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FOXO4 D-Retro-Inverso peptide group buy

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#271 meatsauce

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Posted 24 January 2018 - 06:36 PM

The next batch should be two weeks!


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#272 Moondancer

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Posted 24 January 2018 - 11:19 PM

There has been a brief discussion aboout injecing IM versus SQ. Rocket mentioned injecting a substance IM may be dangerous because of potential blood infection. 

Yet others mentioned injecting IM may increase effectivity. What is the current consensus, injecting SQ rather? Since it is not exactly a cheap peptide ;-) I'd want to be sure not to 'waste' it either.



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#273 extendcel

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Posted 24 January 2018 - 11:27 PM

There has been a brief discussion aboout injecing IM versus SQ. Rocket mentioned injecting a substance IM may be dangerous because of potential blood infection.
Yet others mentioned injecting IM may increase effectivity. What is the current consensus, injecting SQ rather? Since it is not exactly a cheap peptide ;-) I'd want to be sure not to 'waste' it either.


It really doesn't matter much. Either one can result in systemic infection. There is a difference in absorption time with IM and subq. It's always best to test it subq first though, but it should make a negligible difference in absorption.
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#274 framework7

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Posted 26 January 2018 - 12:53 AM

Question.  I have been waiting a bit while doing further research, and it appears that de Keizer's group fused the FOXO4-DRI peptide with an HIV-tat peptide sequence in order to enable it to actually permeate phospholipid bilayer cell membranes in the first place.  It's not clear to me that the FOXO4-DRI sequences published have always included this HIV-tat subsequence.  It doesn't look like it to me.

Here are some references:

http://www.longecity...de/#entry814508

>And they say the FOXO4-DRI was fused with HIV-TAT and then injected into the mice.

 

https://www.ncbi.nlm...pubmed/28340347

>To facilitate cellular uptake, the inverso peptide was fused to a HIV-TAT peptide. The resulting FOXO4-DRI was found to bind p53 with even higher affinity than the corresponding forkhead region of FOXO4 and caused dissociation of the FOXO4-p53 complex. 

http://www.cell.com/...8674(17)30293-3 (original Cell paper) 

>To facilitate cellular uptake of FOXO4-DRI, it was designed as a fusion with HIV-TAT, a basic and hydrophilic sequence that allows energy-independent cellular uptake of cargo through transient pore formation (Herce and Garcia, 2007). Using an antibody against HIV-TAT, we observed FOXO4-DRI to be taken up as soon as 2–4 hr after administration and to remain detectable for at least 72 hr (Figure 2J). Given that the affinity of antibodies is generally low, this indicates FOXO4-DRI effectively enters senescent cells at high intracellular concentrations, which remain abundant and stable over a prolonged period of time. Following its uptake, FOXO4-DRI reduced the number of senescence-induced FOXO4 foci, PML bodies, and 53BP1 DNA-SCARS while not affecting the number of small 53BP1 foci (Figure 2K).

http://www.pnas.org/...nt/104/52/20805 (cited by the Cell paper for HIV-tat): 

>The recombinant HIV-1 Tat protein contains a small region corresponding to residues 47YGRKKRRQRR57R, which is capable of translocating cargoes of different molecular sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner.

The sequences that I have seen for FOXO4-DRI do not include the 'YGRKKRRQRR' HIV-tat peptide subsequence.

Example: this sequence does not include 'YGRKKRRQRR':


https://www.novoprol...ide-318716.html

>H-LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG-OH

Neither does this one:


http://www.buckylabs.com/foxo4dri/

>Sequence: H-D-Leu-D-Thr-D-Leu-D-Arg-D-Lys-D-Glu-D-Pro-D-Ala-D-Ser-D-Glu-D-Ile-D-Ala-D-Gln-D-Ser-D-Ile-D-Leu-D-Glu-D-Ala-D-Tyr-D-Ser-D-Gln-D-Asn-D-Gly-D-Trp-D-Ala-D-Asn-D-Arg-D-Arg-D-Ser-D-Gly-D-Gly-D-Lys-D-Arg-D-Pro-D-Pro-D-Pro-D-Arg-D-Arg-D-Arg-D-Gln-D-Arg-D-Arg-D-Lys-D-Lys-D-Arg-D-Gly-OH


i.e. (https://www.peptide2...d_converter.php ):

>LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG*

Note that YGRKKRRQRR is not comprised of D- amino acids, and all of these listed are D- amino acids, which further supports the idea that these sequences people are quoting around are not including the HIV-tat subsequence to pass through cell membranes.


