2523 replies to this topic

[Nate-2004]

Also recent studies have shown Fisetin to not have affected any senescent cells in any rats. It was a disappointing outcome but tentatively, it may not be worth taking and we may have to wait on something more novel.

 

[resveratrol_guy]

Also recent studies have shown Fisetin to not have affected any senescent cells in any rats. It was a disappointing outcome but tentatively, it may not be worth taking and we may have to wait on something more novel.

 

 

It's interesting that he stopped taking fisetin+quercetin, even though the guy being interviewed in his video explicitly indicated that his team didn't test quercetin. Quercetin is an ionophore, which is perhaps why it was a necessary adjunct to dasatinib in the early senolytic experiments. Might it be the case that fisetin, similarly, needs quercetin to escort it across the bilayer and into the cyotplasm?

 

(I realize that, as the name suggests, ionophores escort ions, and neither dasatinib nor fisetin is an ion. However, perhaps the same mechanism works with, for example, hydrogen bonds under certain circumstances.)

 

I'm not aware of whether or not the successful fisetin rat studies also used quercetin, which would be informative.

[ambivalent]

It is worth noting, that according to the comments the fisetin was administered in water, rendering poor bioavailabilty. 

 

Whilst not being able to prove the removal of senescent cells,  as others have experienced at high doses, there have been effects indicative of it. 

 

Both I and another received temple hair regrowth - weirdly. Delayed wound healing (poster at consistently higher doses had a skin infection which took a couple of course of antibiotics to clear iirc, I had a couple of brief blood clotting difficulties).

 

The most prominent effect, was substantial increased pain in an arthritic knee and then subsequent strengthening. The weakening was immediate after the dose - the following day. At each subsequent round, the effect was less. 

 

The response here in the video was surprising - to presume the other data was fraudulent, or to result from poor experimental design, not be a little more curious, that their admnistration had oversight - the scientist interviewed was a a little sneery. To be wrong is quite a big deal, so why stop?

 

Doing everything the same in each of three independent labs seems smart - smart in producing confidence in the results of that narrow experimental design. But why not administer differently, use differnet experimental conditions in each lab? Then if and when finding something interesting occurs, replicate x 3. It is too narrow 

 

So much of scientific discovery is accidental - yet there is an aversion to creating experimental environments to foster it. It seems the lab is more interested in confidence, than discovery. In this case it seems obvious, where bioavailability is a problem. 

 

In any case, it wasn't able to poo-poo the health benefits of senescent cell removal. Still, it creates doubt so perhaps it might encourage some of us to expand to other senolytics.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Edited by ambivalent, 27 July 2022 - 12:33 PM.

[QuestforLife]

Turnbuckle,

 

Regarding your impressive epigenetic age reversal (was it 28 years?!), have you thought about ceasing your protocol for a time and retesting, to see how long the benefits last?

 

If, as we hope, you are influencing some primitive stem cell reserve like VSELS, then the benefits should last for a long time. But if you are only influencing stem cells that happen to be in circulation at the time of treatment, then the age reversal would quickly be lost and this would show up in an epigenetic age test.

 

Just a thought.

[Turnbuckle]

Turnbuckle,

 

Regarding your impressive epigenetic age reversal (was it 28 years?!), have you thought about ceasing your protocol for a time and retesting, to see how long the benefits last?

 

If, as we hope, you are influencing some primitive stem cell reserve like VSELS, then the benefits should last for a long time. But if you are only influencing stem cells that happen to be in circulation at the time of treatment, then the age reversal would quickly be lost and this would show up in an epigenetic age test.

 

Just a thought.

 

Short answer: I haven't done it.

 

Long answer: VSELS aren’t necessary for age regression, and most stem cells aren’t “in circulation.” Most reside in niches for the 200 different cell types. The epigenetics of SCs aren’t being measured in retail epigenetic tests, only the epigenetics of particular types of somatic cells. In many or most cases stem cells don’t go directly from SCs to somatic cells without passing through a transit amplifying stage. 

 

This is a simplified diagram of how it works.

 

SC ® TAC ® Somatic cells

 

Stem cells begat transit amplifying cells that began somatic cells.

 

SCs are mostly quiescent until needed to replace aged out TACs, while the very active TACs replace aged out somatic cells. In epidermal cells, aging must take place at the TAC level, as the somatic cells only last a matter of weeks. You could eliminate all the SCs, and the TACs would keep things going, but the rate of aging would increase more rapidly than normal.

