1033 replies to this topic

[QuestforLife]

If you really are de-differentiating somatic cells, that should definitely lower your epigenetic age, no matter how old you are chronologically. It would be a fantastic breakthrough.

 

BTW, I see that one ROCK inhibitor--fasudil--promotes gliogenesis of neural stem cells, which isn't a good thing.

 

Exposure to the ROCK inhibitor fasudil promotes gliogenesis of neural stem cells in vitro.

 

According to the first paper I posted for in vitro 'conditional reprogramming', which basically allows lab scientists to culture normal somatic cells indefinitely without immortalising them, the cells differentiate normally (with roughly the same number of remaining divisions as they had beforehand) after the ROCK inhibitor is removed. The question is if you take an epigenetically old endothelial cell (say), de-differentiate it into a progenitor-like state over about a month, and then let it differentiate again, what will the resulting epigenetic state be? According to the Janic et al. study where they took a low dose statin for a month, the benefits on arterial function lasted for 6 months...

[HBRU]

Same opinion of you about statins.... Do you know is same benefits come from Pravastatin ? This is the safests for Brain and also the only taht does not increase diabet risiko but actually (as I know) decrease it.....

[Turnbuckle]

According to the first paper I posted for in vitro 'conditional reprogramming', which basically allows lab scientists to culture normal somatic cells indefinitely without immortalising them, [1] the cells differentiate normally (with roughly the same number of remaining divisions as they had beforehand) after the ROCK inhibitor is removed. The question is [2] if you take an epigenetically old endothelial cell (say), de-differentiate it into a progenitor-like state over about a month, and then let it differentiate again, what will the resulting epigenetic state be? According to the Janic et al. study where they took a low dose statin for a month, the benefits on arterial function lasted for 6 months...

 

 

[1] This suggests the elimination of telomere erosion during ROCK inhibition, but not extension.

 

[2] Induced pluripotent cells would ideally show zero epigenetic age, but of course underlying genetic mutations are not reset, and some of epigenetic pattern may be retained as well. That's for in vitro. For in vivo, who knows. I don't see anyone doing whole body in vivo dedifferentiation.

[QuestforLife]

[1] This suggests the elimination of telomere erosion during ROCK inhibition, but not extension.

[2] Induced pluripotent cells would ideally show zero epigenetic age, but of course underlying genetic mutations are not reset, and some of epigenetic pattern may be retained as well. That's for in vitro. For in vivo, who knows. I don't see anyone doing whole body in vivo dedifferentiation.

Just to clarify a number of points: ROCK inhibitors do activate telomerase - a little (probably due to the fact progenitors have less regulation of the telomerase gene than fully differentiated cells). But for indefinite proliferation another, stronger source of telomerase is required and this is achieved various ways in vitro. And even then, as you point out, telomeres don't get longer, they are only held constant. But that's in vitro in hyperoxia and with massive growth factors when the cells are dividing at a great rate.

Also, ROCK inhibitors can only de-differentiate a little way, i.e. somatic to progenitor cell (and maybe from adult stem cell back a little further too). I wouldn't expect pluripotency. But it would be great if somatic cells could benefit from partial and temporary de-differentiation by resetting any harmful methylation changes they've accumulated, for example.

[QuestforLife]

Same opinion of you about statins.... Do you know is same benefits come from Pravastatin ? This is the safests for Brain and also the only taht does not increase diabet risiko but actually (as I know) decrease it.....

The quick answer is I don't know. All statins as far as I have seen inhibit ROCK, and their effectiveness isn't necessarily related to their ability to lower chloresterol (although this last point is contentious). Pravastatin is hydrophilic so you'd expect less penetration throughout the body than the fat soluble ones. It is one of the less effective statins from a chloresterol lowering point of view.

Janic used fluvastatin, which is also one of the less powerful statins, with much less reported side effects than some of the others, and it worked well in his study. If you can get hold of it fluvastatin is what is recommend. I've used atorvastatin and at 5mg for 20 days it's fine. At 10mg I had some muscle cramps.

