all, it took quite a while for all the paperwork to go through. So, the check cleared and the student was officially hired only in the first week of December. As mentioned before, that does not mean that we have done nothing - but it does mean we got less done than we had anticipated.

To make up for the late start Fong Sheng agreed to extend the work period until June - so we will still have almost 6 months of full-time work - everything up to now then can be considered "bonus".

Ok - that being said, I have asked Fong Sheng (the Student hired on the project) to quickly summarize his work up to now. Apart from reading the literature and getting up to speed with the details of the project he has been working on a new method (new for our lab) that we hope will add another dimension to the data generated. Without further introduction - here is Fong Sheng's summary:

>>>
The mitochondrial free radical theory of aging (MFRTA) proposes that reactive oxygen species (ROS) and ROS associated damage to macromolecules are a major cause of aging (Harman 1956; 1972). During aerobic respiration, protons are pumped out of the mitochondrial matrix into the intermembrane space by respiratory chain complexes sited in the mitochondrial inner membrane. This generates a protonmotive force, composed of a pH gradient and membrane potential, that is utilized for ATP synthesis, and which positively correlates with ROS production in mitochondria. In the context of the MFRTA, the ‘uncoupling to survive’ hypothesis (Brand, 2000) suggests that the induction of mild proton leakage across the mitochondrial inner membrane, for example using chemical uncouplers such as 2,4-dinitrophenol (DNP) or carbonylcyanide-3-chloro-phenylhydrazone (CCCP), will slightly decrease the mitochondrial membrane potential and significantly reduce ROS production, and might even increase organismal lifespan. Due to this non-linear relationship between ROS and membrane potential, very accurate methods of measuring mitochondrial membrane potential are therefore needed.

While methods to measure ROS production and lifespan of Caenorhabditis elegans are routinely employed by our lab, a reliable method for measuring the mitochondrial membrane potential has not been established in our lab. Developing a method to accurately measure the membrane potential, and that is sensitive to small changes in the membrane potential, is difficult and intrinsically challenging. We are currently optimizing and validating a membrane potential method based on that of Gaskova et al. (2007). This method utilizes a cationic, lipophilic carbocyanine dye, 3,3’-dipropylthiodicarbocyanine (diS-C3(3)), that selectively accumulates in the mitochondria and whose fluorescence emission spectrum changes, both in a membrane potential-dependent manner. A major advantage of this method is that accurate quantification of the mitochondrial membrane potential need not depend on the peak intensity value of the fluorescence emission spectrum.

Using diS-C3(3), we found that membrane potential measurements can be performed in as few as approximately 300 worms in a 96-well microtiter plate using a microplate spectrofluorometer. To determine whether the assay could detect a change in the mitochondrial membrane potential, as assessed by a shift in the peak wavelength of the fluorescence emission spectrum, we used the chemical uncoupler CCCP as positive control. CCCP has been shown to decrease the mitochondrial membrane potential in C. elegans, measured using diS-C3(3) (Gaskova et al., 2007). As expected, we found that 4 µM CCCP caused a 3 nanometer shift in the peak wavelength, although subsequent experiments have yielded sometimes inconsistent results.

Our next experiment aims to assess the suitability of DNP as a positive control for this assay instead, using doses previously established in our DNP dose-response curve for ROS production.

<<<

We are going to start the lifespan and other cohort experiments only after the Christmas break - but, since we have until june, there will be plenty of time to do that. In the meantime we hope to get the membrane potential method to work reliably, so that we can use that to guide the more time consuming experiments rather than the other way around.

Jan
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#10 brokenportal

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Posted 23 December 2010 - 12:44 AM

Here is the interview, and thanks again to everybody who made this past mitochondrial uncoupling research project fundraiser a success. Imminst success=all of your success.





The mitochondrial uncoupling project was our 2nd, and we are now on to our 3rd research project fundraiser which is here.

#11 Jan Gruber

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Posted 29 March 2011 - 09:23 AM

OK - I have been asked to prepare another update. Since Fong Sheng is doing all the hard work, he has prepared most of the summary below. We are now busy with all the repeats and mechanism studies:

>>>

We have recently completed phase I of the research project and established an ideal 2,4-dinitrophenol (DNP) dose in Caenorhabditis elegans. In accordance with our original proposal, we first attempted to determine the ideal DNP dose by performing an in vivo reactive oxygen species (ROS) production assay, whereby whole worms in liquid media are exposed to DNP and generalized ROS production is measured using the fluorescent dye, dichlorofluorescein diacetate. We, however, soon encountered some problems suggesting different uptake of DNP by worms in liquid media vs. plates.

