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Brainstorming for a potential c60 mouse study

c60 mouse longevity aging supplementation neurotrophic

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#1 kmoody

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Posted 21 April 2014 - 02:47 PM


Hello all, I would like your help in brainstorming a potential c60 mouse study.

 

It seems as though there has been a lot of interest in c60 as a potential lifespan extending agent, based in large part by Baati et all (2012, see attached). My company (www.ichortherapeutics.com) has a small SCID vivarium that is not yet filled to capacity, and we have been considering the possibility of taking on a small c60 (longevity?) study in mice.

 

I would like your input regarding what specific questions would be best to ask in such a study, whether it be strictly a longevity study, or model-based. For example, Baati et all suggest that C60 can cross the blood brain barrier. It would be interesting to see whether or not neuroprotection was conveyed in a neurodegenerative mouse model, though obviously a study of this type would be far more challenging (and expensive) than a basic longevity study.

 

Our laboratory is already equipped with a flow cytometer and CBC analyzer, and it would be very inexpensive to pick up a chemical analyzer. Observational (behavioral) data, mass, CBC, and a chemistry panel should give us a decent picture of each animal's overall health. What other metrics, if any, do you think would be important to include?

 

My team is willing to conduct this study at cost, but even so we will need to raise a minimum of $20,000 for the project to do it properly. Even then, we may need to raise more depending on what we eventually decide on for experimental parameters. How might we as a community raise such a sum? Do you think there is sufficient interest?

 

Let's hear those ideas!

 

Kelsey


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#2 Logic

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Posted 21 April 2014 - 05:20 PM

I have invited AgeVivo and Niner to this thread Kelsey.
I'm sure they will be very happy to hear of your idea and have plenty of suggestions


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#3 YOLF

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Posted 22 April 2014 - 10:44 PM

Sounds very interesting! This would be a great candidate for the Community Fundraisers. I'd be very excited to see this happen! I'd definitely be interested in helping with the video sales pitch for the fundraiser.


Edited by cryonicsculture, 22 April 2014 - 10:46 PM.

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#4 AgeVivo

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Posted 24 April 2014 - 01:38 AM

Dear Kmoody, does the vivarium allow to do a rat lifespan study? the best thing to do by far is to try to reproduce the life extension of the initial experiment


Edited by AgeVivo, 24 April 2014 - 01:38 AM.

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#5 PWAIN

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Posted 24 April 2014 - 02:11 AM

I agree with AgeVivo. Even a similar size study to what Baati did would be affirmation of his work.

 

If going beyond Baati, I think mid life to end of life administration of C60oo would also be of great interest.


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#6 niner

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Posted 24 April 2014 - 04:04 AM

Kelsey, are these all SCID mice?  If so, it seems like that would complicate the results.  I wouldn't anticipate that c60-oo would compensate for immunodeficiency.  It does appear to improve allergy/atopy, with altering of mast cell properties and/or shifting Th1-Th2 balance as possible mechanisms, based on published results with fullerene analogs. 

 

Would your lab by any chance be interested in doing some bench biochemical work on this?  It would be very interesting to look at the effect of c60-oo on respiration and mitochondrial parameters under both normal and hypoxic conditions.   Based on human experience with the compound, it looks very much like it is significantly improving OXPHOS efficiency.   It would also be interesting to look at redox status of the cell.

 

I'm going to break with the crowd and suggest that precise replication of Baati is not as important as simply showing significant life extension in a genetically normal mammal.   Replicating Baati in all details would be very difficult if not impossible, and would take a very long time.  I'd rather go with a shorter-lived species in order to get results sooner.  One area that represents a major hole in our knowledge is the effect of c60-oo on development.  It would be wonderful if we could run a reproductive tox study, and also look at the effects in animals ranging from neonates through sexual maturity. 

 

We have some suspicions that c60-oo assists in some way in stem cell differentiation, possibly by rescuing stalled or failed differentiations through a mitochodrial effect.  I understand that you guys have stem cell expertise, so perhaps you have some existing assays that you could deploy rapidly?  (we can but hope...) 

 

For in vitro work, you would probably want to use an ethyl oleate adduct instead of a triglyceride adduct.  My thinking here is that cells are likely to have generic esterases that would cleave it to the fatty acid, but I worry that triglycerides either wouldn't get in or the cell wouldn't have the appropriate lipase.  (I might be wrong on either count, though)  One of our members has already played around with the ethyl oleate synthesis, and it requires no modifications from Baati.  Ethyl oleate will form a more concentrated product than will olive oil. 


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#7 YOLF

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Posted 24 April 2014 - 06:45 AM

This says Ethyl oleate is rapidly degraded in the GI tract. Still, I like the idea of finding an oil with higher affinity to C60 so we can fit weekly doses into a gelcap.