I'm hoping two supplements I've identified will be effective ROCK inhibitors that will allow me to avoid chloresterol lowering altogether. I'll let you know how it pans out.

[QuestforLife]

I wanted to summarise the results of my intervention so far. I want to stress than these results are quite preliminary but encouraging, particularly in the light of my null results previous to 2018.  I am committed to continuing testing in 2020 to gather further evidence. Top of my priority list is to determine if the SES protocol really is reversing epigenetic age, and if epitalon really is a powerful telomerase activator.
Results summary
July 2018 to April 2019: DNAm age reverse 1.5 years
July 2015 to November 2018: Blood panel comparison indicates a 2 year reversal in Pheno and DNAm Age when chronological age is held constant (Pheno and DNAm Age results (~30) are far below my chronological age, and the DNAm age estimate from Nov 2019 is much less than my April 2019 DNAm age result, which seems strange)
July 2018 to November 2019 indicates a 2.4 year reversal in biological age as measured by median telomere length; improvement in the shortest telomeres is much greater.

 

Merry Christmas and a Happy New Year to all. May we all get younger in 2020!

[HBRU]

This may interest you....

Combined Inhibitory Effects of Celecoxiband Fluvastatin on the Growth of HumanHepatocellular Carcinoma Xenografts inNude Mice

https://journals.sag...323001003800423

[QuestforLife]

This may interest you....
Combined Inhibitory Effects of Celecoxiband Fluvastatin on the Growth of HumanHepatocellular Carcinoma Xenografts inNude Mice

https://journals.sag...323001003800423

I posted this a while back:
https://www.longecit...e-3#entry870530

The paper I referenced there shows you can de-differentiate glioblastoma cancer cells to neural progenitors using a combination of a ROCK and mTOR inhibitor, which then differentiate normally into neurons.

More specifically statin use seems to be associated with a lower risk of some but not all cancers.

https://www.wjgnet.c...ll/v5/i2/41.htm

Edited by QuestforLife, 23 December 2019 - 02:01 PM.

[dlewis1453]

Hi QuestorLife

 

What are your thoughts now on the extent of the de-differentiation and rejuvenation that is possible with your ROCK protocol? 

 

At one point in this thread I believe you said the effect would be limited to the circulatory system and would be useful mainly as a protocol for atherosclerosis, but elsewhere I believe you said it could have a wider effect. 

[QuestforLife]

Hi QuestorLife

What are your thoughts now on the extent of the de-differentiation and rejuvenation that is possible with your ROCK protocol?

At one point in this thread I believe you said the effect would be limited to the circulatory system and would be useful mainly as a protocol for atherosclerosis, but elsewhere I believe you said it could have a wider effect.

There's a couple of possible answers to that question, depending on exactly what you mean.

ROCK inhibitors only influence differentiation via one of the Yamanaka factors (K), so they'll only partially de-differentiate a given cell from its starting epigenetic position; to my mind that's a good thing.

But what I think you mean is more to do with how much of the body can be affected. I originally thought it would only be the arterial system, as statins are known to increase endothelial progenitors, and those cells are restricted to that system. But now that I know more about how ROCK inhibitors work, and that they increase stemness in all cells, and many other types of cells are in circulation at a given time (especially if you simultaneously use stem cell stimulants to release more from the bone marrow, and telomerase activators to increase their number), I expect the effects will be wider. After all, young factors in the blood plasma have been shown to have rejuvenating effects in parabiosis experiments. And then we have some statins that penetrate further into the body than others, so will have an effect there; it's an open question at this point whether that is a good or bad thing. We may not need a ROCK inhibitors to go further than the blood stream to have systemic beneficial effects.

Edited by QuestforLife, 27 December 2019 - 09:37 AM.

[dlewis1453]

But what I think you mean is more to do with how much of the body can be affected. 

 

Yes! this is what I meant. Thanks for the enlightening response. 