 

Also, there is two-way paracrine signaling between these generations:

 

SC « TAC « Somatic cells

 

When a somatic cell is damaged or commits suicide, it sends a paracrine signal back to the TACs to indicate it needs replacement. And the same thing with TACs to SCs. These signals move by diffusion through the extracellular matrix, so the cellular actors must be very close.

 

Likewise, SCs and TACs must logically send signals in the opposite direction, giving permission to somatic cells to commit suicide. This is not established science, but without such signals, you could easily develop holes in tissues.

 

Feedback signals from daughter cells to stem cells are well studied and known to provide important feedback cues. Emerging evidence shows that stem cells also send feedforward signals to their progeny. Complex circuits involving both negative and positive feedback and feedforward signals likely contribute to robust tissue maintenance and regeneration.

https://www.ncbi.nlm...les/PMC5677519/

 

 

 

The object of all this signaling is to keep tissues in a state of homeostasis, as much as possible. You see plots of epigenetic age correlating very nicely with chronological age in a straight line. But that is due to averaging over many individuals. Individual age can go up or down depending on personal habits over the short term. Long term it goes up because of damage to SCs, which include physical loss and epigenetic damage that shuts down their ability to act as stem cells. This protocol takes the viable SCs you have left and magnifies them, producing a niche population you had years ago.

 

 

So if you stop the process, you will begin to age again, and the rate of that aging is unknown, but there is no reason to believe it would be faster on average than normal aging. I haven’t done that experiment, as I have only one me and limited time, but I have seen that certain supplements can make you age epigenetically much faster than with normal aging. Taking telomerase supplements every day will prevent TACs from aging out, and can result in epigenetic aging with no limit.

Edited by Turnbuckle, 28 July 2022 - 07:31 PM.

[QuestforLife]

Thankyou for your detailed answer, Turnbuckle.

The epigenetics of SCs aren’t being measured in retail epigenetic tests, only the epigenetics of particular types of somatic cells...

Actually I don't think epigenetic aging tests are cell specific, IMO the result from the test will be an average of many cells, including any stem and progenitor cells that happen to be present in the sample. It is plausible that the increase in epigenetic age with chronological age is due to a change in the composition of cells, rather than a step-wise advance of methylation based changes in all cells.

As cellular reservoirs of tissues, stem cell involvement in aging is intuitive. To interrogate the relationship between stem cells and epigenetic aging, we enriched HDK stem cells from neonatal skins of two donors. The stem cell enriched fraction expressed significantly greater amounts of stem cell markers, ITGA6, COL17A1 and p63 (Fig. 3f) and was younger than the stem cell-depleted fraction (Fig. 3g and Extended Data Fig. 7). This age difference was further increased when these cell fractions isolated from another four independent donors were put to culture
in vitro until confluence (Fig. 3h and Extended Data Fig. 7). The consistency of these results makes a strong case that EpiAge of a tissue is a result of the combined methylation profiles of its constituent cells, with stem cells exhibiting younger age than non-stem cells. Source:https://doi.org/10.1...587-022-00220-0

Edited by QuestforLife, 28 July 2022 - 09:23 PM.

[Turnbuckle]

Thankyou for your detailed answer, Turnbuckle.

Actually I don't think epigenetic aging tests are cell specific, IMO the result from the test will be an average of many cells, including any stem and progenitor cells that happen to be present in the sample. It is plausible that the increase in epigenetic age with chronological age is due to a change in the composition of cells, rather than a step-wise advance of methylation based changes in all cells.
 

 

Of course they are specific. There are two hundred different epigenetic codes in the body, so you have to be careful about what part of the code you are looking at. The TruMe test using spit looks at 2 types of cells, so you have to pick portions of the code that are the same with each. If all the codes were the same, there would be absolutely no use for them.

[QuestforLife]

Of course they are specific. There are two hundred different epigenetic codes in the body, so you have to be careful about what part of the code you are looking at. The TruMe test using spit looks at 2 types of cells, so you have to pick portions of the code that are the same with each. If all the codes were the same, there would be absolutely no use for them.

Different cell types have their own epigenetic codes, sure. But that isn't necessarily related to what aging clocks are measuring. They, I expect, will be mostly cell type agnostic, with some exceptions.