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#8 niner

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Posted 25 April 2014 - 01:37 AM

This says Ethyl oleate is rapidly degraded in the GI tract. Still, I like the idea of finding an oil with higher affinity to C60 so we can fit weekly doses into a gelcap.


I'm not thinking of using it as an oral drug, although it would probably work. I was just thinking that it would be appropriate for in vitro experiments (e.g. cells in a Petri dish) because olive oil might not work there. The ethyl oleate adduct is also much better defined chemically. (At least there's only one fatty acid to worry about; there's still the potential for mono, bis, tris, and possibly higher forms, and the associated complication of regioisomerism. Fortunately we don't need to define a specific molecular entity in order to understand what it's doing. If we were trying to get a drug approved, then we would.)
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#9 kmoody

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Posted 25 April 2014 - 07:23 PM

Thanks for these initial thoughts. See replies below.

 

Dear Kmoody, does the vivarium allow to do a rat lifespan study? the best thing to do by far is to try to reproduce the life extension of the initial experiment

 

Strictly speaking the vivarium allows for anything. I do have housing that will support a rat study, but no one on my team has experience working with rat models, only mice. The learning curve for us to do a rat study properly may be prohibitive. All of our analytical instruments are already setup to do research with mice, hence why we could afford to do a more comprehensive study in mice for so cheap. Rats also live longer so the study would be more expensive and drawn out for that reason. Finally, we can order pre-aged mice (can we do that with rats?) and a mouse study may allow us to compete in the Mprize if that is still around. Overall I do not think a rat study would be a good fit for our group though I agree that a rat study would have some advantages over mouse studies.

 

Kelsey, are these all SCID mice?  If so, it seems like that would complicate the results.  I wouldn't anticipate that c60-oo would compensate for immunodeficiency.  It does appear to improve allergy/atopy, with altering of mast cell properties and/or shifting Th1-Th2 balance as possible mechanisms, based on published results with fullerene analogs. 

 

Would your lab by any chance be interested in doing some bench biochemical work on this?  It would be very interesting to look at the effect of c60-oo on respiration and mitochondrial parameters under both normal and hypoxic conditions.   Based on human experience with the compound, it looks very much like it is significantly improving OXPHOS efficiency.   It would also be interesting to look at redox status of the cell.

 

I'm going to break with the crowd and suggest that precise replication of Baati is not as important as simply showing significant life extension in a genetically normal mammal.   Replicating Baati in all details would be very difficult if not impossible, and would take a very long time.  I'd rather go with a shorter-lived species in order to get results sooner.  One area that represents a major hole in our knowledge is the effect of c60-oo on development.  It would be wonderful if we could run a reproductive tox study, and also look at the effects in animals ranging from neonates through sexual maturity. 

 

We have some suspicions that c60-oo assists in some way in stem cell differentiation, possibly by rescuing stalled or failed differentiations through a mitochodrial effect.  I understand that you guys have stem cell expertise, so perhaps you have some existing assays that you could deploy rapidly?  (we can but hope...) 

 

For in vitro work, you would probably want to use an ethyl oleate adduct instead of a triglyceride adduct.  My thinking here is that cells are likely to have generic esterases that would cleave it to the fatty acid, but I worry that triglycerides either wouldn't get in or the cell wouldn't have the appropriate lipase.  (I might be wrong on either count, though)  One of our members has already played around with the ethyl oleate synthesis, and it requires no modifications from Baati.  Ethyl oleate will form a more concentrated product than will olive oil. 

 

I do not see an advantage to using SCID mice in such a study. The point I wanted to make is that we would be conducting our study in a proper environment, not someone's closet. :) I agree with your thoughts on replication of Baati's study. I am 100% skeptical of anyone who claims such profound life extension effects until it has been confirmed many times over, preferably in my own hands. If my mice live longer on C60, well, then I will have a difficult time not believing those results won't I?  I have infrastructure for a limited tox study. We don't have the pathology expertise in house to assess histology but I might be able to find a collaborator for that. But CBC, chemistry profiles, etc. are all easy to do on a regular basis.

 

I would be interested in some biochemical work, depending on the complexity of the assays and learning curve. Why don't you PM me and we can chat about what you had in mind in greater detail?

 

 

This says Ethyl oleate is rapidly degraded in the GI tract. Still, I like the idea of finding an oil with higher affinity to C60 so we can fit weekly doses into a gelcap.