To meatsauce, and anyone else who has looked into synthesizing FOXO4-DRI for injection:

Could you state the specific peptide sequence that you are having synthesized right now?  Does it include the HIV-tat subsequence?

EDIT: are they using some sort of modified form of HIV-tat that does not have the same sequence...?  I am skeptical but could be persuaded by sufficiently strong evidence if that were the claim being made.  I just don't see the HIV-tat sequence as stated in these FOXO4-DRI sequences.

Edited by framework7, 26 January 2018 - 12:56 AM.

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#275 framework7

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Posted 26 January 2018 - 02:34 AM

The patent on FOXO4-DRI does not include the exact subsequence 'YGRKKRRQRR' either, or allude to HIV-Tat by name:

https://www.google.c...6118014A2?cl=en

>The invention relates to a peptide comprising the amino acid sequence LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP, wherein the amino acids in said amino acid sequence are D-amino acid residues, and to methods for the use of this peptide in the treatment of age-related disorders

The sequence from Bucky Labs grafts on an additional ''PPRRRQRRKKRG" but it is made of D- amino acids and does not appear to match the HIV-Tat sequence from Herce and Garcia 2007 which stated L- amino acids "YGRKKRRQRR" instead ( http://www.pnas.org/...nt/104/52/20805 ).  It definitely contains most of the HIV-Tat sequence (just written in reverse order), but it is missing the "Y", includes an extra "RPP" afterward, and appears to be made of D- amino acids.  Is this an alternate form of HIV-Tat sequence?  Is there a paper somewhere justifying this, or confirming that this is indeed the correct variant of HIV-Tat that was used in the Cell paper?  Needs to be validated.
 

EDIT: nevermind -- the patent does state:
 
>3. A peptide according to claim 1 or 2, further comprising a cell- penetrating peptide sequence, preferably said cell-penetrating peptide sequence has the amino acid sequence PPRRRQRRKKRG, preferably wherein the amino acids in said cell-penetrating peptide sequence are D- amino acid residues.
 
>4. Peptide according to claim 3, wherein said cell-penetrating peptide is fused to the C-terminal part of said peptide.
 
So this appears to be correct from the de Keizer patent application.  They are indeed using a modified version of HIV-Tat and making it out of D- amino acids.

This is the exact sequence being used for the group buy, right?

Edited by framework7, 26 January 2018 - 02:49 AM.

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#276 meatsauce

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Posted 26 January 2018 - 04:28 AM

 

The patent on FOXO4-DRI  [...] does state:

 
>3. A peptide according to claim 1 or 2, further comprising a cell- penetrating peptide sequence, preferably said cell-penetrating peptide sequence has the amino acid sequence PPRRRQRRKKRG, preferably wherein the amino acids in said cell-penetrating peptide sequence are D- amino acid residues.
 
>4. Peptide according to claim 3, wherein said cell-penetrating peptide is fused to the C-terminal part of said peptide.
 
So this appears ... They are indeed using a modified version of HIV-Tat and making it out of D- amino acids.

This is the exact sequence being used for the group buy, right?

 

 

I covered this in page 3 of the thread 

 

This is from the tat hiv wikipedia page

 

"Tat contains a protein transduction domain, and is therefore known as a cell-penetrating peptide.[14] Originally[15] characterised by Frankel and Pabo (1988)[16] and Green and Loewenstein (1988),[17] this domain allows Tat to enter cells by crossing the cell membrane. The amino acid sequence of the protein transduction domain is YGRKKRRQRRR.[14] The nuclear localisation signal found within the domain, GRKKR, mediates further translocation of Tat into the cell nucleus.[18][19] As of 2000 The biological role of this domain and exact mechanism of transfer is unknown.[14]

 

The FOXO4 DRI sequence is: ltlrkepaseiaqsileaysqngwanrrsggkrppprrrqrrkkrg. The purple sequences matching up are going to be in reverse order.  This is a retro-inverso peptide so that means the sequence is reversed and the l amino acids are substituted with D amino acids. 