[QuestforLife]

I’ve started the latest iteration of my protocol. The main aim of this iteration is to trial alternatives to atorvastatin and losartan.
• Epitalon SubQ 10mg every other day AM
• Tongkat Ali (TA) (12mg eurycomanone ~3%) 1 capsule in the AM as an alternative to Atorvastatin for ROCK inhibition
• Boswellia Extract (BE) (20% Boswellic Acid) 1 capsule in the AM as an alternative to Losartan for Tgf-B inhibition
• AFA or Sea Buckthorne Extract (SBE) for stem cell release 1 capsule in the AM; AFA has proven effective at this dose, I’ll trial SBE for half the cycle to compare
Observations on the cycle so far:
I was originally taking epitalon in the evening but it was causing disturbances in my sleep (this didn’t happen in previous cycles); no such issues since I switched to AM injections
I trialled both TA and BE between cycles, each on their own to determine their effects. Tongkat Ali had aphrodisiac properties after a few days, as advertised. Boswellia extract turned out to be a very valuable supplement for controlling aches and pains due to weightlifting. I generally find that I ‘run out of steam’ during the month following the conclusion of a cycle and struggle to lift as much or as often. BE remarkably seems to be able to prevent this and has enabled me to keep making progress between cycles.
Other notes
Depending on the success or otherwise of the new additions to the protocol I may extend this one to longer than 20 days, particularly if I need more time to isolate effects.  
Other supplements taken in the same period: Zinc, B6, Vit D+K2, whey protein.
I am still in discussion with Lifelength. I pointed out to them that the telomere length graph they suppled in 2018 (my baseline) had some discrepancies in it, particularly noticeable when compared to the 2019 graph that appears to be correct (and also shows longer telomeres). They have acknowledged there is a problem with the 2018 graph and hope to have it resolved and clarified in January. I do not believe this will affect the reported telomere lengths, but it will enable me to compare 2018 and 2019 across all telomeres measured, not just the figures they have reported (20 percentile, 50 percentile and mean).

[QuestforLife]

This point you made, which I highlighted in bold, makes a lot of sense to me and I think its relevant to the discussion.

I have seen you allude to a positive experience with AFA and Sea Buckthorn in one of your posts on your Alternative Telomere Lengthening protocol thread. Could you share your experiences with those substances? It could give Turnbuckle some ideas for future iterations of his protocol.

I've had good results with AFA and SBBE. But I'm aware that too much asymmetric division will result in the remaining stem cell pool being dominated with AFA and SBBE insensitive cells. I cycle them (don't use them at the same time)and the whole protocol is cyclical anyway, so I'm not too worried about this problem. I do know of people who've used AFA with good results for many years (and they take it every day).

Because I also use epitalon I increase the freed stem cells' ability to increase in number in the blood stream. And I use ROCK inhibitors as well, which might mitigate the effect of stem cell insensitivity by making them more pluripotent. But it's largely guesswork at this time.

[QuestforLife]

A couple of interesting new telomere studies

Khavinson et al. has looked at the effects of epitalon on activated T-cells taken from young and middle-aged subjects. The effects initially look quite confusing with both increases and (a few) decreases in telomere length, and no stand-out effects on the mitotic rate of division. That being said as these leukocytes are already being stimulated to divide, the default result should be telomere loss. I was able to look at the mitotic division rate changes (with and without epitalon) and predict in 10/11 cases whether the telomeres would get shorter or longer, and vice versa was able to look at the telomere length changes with the addition of epitalon and again predict in 10/11 cases whether the mitotic rate was going to go up or down. With the exception of 1 out of the 11, the results were as you would expect; if epitalon ‘made’ (in addition to the other stimulant used) the cells divide faster it could end up decreasing telomere length, but if the result was slower division than before then the telomeres would get significantly longer. It’s such a simple and obvious analysis all the more remarkable for the fact the paper authors do not see it. In any case it is more evidence that epitalon increases telomerase* and can increase telomere length in T-cells (depending on how fast they are dividing) and adds to the previous evidence for telomerase activation in fibroblasts. Interestingly they used 200x the concentration used in fibroblasts, although this concentration (1uM/ml in the blood) is what you’d likely get from a 5mg injection.  That the effect is less impressive than for fibroblasts (who got longer telomeres AND a faster rate of division) might be due to the greater rate of division of T-cells who were stimulated by PHA; *it is a pity they didn’t measure telomerase activity directly so we could see a direct comparison.
Source: https://link.springe...517-019-04664-0