 

Lets see if we can reliably obtain results that suggest a positive effect of C60 before investing the time thinking about how to improve delivery. I would love to find myself in a situation where that is the most important problem that needs addressing. :)


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#10 kmoody

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Posted 25 April 2014 - 07:29 PM

A few people have bounced around ideas with me relating to clinical translation. That is the basis of my interest in assessing the effects of C60 in a neurodegenerative model. The feedback cycles for any human aging study would be very long... it may take a decade to see a lifespan effect even if the drug worked really well. In my mind we need to 1) replicate Baati's study to the extent that we confirm C60 causes a longevity effect in mammals, 2) take some educated guesses about what specific, non-aging indications may benefit from C60 administration and test them, then 3) move into clinical translation.

 

Importantly, these steps should progress step-wise. The absolute most important thing is to verify that C60 works at all. Many times wild claims like these simply do not hold up, or have some major catch (i.e. the intervention fixes X, but at the expense of Y).



#11 YOLF

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Posted 26 April 2014 - 12:18 AM

Good point Kelsey. Sent you a PM.



#12 caliban

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Posted 18 June 2014 - 11:35 PM

Please note that Kelsey has send a draft proposal  (Members only) 

 

 


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#13 AgeVivo

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Posted 16 August 2014 - 11:42 PM

Hello, for information the mouse lifespan experiment in Kiev will start end of August.

- 3 groups: C60oo, oo, water; by gavage with the same frequency as in the original study

- for each group, N=21 for males and 14 for females. Age at start: 12.5 months (were looking for 11 months but preparation took quite some time)
- A few months ago we have checked the absorbtion of C60 when administered orally to mice

- C60 in oil and oil itself have been kindly prepared by the team who reported the life extension in rats, in order to avoid later interpretations on that matter.

- apart from lifespan and gross anatomopathology, various health measures will be monitored (behavior with open field, fur, blood analysis for some animals based on what is available in the lab)
 
If all goes well we should start to know at the beginning of 2016 if it is seems that there is a life extension or not.
Regarding Creveterbelle's experiments at home, we should disclose what is what by the end of 2014.

 


Edited by AgeVivo, 16 August 2014 - 11:44 PM.

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#14 pone11

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Posted 18 March 2015 - 11:07 PM

Here is a study that a small lab like yours might be able to do, that would have enormous benefit for everyone.   We need a dosing study of C60.   Similarly constructed antioxidants like IAC end up having a very narrow range of benefit.  See Column A in Table 1:

http://biomedgeronto...na.glu160.short

 

There is literally no dosing information that lets us build a dose response curve for mice for C60.  You have humans taking megadoses of C60 on the probably-wrong assumption that more is better.   What Table 1 suggests is that these types of antioxidants may have enormous life extension at very narrow ranges, and that quickly tapers off.  At megadoses there is not a small shortening of life, but a huge outsized shortening of life.   It is therefore pretty critical that someone start to develop a similar type of dose response curve for C60.

 

 

 


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#15 kmoody

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Posted 26 March 2015 - 01:59 PM

This paper was recently brought to my attention by niner. This is a very good idea. What are your thoughts on study endpoints? Dosing mice with varying concentrations of c60 is easy enough, but what biomarker(s) would you use? Something general like GSSG/GSH ratio might not be sensitive or specific enough to see easily. We do have the ongoing c60oo leukemia study and it may be interesting to look across a wide range of doses, but there is a huge difference between assessing c60 for health maintenance vs. assessing its ability to protect against a large bolus of foreign cancer cells. The ideal of course would be to look at several biomarkers, but then prices start to creep up unless you strictly limit the time points. I.e. you could just run metrics at the end of the study, but errors and variance would be hard to see in such a data set. Do we look at acute treatment or chronic? Presumably you would want chronic, but that means a long study.

 

Dear Longecity community... lets bounce some ideas around and see where the conversation goes.  :)



#16 pone11

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Posted 26 March 2015 - 06:39 PM

This paper was recently brought to my attention by niner. This is a very good idea. What are your thoughts on study endpoints? Dosing mice with varying concentrations of c60 is easy enough, but what biomarker(s) would you use? Something general like GSSG/GSH ratio might not be sensitive or specific enough to see easily. We do have the ongoing c60oo leukemia study and it may be interesting to look across a wide range of doses, but there is a huge difference between assessing c60 for health maintenance vs. assessing its ability to protect against a large bolus of foreign cancer cells. The ideal of course would be to look at several biomarkers, but then prices start to creep up unless you strictly limit the time points. I.e. you could just run metrics at the end of the study, but errors and variance would be hard to see in such a data set. Do we look at acute treatment or chronic? Presumably you would want chronic, but that means a long study.

 

Dear Longecity community... lets bounce some ideas around and see where the conversation goes.  :)

 

The only thing that really matters is lifespan.   Biomarkers are interesting but people will argue about their meanings, and sceptics will never accept that as a proxy for lifespan.