[Turnbuckle]

Horvath looked at a big data set and found common elements that could be used without having to make adjustments based on cell types (about a quarter of them)--

 

The personal story behind the discovery was featured in Nature.[13] The age estimator was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. The major innovation of Horvath's epigenetic clock lies in its wide applicability: the same set of 353 CpGs and the same prediction algorithm is used irrespective of the DNA source within the organism, i.e. it does not require any adjustments or offsets.[10] This property allows one to compare the ages of different areas of the human body using the same aging clock.

https://en.wikipedia...pigenetic_clock

 

 

You have to sift through the very large codes to find areas that have this commonality, as cell type epigenetics vary like sheets of music for different songs.

 

Other similar clocks:

 

1) Weidner et al. (2014) describe an age estimator for DNA from blood that uses only three CpG sites of genes hardly affected by aging

2) Hannum et al. (2013)[9] report several age estimators: one for each tissue type. 

 

 

Five more types are listed in the above reference. Keep in mind that these clocks are all created by humans. They don't exist in the cell. Cells use the much simpler telomeric clock to decide when cells need replacement. That's why you don't want to screw with it.

Edited by Turnbuckle, 28 July 2022 - 11:16 PM.

[QuestforLife]

 

Five more types are listed in the above reference. Keep in mind that these clocks are all created by humans. They don't exist in the cell. Cells use the much simpler telomeric clock to decide when cells need replacement. That's why you don't want to screw with it.

 

I still think that even stem cells will eventually telomerically age out, starving all downstream cells of replacement, so we will be forced to activate telomerase to increase telomeres in stem cells. The question is how to do it, without as you say, messing with the whole replacement scheme.  

 

There are claims that VSELs have high telomerase levels, but the data is only alluded to and not published. I have looked through numerous papers for confirmation but to no avail.

 

VSELs identified in UCB (Figure 1 panel A), like their murine counterparts, i) highly express telomerase (not published).. source: https://www.ncbi.nlm...les/PMC3371759/

[Turnbuckle]

I still think that even stem cells will eventually telomerically age out, starving all downstream cells of replacement, so we will be forced to activate telomerase to increase telomeres in stem cells. The question is how to do it, without as you say, messing with the whole replacement scheme.  

 

 

 

 

The time to do it is during SC proliferation, as during this process. With that short window, the effect on other cells is minimized.

Edited by Turnbuckle, 29 July 2022 - 02:39 PM.

[Kelvin]

Might this protocol be effective as a treatment for autism?

[Turnbuckle]

Might this protocol be effective as a treatment for autism?

 

According to this website, one might think so.

 

The outcome of stem cell therapy in autism is remarkably positive. 91% of individuals with autism have shown clinical improvements. Decrease in aggressive behaviour & hyperactivity, improved eye contact & attention span, improvements in communication & social skills are observed.

 

While on PubMed you can find papers saying there isn't enough evidence. Sometimes the market leads the science, as people cannot wait for years.

Edited by Turnbuckle, 02 August 2022 - 03:22 PM.

[FWP]

I am wondering how autism in for example children could be fixed with stem cells. Have they already depleted those? If not, one could argue stem cells are not the cure? I am thinking of this a lot as my 2 year old is recently diagnosed with diabetes type 1 and one would think there still would be enough stem cells. Perhaps the underlining issue in autism and diabetes, auto immuum disease? must be tackled first.

Don't worry I will not experiment with a child just trying to understand

Edited by FWP, 03 August 2022 - 05:35 AM.

[Turnbuckle]

I am wondering how autism in for example children could be fixed with stem cells. Have they already depleted those? If not, one could argue stem cells are not the cure? I am thinking of this a lot as my 2 year old is recently diagnosed with diabetes type 1 and one would think there still would be enough stem cells. Perhaps the underlining issue in autism and diabetes, auto immuum disease? must be tackled first.

Don't worry I will not experiment with a child just trying to understand

 

 

Another possibility: Meet the ‘mitomaniacs’ who say mitochondria matter in autism

 

Experimenting with my mito protocol would be substantially less problematical, though I would substitute 2-3 grams of dihydromyricetin for the GMS.

[FWP]

"For one thing, variations in mtDNA long after birth may not be meaningful, given that mitochondria are constantly fusing and dividing, creating new organelles that may differ from the previous ones, experts say. So the mitochondria an adult or child has are not necessarily the same as those she had in the womb, when autism is thought to develop."