Shay et al. have got in on the ROCK inhibition act, claiming ‘Proliferation of adult human bronchial epithelial cells without a telomere maintenance mechanism for over 200 population doublings’. The fact that they are using a ROCK inhibitor as well as irradiated feeder cells clearly indicates to me that there is a ‘telomere maintenance mechanism’. I’ve always had a great respect for Shay (and Wright), but they’ve been comfortably scooped here by other teams as reported at numerous points earlier in this thread. There are some other comments of interest in the paper on clonally expanding out certain selected cells once the culture did eventually begin to senesce, but I haven’t had time to analyse these results yet.
Source: https://onlinelibrar...6/fj.201902376R

[RWhigham]

• Yamanaka OSKM factors = Oct3/4, Sox2, Klf4 and c-Myc  Added to culture produce a few low-quality embryonic cells. (Embryos grown from them are usually defective)
• The quality is improved by omitting Oct3/4. Fewer (30%) reprogrammed cells occur but they grow healthy embryos
• Adding a TGF-b inhibitor restores the efficiency (more cells occur) ,and reduces the amount of Sox2 required
• Creating fully reprogrammed (embryonic) cells at random in a fully formed organism will likely cause cancer. When embryonic cells are injected into mice they produce tumors/cancer
• When c-Myc is omitted partially reprogrammed cells are obtained that retain their identity while reverting to young cells. When these are injected into mice they do not cause tumors
• So for rejuvenation we only need Sox2 and Klf4.
• ALK5/TGF-b inhibitors are thought to cause the transcription of Sox2
• ROCK inhibitors are thought to cause the transcription of Klf4
• Mice have been rejuvenated with just ALK5-inhibitor, but it required dangerous amounts (ALK5 is immediately upstream of TGF-b). ALK5-inhibitors can damage heart valves (likely by stopping cell replacement--heart valves seem to require constant cell replacement)
• By injecting mice with 1ug/g of oxytocin daily for 7 days rejuvenation occurred with a safe amount of ALK5 inhibitor. The mice continued to rejuvenate for 30days. Human equivalent dose of 1ug/g is impractical. By adding ROCK inhibitors perhaps a reasonable dose like 10 iu (1cc) of oxytocin subq would be useful
• ROCK inhibitors - Statins, Pholretin (LEF Applewise), Tongkat-Ali, Skullcap, Reishi, Cialis, Gotu-Kola
• TGF-b inhibitors - Boswellia, Curcumin, AMPK activator, Milk Thistle, DHA, Delta-tocotrienols, Ginger, Pomegranate, Red Sage Root (Danshen), Honokiol, Ursolic acid, Fucoidan, EGCG

 

Edited by RWhigham, 24 January 2020 - 05:57 PM.

[Andey]

 

• Yamanaka OSKM factors = Oct3/4, Sox2, Klf4 and c-Myc  Added to culture produce a few low-quality embryonic cells. (Embryos grown from them are usually defective)
• The quality is improved by omitting Oct3/4. Fewer (30%) reprogrammed cells occur but they grow healthy embryos
• Adding a TGF-b inhibitor restores the efficiency (more cells occur) ,and reduces the amount of Sox2 required
• Creating fully reprogrammed (embryonic) cells at random in a fully formed organism will likely cause cancer. When embryonic cells are injected into mice they produce tumors/cancer
• When c-Myc is omitted partially reprogrammed cells are obtained that retain their identity while reverting to young cells. When these are injected into mice they do not cause tumors
• So for rejuvenation we only need Sox2 and Klf4.
• ALK5/TGF-b inhibitors are thought to cause the transcription of Sox2
• ROCK inhibitors are thought to cause the transcription of Klf4
• Mice have been rejuvenated with just ALK5-inhibitor, but it required dangerous amounts (ALK5 is immediately upstream of TGF-b). ALK5-inhibitors can damage heart valves (likely by stopping cell replacement--heart valves seem to require constant cell replacement)
• By injecting mice with 1ug/g of oxytocin daily for 7 days rejuvenation occurred with a safe amount of ALK5 inhibitor. The mice continued to rejuvenate for 30days. Human equivalent dose of 1ug/g is impractical. By adding ROCK inhibitors perhaps a reasonable dose like 10 iu (1cc) of oxytocin subq would be useful
• ROCK inhibitors - Statins, Pholretin (LEF Applewise), Tongkat-Ali, Skullcap, Reishi, Cialis, Gotu-Kola
• TGF-b inhibitors - Boswellia, Curcumin, AMPK activator, Milk Thistle, DHA, Delta-tocotrienols, Ginger, Pomegranate, Red Sage Root (Danshen), Honokiol, Ursolic acid, Fucoidan, EGCG