 

And the really critical thing is are human beings potentially shortening their lives significantly by taking too much C60.   Having a mouse dose-response study would be helpful to understanding if there might be overdosing risks.



#17 pone11

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Posted 28 March 2015 - 06:08 PM

 

This paper was recently brought to my attention by niner. This is a very good idea. What are your thoughts on study endpoints? Dosing mice with varying concentrations of c60 is easy enough, but what biomarker(s) would you use? Something general like GSSG/GSH ratio might not be sensitive or specific enough to see easily. We do have the ongoing c60oo leukemia study and it may be interesting to look across a wide range of doses, but there is a huge difference between assessing c60 for health maintenance vs. assessing its ability to protect against a large bolus of foreign cancer cells. The ideal of course would be to look at several biomarkers, but then prices start to creep up unless you strictly limit the time points. I.e. you could just run metrics at the end of the study, but errors and variance would be hard to see in such a data set. Do we look at acute treatment or chronic? Presumably you would want chronic, but that means a long study.

 

Dear Longecity community... lets bounce some ideas around and see where the conversation goes.  :)

 

The only thing that really matters is lifespan.   Biomarkers are interesting but people will argue about their meanings, and sceptics will never accept that as a proxy for lifespan.

 

And the really critical thing is are human beings potentially shortening their lives significantly by taking too much C60.   Having a mouse dose-response study would be helpful to understanding if there might be overdosing risks.

 

 

Another thing worth mentioning is that it would be nice to find biomarkers of different levels of C60 activity that would translate to human use of C60.

 

I talk about this issue in the last paragraph of this post:

http://www.longecity...on/#entry721007



#18 seivtcho

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Posted 28 March 2015 - 07:40 PM

@kmody, I think, that the most important for your study is to use animals with a very clearly known life span. Then you will be able first to compare very well the effect, and second your study will not raise suspicions about the accuracy of the results. For example if you say this brand of mice lives this much, we gave them C60 and they lived that much, your results will be comparable and stable. If the life span of the mice, that you will use is not clear, then there will be a lot of questions about the percentages of the life span increasement (if such appears to exist) and why are you so sure, that it is due to the C60.
 


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#19 kmoody

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Posted 01 April 2015 - 12:37 PM

 

The only thing that really matters is lifespan.   Biomarkers are interesting but people will argue about their meanings, and sceptics will never accept that as a proxy for lifespan.

 

And the really critical thing is are human beings potentially shortening their lives significantly by taking too much C60.   Having a mouse dose-response study would be helpful to understanding if there might be overdosing risks.

 

Any study that investigates lifespan outcomes is going to be very expensive. Not that some aren't affordable, but each permutation (i.e. strain, dose, other interventions) is going to cost lots of $$. How do we avoid this? Big pharma has made a compelling case for some biomarkers, at least to the FDA. :)

 

@kmody, I think, that the most important for your study is to use animals with a very clearly known life span. Then you will be able first to compare very well the effect, and second your study will not raise suspicions about the accuracy of the results. For example if you say this brand of mice lives this much, we gave them C60 and they lived that much, your results will be comparable and stable. If the life span of the mice, that you will use is not clear, then there will be a lot of questions about the percentages of the life span increasement (if such appears to exist) and why are you so sure, that it is due to the C60.

 

Excellent point. For our lifespan studies we are using a known strain with ample lifespan data available. Of course, we are also including appropriate untreated controls in parallel with our treatment group. And I think what you're hinting at here is the need to conduct a power analysis so you can determine what effect on lifespan you are able to reliably detect, which we have also done. :)



#20 Kalliste

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Posted 01 April 2015 - 02:48 PM

I have a few ideas that I hope are not completely hopeless due to cost and so on. Each of them is related to C60's likely mechanism of action (being a really bioavailable antioxidant)

 

Furthermore it would be possible to use this stuff as good background material for a C60-mouse study to kind of anchor it within the pre-existing free radical biological-scientific framework.

I will present them below with links to the relevant articles.

 

Evaluating endothelial function / dysfuntion:

 

 

J Physiol. 2014 Jun 15;592(Pt 12):2549-61. doi: 10.1113/jphysiol.2013.268680. Epub 2014 Mar 24.
Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice. Abstract
Abstract

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD.

© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

http://onlinelibrary...268680/abstract

 

Looking at levels of 8-oxodG, which seems to be a fairly established biomarker:

 

 

Cruciferous vegetables contain compounds with antioxidant properties (e.g. carotenoids, vitamin C and folates) and can alter the activity of xenobiotic metabolism (i.e. isothiocyanates). These constituents may be particularly important for subjects who are exposed to free radicals and genotoxic compounds, including smokers. The aim of the study was to evaluate the effect of broccoli intake on biomarkers of DNA damage and repair. Twenty-seven young healthy smokers consumed a portion of steamed broccoli (250 g/day) or a control diet for 10 days each within a crossover design with a washout period. Blood was collected before and after each period. The level of oxidatively damaged DNA lesions (formamidopyrimidine DNA glycosylase-sensitive sites), resistance to ex vivo H2O2 treatment and repair of oxidised DNA lesions were measured in peripheral blood mononuclear cells (PBMCs). We also measured mRNA expression levels of repair and defence enzymes: 8-oxoguanine DNA glycosylase (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1) and heme oxygenase 1 (HO-1). After broccoli consumption, the level of oxidised DNA lesions decreased by 41% (95% confidence interval: 10%, 72%) and the resistance to H2O2-induced DNA strand breaks increased by 23% (95% CI: 13%, 34%). Following broccoli intake, a higher protection was observed in subjects with glutathione S-transferase (GST) M1-null genotype. The expression level and activity of repair enzymes was unaltered. In conclusion, broccoli intake was associated with increased protection against H2O2-induced DNA strand breaks and lower levels of oxidised DNA bases in PBMCs from smokers. This protective effect could be related to an overall improved antioxidant status.

 

 

Mutagenesis. 2010 Nov;25(6):595-602. doi: 10.1093/mutage/geq045. Epub 2010 Aug 16.

DNA damage and repair activity after broccoli intake in young healthy smokers.
Abstract

Cruciferous vegetables contain compounds with antioxidant properties (e.g. carotenoids, vitamin C and folates) and can alter the activity of xenobiotic metabolism (i.e. isothiocyanates). These constituents may be particularly important for subjects who are exposed to free radicals and genotoxic compounds, including smokers. The aim of the study was to evaluate the effect of broccoli intake on biomarkers of DNA damage and repair. Twenty-seven young healthy smokers consumed a portion of steamed broccoli (250 g/day) or a control diet for 10 days each within a crossover design with a washout period. Blood was collected before and after each period. The level of oxidatively damaged DNA lesions (formamidopyrimidine DNA glycosylase-sensitive sites), resistance to ex vivo H(2)O(2) treatment and repair of oxidised DNA lesions were measured in peripheral blood mononuclear cells (PBMCs). We also measured mRNA expression levels of repair and defence enzymes: 8-oxoguanine DNA glycosylase (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1) and heme oxygenase 1 (HO-1). After broccoli consumption, the level of oxidised DNA lesions decreased by 41% (95% confidence interval: 10%, 72%) and the resistance to H(2)O(2)-induced DNA strand breaks increased by 23% (95% CI: 13%, 34%). Following broccoli intake, a higher protection was observed in subjects with glutathione S-transferase (GST) M1-null genotype. The expression level and activity of repair enzymes was unaltered. In conclusion, broccoli intake was associated with increased protection against H(2)O(2)-induced DNA strand breaks and lower levels of oxidised DNA bases in PBMCs from smokers. This protective effect could be related to an overall improved antioxidant status.

http://www.ncbi.nlm....pubmed/20713433

 

 Ischemia-Reperfusion injury:

 

 

Plast Reconstr Surg. 2003 Jan;111(1):251-5; discussion 256-7.
Protective effects of superoxide dismutase against ischemia-reperfusion injury: development and application of a transgenic animal model.
Abstract

Reperfusion of ischemic tissues can be associated with structural and functional injury, which is referred to as ischemia-reperfusion injury. Superoxide dismutase is an endogenous free radical scavenger that converts toxic oxygen derived free radicals to hydrogen peroxide. With the development of gene cloning technology, the potential of manipulating cells to overexpress endogenous proteins has been realized. Transgenic mice capable of overexpressing superoxide dismutase, and knockout mice in which the gene responsible for its production has been deleted, were used as a model to examine the protective effects of superoxide dismutase against ischemia-reperfusion injury. Epigastric island flaps were elevated in wild-type (control), transgenic superoxide dismutase 1, and knockout superoxide dismutase 1 mice and subjected to ischemic intervals of 0, 3, 6, 9, or 12 hours. Five animals were studied at each time point in each study group. Flap viability was assessed on postoperative day 7. Baseline wild-type flap survival was 100 percent after 3 hours of ischemia and subsequent reperfusion; survival decreased to 21 percent after 9 hours of ischemia. Transgenic mice had significantly higher flap survival than wild-type animals after 6 hours of ischemia and subsequent reperfusion (97.0 versus 85.2 percent) and after 9 hours of ischemia (82 versus 21 percent, p < 0.01). In knockout mice, there was complete flap necrosis after as little as 3 hours of ischemia. This study confirms the protective effects of superoxide dismutase against ischemia-reperfusion injury. In addition, its deficiency results in a dramatic susceptibility to ischemic injury.