So reading it I understand that in one point in time there could have been a problem with mitochondria output and potentially not noticable on a latter point in time when the damage is already done. Hopefully the mito protocol could still provide benefit for those who have autisme. I am still quite curious if the stem cell protocol could resolve diabetes type 1. https://asweetlife.o...ype-1-diabetes/

[Turnbuckle]

"For one thing, variations in mtDNA long after birth may not be meaningful, given that mitochondria are constantly fusing and dividing, creating new organelles that may differ from the previous ones, experts say. So the mitochondria an adult or child has are not necessarily the same as those she had in the womb, when autism is thought to develop."
 

 

 

That statement shows a lack of understanding by the author. The mitochondrial organelles are not generally the source of the problem, only the mtDNA within them. These mtDNA are constantly being duplicated by biogenesis and defective copies destroyed by mito QC (via PINK1/Parkin mitophagy). For QC to eliminate defective mtDNA, mitochondria must first be fissioned so that each mitochondrion has only one loop of mtDNA, and this fissioned mitochondrion must be non-functional (ΔΨm = 0). Partially functional mtDNA (ΔΨm > 0) get a pass, and that could be the problem here, where cells try to make up for large numbers of only partially functional mitochondria by increasing mtDNA mass. The process I developed forces the membrane potential (ΔΨm) of fissioned mitochondria with low ΔΨm to zero, exposing them to mitophagy.

 

That said, an initial problem with mitochondria could correct itself but too late for brain development. But the presence of excessive numbers of mitochondria points to an ongoing issue.

Edited by Turnbuckle, 03 August 2022 - 02:50 PM.

[FWP]

Great reply So you would think that also an inherited mitochondrial disease could be fixed?

[Turnbuckle]

Great reply So you would think that also an inherited mitochondrial disease could be fixed?

 

 

Mitochondrial DNA have 37 genes, all of which are essential. Even one nonfunctional gene will expose it to mitophagy. But it's possible that a mutation might occur that produces only partial function. If all mtDNA were the same, then it would not be possible to fix the problem as there would be no fully functional mtDNA to promote. But if only some were partially functional, then those partially functional mtDNA would tend to dominate over time, as they produce less ROS and thus would be less likely to pick up mutations that would eliminate them compared to fully functional mtDNA. The same thing with methylation of mtDNA that reduces function. This methylation is copied during biogenesis and will give these low functioning mtDNA a survival advantage.

 

This is totally hypothetical and assumes a mitochondrial etiology, but it could explain the delay in the development in symptoms, at least in some cases.

[kurt9]

How long will the C60 in Olive oil last if you keep it in a refrigerator as opposed to keeping it in a dark cabinet? Since C60 is light sensitive, I will keep it in a black container. I  plan to do only 2-3 cycles this fall, then wait 6 months, then a couple more in Feb-March of next year. i want to keep all of the compounds, including the C60, long term. 

Edited by kurt9, 08 August 2022 - 04:12 AM.

[RWhigham]

Expansion of VSELs in-vitro requires SIRT-1 inhibition. SIRT-1 silences a gene that VSELs require for division..

 VSELs and SIRT1 inhibition.pdf   256.99KB   8 downloads

Edited by RWhigham, 10 August 2022 - 11:51 PM.

[RWhigham]

SIRT1 normally silences genes {which I assumed in the previous post), but the paper says that SIRT-1 "inhibits the activity of the de novo DNA methylotransferase DnmT3L" and that prevents VSELs from proliferating in-vitro unless SIRT-1 is inhibited.

Edited by RWhigham, 11 August 2022 - 02:03 AM.

[Turnbuckle]

SIRT1 normally silences genes {which I assumed in the previous post), but the paper says that SIRT-1 "inhibits the activity of the de novo DNA methylotransferase DnmT3L" and that prevents VSELs from proliferating in-vitro unless SIRT-1 is inhibited.

 

 

One user of the protocol failed to get results with the protocol a while back, but turned out he had added resveratrol to it. (See this post.) So that was a double whammy, as not only does resveratrol increase telomerase (thus blocking senescent cell replacement), but it also stimulates SIRT-1, thereby inhibiting de novo methyltransferase as noted above.

[rarefried]

Expansion of VSELs in-vitro requires SIRT-1 inhibition. SIRT-1 silences a gene that VSELs require for division..

VSELs and SIRT1 inhibition.pdf

 

Searched for natural compounds that might inhibit sirt1.  A few came up, eg amurensin G (https://molpharm.asp...ab-figures-data) but not many candidates as supplements to take now. Licorice might be one such (if it doesn't contain other flavonoids, or, triterpenoids which might negate the putative effect).  