 

 

Thank you, that's quite a food for thoughts

Am I wrong that pioglitazone is also a TGF-b inhibitor? 

 

[RWhigham]

The same researcher that found ALK5 + oxytocin rejuvenates mice, also concluded that VCAM1 (vascular cell adhesion molecule) is an important aging factor in old blood, and deta-tocotrienols antidote it.

 

Am I wrong that pioglitazone is also a TGF-b inhibitor? 

I don't know

Edited by RWhigham, 24 January 2020 - 08:25 PM.

[RWhigham]

• The purpose of oxytocin is to greatly reduce the amt of TGF-b required
• This is needed because too much TGF-b damages heart valves
• The mouse study used 1ug/g = 1mg/kg which is way impractical for humans
• Most injectable oxytocin has 1 to 20 iu/cc  (for both human & veterinary use)
• 1 iu = 1.68 ug
• OxyLuv nasal spray comes in a 1oz (29.57cc) bottle containing 500 iu of pure oxytocin without preservatives.
• OxyLuv has 500iu/29.57cc = 16.9 iu/cc = (1.68)(16.9)= 28.4 ug/cc = 28.2 nMol/cc ( molar wt is 1007)
• OxyLuv can be prepared for injection by passing through a 0.22 micron PES filter
• 1cc subq stings, makes a small bump, leaves a reddened spot for a couple of days, may cause an urgent BM, and may cause uterine contractions in a female.
• About 60% of body weight is fluid, 40% inside cells and 20% extracellular. 
• A 60kg human has about (20%)(60kg) - 5L (blood) = 7L of nonblood extracellular fluid & 24L inside cells.
• Oxytocin does not  appear to remain in blood (it's almost immediately indetectable)
• 1 cc of OxyLuv could give 28.2nMol/7L = 4 nMol concentration if it stays in the nonblood extracellular compartment (per L is understood when talking about nMol concentrations)
• 1 cc of OxyLuv could give 28.2nMol/24L = 1.18 nMol concentration if it all goes inside cells.
• The patent on mouse rejuvenation specifies an oxytocin concentration of 7.5-30 nMol
   

Edited by RWhigham, 24 January 2020 - 10:33 PM.

[QuestforLife]

Great research RWhigham!

We know a low dose statin (ROCK inhibitor) and Sartan (TGF-B inhibitor) works to rejuvenate the cardiovascular system, as evidenced by the papers by Janic et al. Interestingly they showed a cyclical approach was best. One month on, five or six off (or three of you're diabetic). This suggests that most people on these drugs would do better on a cyclical protocol. Also I've reversed the methylation and telomere clocks somewhat using a similar protocol (also includes epitalon and stem cell activators) even though I'm only 41.

The point of my latest experiment was to see if I could replace the statin and sartan. I identified a 5-LOX inhibitor from boswellia extract to replace the sartan. I trialled it alone and it definitely works for inflammation. For the statin I identified three potential ROCK inhibitors - tongkat ali, baicalein (from skullcap) and phloretin. Baicalein looked too improbable from a bioavailability point of view. Tongkat Ali and phloretin looked more promising.

I trialled Tongkat Ali for the first two weeks of my three week protocol. Interestingly it lowered my systolic BP (by 10-15 points). This last week I've been taking 98% phloretin instead. It has lowered my diastolic BP (by 10-15 points)! It also seems to have a glitazone like effect on adipocytes as I've put on body fat. I'll report more fully when my protocol is complete.