http://www.ncbi.nlm....pubmed/12496586

 

 

Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2343-8. doi: 10.1073/pnas.1417047112. Epub 2015 Feb 9.
Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters.
Abstract

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2 (•-)) dismutase-like properties yet were inert to nitric oxide (NO(•)) as well as peroxynitrite (ONOO(-)). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2 (•-) to O2 by PEG-HCCs at >20,000 s(-1). The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

 

Nanoparticles reboot blood flow in brain

Nanoantioxidants reboot brain blood-flow
Rice University, Baylor College of Medicine discovery might aid emergency care of traumatic brain-injury victims

A nanoparticle developed at Rice University and tested in collaboration with Baylor College of Medicine (BCM) may bring great benefits to the emergency treatment of brain-injury victims, even those with mild injuries.

Combined polyethylene glycol-hydrophilic carbon clusters (PEG-HCC), already being tested to enhance cancer treatment, are also adept antioxidants. In animal studies, injections of PEG-HCC during initial treatment after an injury helped restore balance to the brain’s vascular system.

The results were reported this month in the American Chemical Society journal ACS Nano.

A PEG-HCC infusion that quickly stabilizes blood flow in the brain would be a significant advance for emergency care workers and battlefield medics, said Rice chemist and co-author James Tour.

“This might be a first line of defense against reactive oxygen species (ROS) that are always overstimulated during a medical trauma, whether that be to an accident victim or an injured soldier,” said Tour, Rice’s T.T. and W.F. Chao Chair in Chemistry as well as a professor of mechanical engineering and materials science and of computer science. “They’re certainly exacerbated when there’s trauma with massive blood loss.”

In a traumatic brain injury, cells release an excessive amount of an ROS known as superoxide (SO) into the blood. Superoxides are toxic free radicals, molecules with one unpaired electron, that the immune system normally uses to kill invading microorganisms. Healthy organisms balance SO with superoxide dismutase (SOD), an enzyme that neutralizes it. But even mild brain trauma can release superoxides at levels that overwhelm the brain’s natural defenses.

“Superoxide is the most deleterious of the reactive oxygen species, as it’s the progenitor of many of the others,” Tour said. “If you don’t deal with SO, it forms peroxynitrite and hydrogen peroxide. SO is the upstream precursor to many of the downstream problems.”

SO affects the autoregulatory mechanism that manages the sensitive circulation system in the brain. Normally, vessels dilate when blood pressure is low and constrict when high to maintain an equilibrium, but a lack of regulation can lead to brain damage beyond what may have been caused by the initial trauma.

“There are many facets of brain injury that ultimately determine how much damage there will be,” said Thomas Kent, the paper’s co-author, a BCM professor of neurology and chief of neurology at the Michael E. DeBakey Veterans Affairs Medical Center in Houston. “One is the initial injury, and that’s pretty much done in minutes. But a number of things that happen later often make things worse, and that’s when we can intervene.”

Kent cited as an example the second burst of free radicals that can occur after post-injury resuscitation. “That’s what we can treat: the further injury that happens because of the necessity of restoring somebody’s blood pressure, which provides oxygen that leads to more damaging free radicals.”

In tests, the researchers found PEG-HCC nanoparticles immediately and completely quenched superoxide activity and allowed the autoregulatory system to quickly regain its balance. Tour said ROS molecules readily combine with PEG-HCCs, generating “an innocuous carbon double bond, so it’s really radical annihilation. There’s no such mechanism in biology.” While an SOD enzyme can alter only one superoxide molecule at a time, a single PEG-HCC about the size of a large protein at 2-3 nanometers wide and 30-40 nanometers long can quench hundreds or thousands. “This is an occasion where a nano-sized package is doing something that no small drug or protein could do, underscoring the efficacy of active nano-based drugs.”

“This is the most remarkably effective thing I’ve ever seen,” Kent said. “Literally within minutes of injecting it, the cerebral blood flow is back to normal, and we can keep it there with just a simple second injection. In the end, we’ve normalized the free radicals while preserving nitric oxide (which is essential to autoregulation). These particles showed the antioxidant mechanism we had previously identified as predictive of effectiveness.”

The first clues to PEG-HCC’s antioxidant powers came during nanoparticle toxicity studies with the MD Anderson Cancer Center. “We noticed they lowered alkaline phosphatase in the liver,” Tour said. “One of our Baylor colleagues saw this and said, ‘Hey, this looks like it’s actually causing the liver cells to live longer than normal.’