Generally, significant differences in lifespan, metabolism and related metabolic parameters between worms cultured in liquid media and on solid agar exist. While the reasons for these differences remain unclear, we were therefore worried that the optimized DNP dose might differ between worms grown in liquid media and on agar plates. Hence optimization of the ideal DNP dose using the DCF-DA assay, as originally proposed, was deemed unsuitable and was abandoned. Most of our phase II biomarker and lifespan assays are optimized for use with worms grown on solid agar and we therefore decided to do everything on plates.

Although more demanding in terms of time and material, the ideal DNP dose was established instead by screening for the effects of DNP treatment on modulating C. elegans lifespan over a wide range of DNP concentrations (11 concentrations ranging from zero up to 250 µM). Due to the large number of worms required for this lifespan screen, we employed the C. elegans temperature-sensitive mutant strain, glp-1 (JK1107), which is fertile at 15oC but sterile at 25oC. Following hypochlorite treatment to obtain synchronized worms, worms were incubated at the restrictive temperature of 25oC throughout the entire experiment. Untreated worms (control) were scored for survival daily to determine the number of survivors and this data expressed as the percentage survival (number of survivors / total number of worms in starting population x 100%).

The number of survivors for each of the 10 DNP treatment conditions was determined only on the days for which 50% (Day 10) and 25% (Day 14) of the total starting population of untreated control worms remained (50% and 75% death). The typical maximum lifespan of glp-1 worms at 25 oC is 21 days, and therefore, in this context, the survival data for our synchronized cohort of untreated control worms do not appear unusual. Using these data, we determined the relative changes in survival (at 50% and 25% of surviving control worms) between DNP treated and untreated worms.

With the exception of 250 µM DNP, we found a dose-dependent increase in worm survival with the largest increase occurring at 100 µM DNP (Figure 1). While 250 µM DNP produced similar lifespan extension effects as 50 µM DNP at day 10 (relative to the 50% survival of untreated worms), 250 µM DNP had detrimental, probably toxic, effects on worms later in life (Figure 1).

In our initial lifespan screen we found the most significant increase in survival for worms treated with 100 µM DNP and this is the concentration we have therefore chosen for mechanistic work.

We have since carried out additional lifespan experiments at the optimal dose of 100 µM DNP as part of phase II of the project. This lifespan data at the optimal dose of 100 µM DNP showed a similar extension in worm survival when compared to results from phase I. However, it is important to note that both of these test were in the glp-1 (JK1107) worms at the restrictive temperature of 25oC. We are now repeating this study using N2 WT worms at 20 oC.

In parallel, we also measured steady state adenosine triphosphate (ATP) and activity levels of worms treated with 100 µM DNP. Surprisingly, relative to untreated worms, DNP treated worms had significantly higher steady state ATP levels and were more active, suggesting that DNP treated worms had more energy than untreated worms. These observed effects of 100 µM DNP treatment on C. elegans metabolism are truly surprising, since mitochondrial uncoupling by DNP would be expected to lower ATP levels instead.

Thus far, we find that treatment with 100 µM DNP leads to the greatest increase in C. elegans survival, but surprisingly, unlike the predictions of mitochondrial uncoupling on metabolism, we find that 100 µM DNP treated worms have more energy than untreated worms. Our following series of experiments will attempt to elucidate the underlying mechanism responsible for the significant lifespan extension effects observed in worms treated with 100 µM DNP. As a first step towards answering the above question, we next intend to measure biomarkers of mitochondrial metabolism, as well as ROS production and oxidative damage in 100 µM DNP treated and untreated control worms. This work is currently underway and we will report on it, once the results have been finalized. Repeat measurements of ATP and activity levels in a second cohort of synchronized worms, as well as further lifespan studies, are also currently underway.

Finally, it is important to note that none of this work (or, for that matter, any review of the literature) suggests that taking DNP itself would be beneficial to lifespan in higher organisms – in particular in people (where there is clear evidence that it is in fact toxic). The elevation in ATP levels and activity for instance is totally unexpected, illustrating that DNP might act in ways very different to what standard theory might suggest – even in worms. It remains to be seen by what mechanism DNP affects lifespan in worms – this may yet turn out to be very surprising.

Given the delay with getting this project started, we expect Fong Sheng to work on this until the end of July. We hope to be able to complete our initial studies into possible mechanisms of lifespan extension by then. Oh- the work on the membrane potential method is kind of on hold again ... maybe if we have time at the end ...




Figure 1. Dose-Response evaluation of DNP by determining worm survival. For each treatment condition, the number of survivors was scored on the days when 50% (black) and 25% (white) of the total starting population of untreated worms remained.