Interesting what you say about oxytocin and heart valves. I was considering using it by subQ injection. But Believer reports BP and heart rate side effects from injecting 1mg of oxytocin in the Supplement Forum.

[RWhigham]

1cc OxyLuv subq + TGF-b inh + Rho Kinase inh in the morning dropped my Elite HRV (heart rate variability) from 75 to 49 an hour later, but it fully rebounded the next day. A week ago I repeated this 4 days in a row without noticing much.  Starting today I'm going to do it every other day for a week or so. (I'm trying every other day because Dr. Harold Katcher rejuvenated old rats with an injection given every other day for a total of 4 injections. His rats continued to rejuvenate for a full month, ending as young adults per a number of measurements. He has not revealed the content of the injections pending patent applications). It's been pointed out that a day in the life of a rat corresponds to about a month in the life of a human, so that's an argument for repeating every other month for about 7 months. Rejuvenation that took a month in rats might take 30 months in humans. On the other hand reprogramming cells in culture with Yamanaka factors takes about 20 days, during which their epigenetic age gradually reverses, slowly at first and then faster near the end.

Edited by RWhigham, 24 January 2020 - 11:07 PM.

[RWhigham]

• The purpose of oxytocin is to greatly reduce the amt of TGF-b required
• This is needed because too much TGF-b damages heart valves

"inhibitor" is missing. Should say

• The purpose of oxytocin is to greatly reduce the amt of TGF-b inhibitor required
• This is needed because too much TGF-b inhibitor damages heart valves

Edited by RWhigham, 25 January 2020 - 01:09 AM.

[QuestforLife]

<p>

"inhibitor" is missing. Should say

• The purpose of oxytocin is to greatly reduce the amt of TGF-b inhibitor required
• This is needed because too much TGF-b inhibitor damages heart valves

Do you have a reference for your claim on heart valves?

You can buy 5mg of oxytocin from various peptide vendors. If you used 0.25mg a shot that would be ~12nM in the cells.

Have you got a link to the patent as well?

I'm not sure it's worth filtering and injecting intranasal oxytocin. Can't you get nM levels (in serum) just by snorting (more of) it? Pretty sure I read that in various studies. If it's safe though, I'd prefer to inject proper oxytocin peptide intended for that purpose.

[Turnbuckle]

 

Cardiac Safety of TGF-β Receptor I Kinase Inhibitor LY2157299 Monohydrate in Cancer Patients in a First-in-Human Dose Study

 

The transthoracic 2D echocardiography/color Spectral Doppler examination assessed overall cardiac structure and function, and potential toxicity risks related to damage to the heart valves, myocardium, or pericardium. The left ventricular function was not changed, including in patients who received treatments longer than six cycles (Fig. 7). In 15 patients, there was a change from baseline that was considered per protocol and central review as potentially pathological, because the valve function suggested a change from a lower risk to a moderate risk. Another patient experienced a change by two severity grades (absent to mild) (Table 5). All these changes were not considered medically significant...

https://www.ncbi.nlm...les/PMC4575352/

 

 

 

Not significant for cancer patients, perhaps, as side effects are expected and balanced against the alternative.

[RWhigham]

Have you got a link to the patent as well?

U.S. Patent number: 10265372 "Molecular composition for enhancing and rejuvenating maintenance and repair of mammalian tissues"  - rejuvenation of tissues by oxytocin together with an ALK5 antagonist. This patent is the work of Hanadie Yousef at Irina Conboy's lab at Stanford. Hanadie has since started her own company (Juvena Theraeputics) to develop rejuvenation therapies.

 

Edited by RWhigham, 26 January 2020 - 06:02 PM.

[dlewis1453]

The point of my latest experiment was to see if I could replace the statin and sartan. I identified a 5-LOX inhibitor from boswellia extract to replace the sartan. I trialled it alone and it definitely works for inflammation. For the statin I identified three potential ROCK inhibitors - tongkat ali, baicalein (from skullcap) and phloretin. Baicalein looked too improbable from a bioavailability point of view.