“Oxidative destruction of liver cells is normal, so that got us to thinking these might be really good radical scavengers,” Tour said.

Kent said the nanoparticles as tested showed no signs of toxicity, but any remaining concerns should be answered by further tests. The researchers found the half-life of PEG-HCCs in the blood – the amount of time it takes for half the particles to leave the body – to be between two and three hours. Tests with different cell types in vitro showed no toxicity, he said.

The research has implications for stroke victims and organ transplant patients as well, Tour said.

Next, the team hopes to have another lab replicate its positive results. “We’ve repeated it now three times, and we got the same results, so we’re sure this works in our hands,” Kent said.

First authors of the paper are BCM graduate student Brittany Bitner, Rice graduate student Daniela Marcano and former Rice postdoctoral researcher Jacob Berlin, now an assistant professor of molecular medicine at the Beckman Research Institute of the City of Hope, Duarte, Calif. Co-authors are all at BCM: Roderic Fabian, associate professor of neurology; Claudia Robertson, professor of neurosurgery; Leela Cherian, research instructor of neurosurgery; Mary Dickinson, associate professor of molecular physiology; Robia Pautler, associate professor of molecular physiology; and James Culver, a graduate student in molecular physiology.

The research was funded by the Department of Defense’s Mission Connect Mild Traumatic Brain Injury Consortium, the National Science Foundation, the National Institutes of Health and the National Heart, Lung and Blood Institute.
- See more at:

http://www.ncbi.nlm....pubmed/25675492

Nanoparticles reboot blood flow in brain
– August 23, 2012Posted in: Current News

Rice University, Baylor College of Medicine discovery might aid emergency care of traumatic brain-injury victims

A nanoparticle developed at Rice University and tested in collaboration with Baylor College of Medicine (BCM) may bring great benefits to the emergency treatment of brain-injury victims, even those with mild injuries.

Combined polyethylene glycol-hydrophilic carbon clusters (PEG-HCC), already being tested to enhance cancer treatment, are also adept antioxidants. In animal studies, injections of PEG-HCC during initial treatment after an injury helped restore balance to the brain’s vascular system.

- See more at: http://news.rice.edu...h.KknZLtJN.dpuf
Nano-antioxidants prove their potential Rice-led study shows how particles quench damaging superoxides 

Injectable nanoparticles that could protect an injured person from further damage due to oxidative stress have proven to be astoundingly effective in tests to study their mechanism.

Scientists at Rice University, Baylor College of Medicine and the University of Texas Health Science Center at Houston (UTHealth) Medical School designed methods to validate their 2012 discovery that combined polyethylene glycol-hydrophilic carbon clusters — known as PEG-HCCs — could quickly stem the process of overoxidation that can cause damage in the minutes and hours after an injury.

 

 

- See more at: http://news.rice.edu...h.pqtaFsJj.dpuf

http://news.rice.edu...low-in-brain-2/

http://news.rice.edu...ir-potential-2/

 

Inhibition of Metastasis by interfering with Mito-superoxide production:

 

 

Tumors are highly heterogeneous in all phenotypic features including glucose metabolism. Although glycolysis is coupled to oxidative phosphorylation (OXPHOS) in oxygenated tumor cells, some malignant cells are subject to fluctuations in oxygen availability and, therefore, rely on glycolysis uncoupled from OXPHOS for energy production (Dewhirst, 2009). From an energetic standpoint, it is surprising that some cancer cells also perform aerobic glycolysis (known as the Warburg effect), characterized by an increased normoxic flux of glucose to lactate (Warburg, 1956). However, this particular metabolic phenotype is shared with nonmalignant proliferating cells and is therefore thought to reflect increased biosynthetic plasticity (Vander Heiden et al., 2009, Ward and Thompson, 2012). Although it sometimes results from mutations (Frezza et al., 2011), there is evidence that mitochondria generally retain full oxidative capacities in solid tumors and that aerobic glycolysis can often be reverted (Faubert et al., 2013, Vaupel and Mayer, 2012), suggesting that this metabolic activity generally results from reversible changes that could be targeted pharmacologically. Thus, mitochondria actively contribute to aerobic glycolysis by producing cataplerotic intermediates and reducing equivalents to sustain cell growth and cell cycling (Anastasiou et al., 2011, Vander Heiden et al., 2009).