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#12 Jan Gruber

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Posted 09 September 2011 - 06:57 AM

Dear All,

first of all, thank you very much for your support - this has been a useful and exciting if not entirely straight forward project (see below). Sorry for the delay with this report - with Fong Sheng off to medical school working on this has been slowed down.


As indicated in our last update, Fong Sheng has finished his summer attachment and left for his medical training – he will be attending DUKE-NUS graduate medical school. Although I hope that he may be willing to return for a research attachment on ageing at some point of this course, this means that this project is, for now, concluded.

Our preliminary results had indicated a promising lifespan extending effect of DNP in glp-1 mutants at the restrictive temperature of 25oC. However, these results were obtained using a screening approach optimised for high throughput.

The glp-1 strain at high temperature is deficient in progeny production and mutations affecting progeny can themselves affect lifespan. High temperatures also carry a higher risk of contamination. We therefore carried out two full lifespan studies (at 10 and 100 mM DNP) using large numbers (n > 100) of N2 wild type (WT) animals and manual transfer of adults to prevent overcrowding by progeny. While this approach is the most labour intensive, it is the preferred protocol for lifespan studies.

Unfortunately, under these more optimal conditions, we observed no lifespan extension by either concentration of DNP. In fact, we found that DNP at the higher concentration was significantly toxic to WT worms.

In parallel to the lifespan studies, we also determined levels of oxidative damage. We measured protein carbonyl levels in whole worm lysate of control (0 mM) as well as treated (10 and 100 mM) worms. Interestingly, we found that, at the lower concentration, DNP treatment significantly elevated oxidative damage. This result is unexpected and opposite to what would be expected from mild uncoupling. We therefore determined oxygen consumption and ATP levels in control and treated animals. Mild uncoupling would be expected to elevate oxygen consumption and maybe lower ATP levels. However, we observed neither elevated oxygen nor lowered ATP levels in either of the treated cultures. These data are consistent with the hypothesis that DNP at the levels tested most likely did not cause the intended mild uncoupling.

While the absence of evidence for uncoupling and lack of lifespan effects were disappointing, there are a number of interesting observations that may be of interest in the context of the free radical theory of ageing. One such observation is that lifespan in the lower treatment group is normal while levels of oxidative damage is significantly elevated.

Fong Sheng has presented these results (in the form of a poster) on an international conference on the biology of ageing:

“The 61st Annual Scientific Meeting of the British Society for Research on Ageing and 14th Congress of the International Association of Biomedical Gerontology: “The Science of Ageing – Global Progress, Brighton, UK”

The poster was well received and a manuscript of this work is also now in preparation. I will post detailed results and data once this manuscript has been published. I will update you on the progress of the manuscript and any additional work that we might be able to do on this project. However, given the results so far I doubt that DNP is the right chemical uncoupler to use in C. elegans.
As an aside, while Fong Sheng received a partial bursary for the conference, he used his own salary out of the ImmInst / Longecity grant to pay for his air ticket and his accommodation in the UK. While the lack of DNP efficacy in terms of lifespan extension is disappointing, the ImmInst / Longecity grant has enabled Fong Sheng to work for almost 6 months on this ageing project and to attend the UK conference. We are hopeful that this work will be published. If it is we will of course credit ImmInst / Longecity as one of the funding agencies (as is customary). Finally, Fong Sheng has certainly gained experience that will be valuable for him, and the Biogerontology community as a whole.

Compared to more traditional funding mechanisms, this is exceptionally cost effective in terms of return on investment. It is in the nature of scientific experiments that they do not always work out the way expected. Nevertheless, I think this project has shown that a relatively small investment can allow for significant synergy if it is flexible and comes at the right time. While the results might be disappointing, I therefore would like to suggest that the funding drive itself was a full success.

Attached:

Picture 1: Getting ready for the large lifespan study. Fong Sheng is pointing at his large pile of NGM culture plates – each stack (orange topped packs) contains about 20 NGM culture plates.

Picture 2: Arrival in Brighton for: “The Science of Ageing – Global Progress” and gratuitous conference group photo at Brighton pier.

Picture 3: Conference in Brighton, UK. Fong Sheng is presenting his poster on DCA, which was widely discussed and received well.


Attached File  Picture1.jpg   769.99KB   10 downloadsAttached File  Picture2.JPG   425.12KB   18 downloadsAttached File  Picture3.JPG   324.87KB   16 downloads

Edited by Jan Gruber, 09 September 2011 - 07:01 AM.

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#13 Jan Gruber

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Posted 12 October 2011 - 02:50 PM

DNP concentrations should be uM not mM in the above (1000x less) ... sorry about the error but I seem to be unable to edit the post.
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#14 Multivitz

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Posted 19 December 2015 - 12:54 AM

Where's Silicas role in all this?





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