 

"Chalcones have shown inhibitory properties against sirt1 and hindered cell growth in HEK293T cells, for example, Kahyo et al., demonstrated that 3,2′,3′,4′-tetrahydroxychalcone produces physiological effects on organisms probably through inhibiting the deacetylation by SIRT1, i.e., by inhibiting the SIRT1-mediated deacetylation of a p53 acetylated peptide and recombinant protein in vitro [117]."

https://www.ncbi.nlm...les/PMC7397027/

 

"Licorice is an important Chinese materia medica frequently used in clinical practice, which contains more than 20 triterpenoids and 300 flavonoids. Chalcone, one of the major classes of flavonoid, has a variety of biological activities and is widely distributed in nature. To date, about 42 chalcones have been isolated and identified from licorice."

https://www.hindawi....m/2020/3821248/

 

​Another might be Codonopsis pilosula 

 

"Three new compounds, pilosulinene A (1), pilosulinols A (2), and B (3), along with seven known compounds, were isolated from the roots of Codonopsis pilosula cultivated in Xundian County of Yunnan Province. The structures of new compounds were established by spectroscopic methods. In particular, the presence of an aromatic ring in the structure of 1 makes it intriguing. The inhibitory activity of compounds against SIRT1 was evaluated. The results showed that 8 could inhibit Sirt1 in a dose-dependent manner."

https://www.scienced...028602022004325

[Kelvin]

Licorice might be hard to integrate into the protocol because it affects a wide variety of processes involved with cortisol levels, vasoconstriction, the kidneys, and more.

Its wide ranging impact across the body make it hard to know how it would benefit or harm the goals of C60 supplementation.

Edited by Kelvin, 11 August 2022 - 11:32 PM.

[Kelvin]

By the way, I came back from my dental cleaning yesterday.

For the first time ever when the nurse used the plaque scrapper I felt no sharpness around my gums.

It was more of a firm, dull, pressure than a sharp sensation.

When I used the fission fusion mito cycle I mostly stopped gum bleeding when the plaque scrapper was used around my gums. But the sharp sensation was still there.

It appears the C60 cycle enhanced my gum health.

I had no signs of cavities at all but that wouldn’t be affected by C60 since tooth enamel is not living tissue.

[Bike_to_120]

Took my first "dose" of this protocol today. What is the frequency people take this?  I've seen once a week and Mr Turnbuckle seemed to take it 3-4 times per week.

[Kelvin]

My protocol frequency was twice a week for three weeks.

Going forward I plan doing two to three cycles every 6 months.

[Turnbuckle]

I'm presently using the protocol (one fusion day followed by 2-3 fission days) every week to three weeks. I let the fission days be my guide. Expect to get flu like symptoms during fission, and if you don't, there probably isn't much more replacement going on than with normal maintenance (in which some 300 billion cells are replaced on a daily basis). I recently made a tweak to the fusion part that resulted in the strongest flu-like symptoms I've yet seen during fission. I will get epigenetic tests done in the coming weeks to see if that was truly something special.

[kurt9]

How many cycles have you done?

 

I have yet to buy all the "stuff" but will in the next couple of weeks. I was looking at doing only like 2-3 cycles, each two weeks apart, then wait 4-6 months for results, then do another 2-3 cycles, say, around February of next year.

 

I have some issues, but realized that most of them were due to nothing more than an extended summer cold rather than aging itself. Since I am nowhere near "geriatric" (however you define it), I am leery of doing too many cycles for fear of stem cell depletion. I am 59 years old, but look and feel significantly younger (it must be your mitochondrial protocol I did last year).

 

When the SARS-CoV-2 first showed up two and a half years ago, I got an email from a friend of mine in the "alternative" medical community who recommended a prophylactic she developed for the first SARS back in 2003. Among many things such as Vitamin A, C, D, and other stuff, was Astragalus Root, which I merrily took 5 days per month for 6 months. At the time I remember having heard of it before but could not place it. It was in here that I read that it was a rather potent telomere elongator, which probably why I felt like dynamite during spring and summer of 2020, then slightly run down in 2021. Anyways, I long ditched that prophylactic and will never return to it again. I don't think the lady who came up with this prophylactic realizes the telomere elongation aspect of this.

 

My main issues was slight hair-loss (I think this was long COVID) that I seem to have reversed by methods I will not go into here and age spots on the skin.