Tumor metastasis is a leading cause of cancer death. The metastatic switch marking the onset of metastatic dissemination corresponds to the acquisition of specific traits by tumor cells, including migration, invasion, and survival in the blood stream (Gupta and Massagué, 2006, Roussos et al., 2011). Three main lines of evidence led us to hypothesize that metastasis is under metabolic control. First, PET with the glucose analog tracer [18]-F-fluorodeoxyglucose (FDG) is routinely used for the clinical detection and imaging of tumor metastasis (Gambhir et al., 2001). This application is based on the observation that the vast majority of metastases trap far more glucose than normal tissues (with the exception of the brain), which has often been related to metabolic characteristics already acquired at the primary tumor site (Vander Heiden et al., 2009). Second, aerobic glycolysis could offer protection against oxidative damage and cell death when tumor cells transit in oxygenated blood (Porporato et al., 2011). Third, a mitochondrial defect corresponding to an inactivating mutation of respiratory chain complex I has been found in a metastatic mouse Lewis lung carcinoma cell line and was absent in weakly metastatic parental cells (Ishikawa et al., 2008a). Experimentally, the transfer of mtDNA from highly to weakly metastatic cells increased their metastatic potential in mice.

It is estimated that on average 106 tumor cells per gram of tumor are shed in the circulation daily (Chang et al., 2000). Of these cells, only a few, termed metastatic progenitors, are able to generate metastases in distant organs. In this study, we tested whether Warburg-phenotype tumor cells constitute a population of metastatic progenitors. We observed that tumor cell migration, invasion, clonogenicity, metastatic take, and spontaneous metastasis in mice are promoted by the natural selection of a mitochondrial phenotype associated with superoxide production, which was phenocopied by experimentally targeting the electron transport chain (ETC). We also report that pharmacological scavenging of mitochondrial superoxide prevents metastatic dissemination from primary orthotopic tumors in mice.

 

http://www.cell.com/...1247(14)00527-0

 

 

 

Good luck and thank you for the hard work Kmoody!

 

 


Edited by Cosmicalstorm, 01 April 2015 - 02:49 PM.

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#21 alc

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Posted 01 April 2015 - 04:24 PM

@kmody - since you guys really want to take things beyond endless discussions and arguments, would your team also consider studies for all these "new" developments that showed promissing results for past couple years?

 

and I'm referring to: NAD+. GDF-11 (parabiosis/plasma), Telomerase activation.

 

If you set up these studies and crowdfunding them, I'm pretty much sure there will be a lot of people donating just to see a clear and independent result.

 

Not to mention that your company will gain a lot of respect and public exposure.

 

By completing such studies, results will stop endless discussions - incuding on this web site - that "it is working, look at this study ..." vs. "no it is not working, look at this study ..."

 

Thanks for reading and considering these suggestions.

 


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#22 YOLF

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Posted 01 April 2015 - 04:43 PM

If we just want to do it with mice, perhaps we could just look do tissue comparisons, then once we see youthful gains or age reversal in tissues we can repeat the experiment and determine what the cause of those gains was. Skin and eyes should be easy enough to do... Not sure about the rest.



#23 alc

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Posted 01 April 2015 - 04:51 PM

@YOLF - are you referring to what I suggested above?



#24 YOLF

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Posted 01 April 2015 - 04:59 PM

We could do some studies on NAD+, but I wasn't suggesting any particular supplement. NAD+ might be good for LC as it's been getting alot of attention.



#25 alc

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Posted 01 April 2015 - 05:08 PM

Me neither, I'm not suggesting any products in particular, but I want to see independent studies done bya trusted party to reach a conclusion on NAD+, parabiosis and telomerase activation. There are so many discussions around and so many sorces cited, but if complee studies are done, then there will be little bit left to discuss and argue. And since I've seen Ichor Therapeutics really wants to make a difference, I suggested they will take on these and clarify things. That will be a huge step forward. Also, crowd-funding these projects will be the way to go.



#26 Kalliste

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Posted 02 April 2015 - 08:43 PM

Lets stick to c60 in this thread?
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#27 kmoody

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Posted 05 April 2015 - 06:27 PM

Me neither, I'm not suggesting any products in particular, but I want to see independent studies done bya trusted party to reach a conclusion on NAD+, parabiosis and telomerase activation. There are so many discussions around and so many sorces cited, but if complee studies are done, then there will be little bit left to discuss and argue. And since I've seen Ichor Therapeutics really wants to make a difference, I suggested they will take on these and clarify things. That will be a huge step forward. Also, crowd-funding these projects will be the way to go.

 

I am very open to discussing alternative projects further. Perhaps you could start a new thread so we can continue the conversation there and leave this thread to c60? I'll make sure to reply ASAP. :)


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#28 alc

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Posted 07 April 2015 - 01:26 AM

 

I am very open to discussing alternative projects further. Perhaps you could start a new thread so we can continue the conversation there and leave this thread to c60? I'll make sure to reply ASAP. :)

 

 

@kmoody - yes, I will start new thread (under Project Ideas) where to suggest things from my previous post. Apologize for "hijacking" this c60 thread.


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