Rather than using the herbal extracts to replace the drugs, you could use them with the drugs. Do you think there could be benefit to taking boswellia and a sartan together, along with tongkat ali and a statin together?

[QuestforLife]

I’ve completed the latest cycle of my protocol.
• I injected Epitalon every other morning for 3 weeks
• I took Boswellia Extract every morning for 3 weeks
• I took Tongkat Ali every morning for the first 2 weeks
• I took Phloretin 98% powder in yogurt every morning for the last week (dropped TA)
• I took Sea Buckthorn Berry Extract every morning for the 1st and 3rd week
• I took AFA every morning for the 2nd week
What have I learnt?
• I couldn’t tell any difference between SBBE and AFA
• Epitalon is very effective for making you sleep more deeply. This time I had to take it in the AM, as in the PM it actually seemed to disrupt my sleep
• Boswellia extract (BE) reduces muscle soreness
• Tongkat Ali (TA) has a fairly strong aphrodisiac effect after a few days
• TA also lowered my systolic BP by ~10-15 points after about a week. This effect disappeared as soon as I stopped taking it.
• Neither BE or TA seemed to have any side effects at the dose I took. I have taken BE for well over a month and TA for 2 weeks (the aphrodisiac effect seemed to wane after about 8 days, possibly related to the drop in BP – the manufacturer does recommend cycling)
• Phloretin had a plethora of effects. I took it about ~100-200mg a day, in hindsight this was too much
• Phloretin made me slightly tired and slightly hungry initially. After a week of dosing I was fatigued and short with my family. It felt like being back on a keto diet. This can be explained by its blocking of glucose receptors (GLUT). Phloretin also seemed to make me put on some fat, which was confirmed by a machine. The effects to me seemed similar to when I took pioglitazone many years ago, and I expect are due to the upregulation of adiponectin and insulin sensitising effects downstream of the blockage of glucose receptors. (See https://www.ncbi.nlm...es/PMC5449513/)
• I expect this also means phloretin will be a powerful autophagy inducer. Phloretin also dropped my diastolic blood pressure by ~10-15 points. This was quite remarkable as my diastolic BP (unlike systolic) is very stable normally. I discontinued phloretin after 7 days and immediately felt fantastic
• I’ve done some further research on phloretin and it appears that the GLUT blockade takes 10uM and upwards (depending on cell type and which GLUT you are referring to). 50% ROCK inhibition only requires 220nM. (See https://iubmb.online...10.1002/iub.315 )
• So if I drop my dose by 3-4x I should still achieve ROCK inhibition but less of the other effects.
Conclusions
I can’t tell without further blood tests whether my alternatives to atorvastatin and losartan are as effective or whether they can be used together perhaps. But the lowering effects on BP and inflammation seem very positive.
Future plans
I’ve discovered that cAMP activation is synergistic with TGF-B inhibition, so next time I will add forskolin to the protocol. (See https://www.ncbi.nlm...pubmed/30711170
I can’t get the full text from the usual places, please let me know if anyone can get hold of it)
I am also considering subQ oxytocin, but there seems to be some discussion over how safe it is. We know it is highly synergistic with TGF-B inhibition however.

Edited by QuestforLife, 29 January 2020 - 10:04 AM.

[dlewis1453]

Thanks for this detailed update! Looking forward to future updates and hopefully continued progress. 

Edited by dlewis1453, 29 January 2020 - 03:28 PM.

[RWhigham]

This Ref from Nov 2019 shows that Sox2 and Klf4 are sufficient to induce pluripotent stem cells that generate teratomas when injected in mice. The cells lost their identity on day 2-4. So perhaps we should not add rho kinase inhibitors (to sitmulate Klf4) when attempting rejuvenation with TGF-b inhibitors (to stimulate Sox2).

[QuestforLife]

This Ref from Nov 2019 shows that Sox2 and Klf4 are sufficient to induce pluripotent stem cells that generate teratomas when injected in mice. The cells lost their identity on day 2-4. So perhaps we should not add rho kinase inhibitors (to sitmulate Klf4) when attempting rejuvenation with TGF-b inhibitors (to stimulate Sox2).

 

Thanks for your post. There are a couple of reasons I think you are wrong to be worried about this. Firstly, in the paper you reference, they actually used SKM, with c-Myc to increase efficiency. I’ll have to search through the paper again to confirm they could do it without c-Myc, but I’ll take your word for it. Even so it’s going to be a very long shot to do this in vitro. Remember in vitro the cells are sitting in culture being hammered by these factors, and even so it takes 2-4 days to start losing their somatic identities, and then the shift to pluripotency is actually at about day 12. But the main reason I’m not worried is this paper: ‘Direct in vivo Application of Induced Pluripotent Stem Cells Is Feasible and Can Be Safe’.

https://pubmed.ncbi....nd-can-be-safe/

 

 

Abstract
Increasing evidence suggests the consensus that direct in vivo application of induced pluripotent stem cells (iPSCs) is infeasible may not be true. Methods: Teratoma formation and fate were examined in 53 normal and disease conditions involving brain, lung, liver, kidney, islet, skin, hind limb, and arteries. Results: Using classic teratoma generation assays, which require iPSCs to be congregated and confined, all mouse, human, and individualized autologous monkey iPSCs tested formed teratoma, while iPSC-derived cells did not. Intravenously or topically-disseminated iPSCs did not form teratomas with doses up to 2.5×108 iPSCs/kg and observation times up to 18 months, regardless of host tissue type; autologous, syngeneic, or immune-deficient host animals; presence or absence of disease; disease type; iPSC induction method; commercial or self-induced iPSCs; mouse, human, or monkey iPSCs; frequency of delivery; and sex. Matrigel-confined, but not PBS-suspended, syngeneic iPSCs delivered into the peritoneal cavity or renal capsule formed teratomas. Intravenously administered iPSCs were therapeutic with a dose as low as 5×106/kg and some iPSCs differentiated into somatic cells in injured organs. Disseminated iPSCs trafficked into injured tissue and survived significantly longer in injured than uninjured organs. In disease-free animals, no intravenously administered cell differentiated into an unwanted long-lasting cell or survived as a quiescent stem cell. In coculture, the stem cell medium and dominant cell-type status were critical for iPSCs to form cell masses. Conclusion: Teratoma can be easily and completely avoided by disseminating the cells. Direct in vivo iPSC application is feasible and can be safe.

 

The bold highlights are mine. In short, so long as the signalling of the surrounding tissue is stronger than that of the iPSC, no teratomas will form. The issue only occurs if many IPSCs are created and kept in a confined area. This seems to me to be highly unlikely. Also, ROCK inhibitors are effective because they give cells a simpler cytoskeleton. This helps them break free from the extra cellular matrix and become more stem-like. So, using ROCK inhibitors is likely to lead to less confined cells not more.

Realistically we are up against it here to make any iPSCs. Even replicating the Conboys study, which I assume only partially de-differentiated cells and didn’t form any iPSCs, using strong tgf-b inhibition and strong MAPK upregulation, is going to be hard (as we don’t have access to the ALK inhibitor).  I have some ideas on how we might attempt to do it. But it will most likely require ROCK inhibitors.

My best guess it will need

• Tgf-b inhibition – it is an open question whether anything we have can repress this sufficiently
• ROCK inhibition – this may address the above somewhat as tgf-b tends to activate ROCK
• cAMP activation – this has lots of helpful cross-talk with both tgf-b inhibition and ROCK inhibition
• MAPK activation – as per the Conboys, but with a smaller/safer dose of Oxytocin

[RWhigham]

Biogaia Osfortis L-reuteri 6475 increases oxytocin (REF1, REf2)

• Drinking L. reuteri leads to higher blood levels of oxytocin in mice
• Oral L. reuteri down-regulates blood levels of stress hormone corticosterone
• Thymus gland size is increased after oral dosing with L. reuteri
• Circulating neutrophil counts are decreased in mice consuming L. reuteri

Edited by RWhigham, 08 February 2020 - 12:39 AM.