 

"Retro-inverso peptides are linear peptides whose amino acid sequence is reversed and the α-center chirality of the amino acid subunits is inverted as well. Usually, these types of peptides are designed by including D-amino acids in the reverse sequence to help maintain side chain topology similar to that of the original L-amino acid peptide and make them more resistant to proteolytic degradation." - biosyn.com

 

 

Yes this is the exact sequence being used! :)

 

 


 


Edited by Michael, 29 July 2018 - 07:59 PM.

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#277 BieraK

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Posted 28 January 2018 - 12:28 AM

I wonder if this peptide could eliminate senescent and damaged microglia, such thing could be great as a preventive of neurodegenerative and neuro-inflammatory conditions.

We need something for get rid off senescent cells and something for reversing DNA transcription changes of cells typical of aging we these two thing we are in the next level of figthing aging and aging related disease


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#278 Orinoco

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Posted 28 January 2018 - 01:07 AM

I would have hoped the first feedback would have been posted. When did the guys of the first group start using FOXO4 DRI inverso? Shouldn't the first effects (if any at all) have been observed within 10 days after treatment with the peptide?


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#279 meatsauce

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Posted 28 January 2018 - 03:31 AM

Im the first one to try it so far. Two other people received theirs with big orders and one has a few family members and friends taking it but I don't think they started yet. 

 

Im relatively young so I don't have many senescent cell to get rid of but I can say that Ill do double takes in the mirror and expect myself to look older than what I see. I think my skin looks nicer. I had no wrinkles before and am pretty healthy so there wasn't much improvement to be had though. 

 

The older people trying it will be the best candidates to judged its effects.

 

Also according to the paper if Anti-inflammatories were used the peptide was not effective and and more inflamed the cells were the better it worked. So when you take it you should be off all Anti-inflammatory, supplements, drugs, herbs, and fish oil. Eat like crap if you want it will only help. 


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#280 meatsauce

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Posted 30 January 2018 - 06:52 PM

We are about to get the rest of the order!


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#281 Daniel Cooper

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Posted 31 January 2018 - 10:24 PM

I wonder if this peptide could eliminate senescent and damaged microglia, such thing could be great as a preventive of neurodegenerative and neuro-inflammatory conditions.

We need something for get rid off senescent cells and something for reversing DNA transcription changes of cells typical of aging we these two thing we are in the next level of figthing aging and aging related disease

 

 

Are we certain that this peptide gets past the BBB?



#282 tunt01

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Posted 01 February 2018 - 05:28 PM

I fell out of the loop on this group purchase/FOXO4-DRI.  Is there any available and what is pricing?

 

Thanks.



#283 meatsauce

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Posted 03 February 2018 - 03:54 AM

I fell out of the loop on this group purchase/FOXO4-DRI.  Is there any available and what is pricing?

 

Thanks.

Yes we are doing another order which should be quicker I am about to get the rest of the first one. I think I pm'd you. You can pm me If I didn't. 



#284 helix66

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Posted 03 February 2018 - 09:06 AM

 

when you say stealth packaging, do you mean not listing what is inside the package or listing it as some other item. If the later what do you recommend listing it as?

 

Stealth as in "reasonable deniability" . You dont want to mislabel something (eg call it "childs doll" )  as that could be a red flag. However you can use vague or tangential references .."ie Beauty Product "  or as is normally put on the numerous vials of RC's and peps I have received -"MS samples"  - whatever that is.

 

PS -I generally make a small animal sacrifice to Mercury the messenger of the Gods every time I order a package - seems to work.. :)

 

 

 

Thanks for your feedback Zed. Did the packages listed as MS Samples usually have a $ cost of packaged items listed? And if yes, what was the ballpark price?

 

Also what animal do you suggest I sacrifice... Would a cockroach do? :)

 


Edited by Michael, 29 July 2018 - 09:52 PM.
Helix original question, trim quotes

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#285 Nate-2004

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Posted 03 February 2018 - 05:23 PM

Also according to the paper if Anti-inflammatories were used the peptide was not effective and and more inflamed the cells were the better it worked. So when you take it you should be off all Anti-inflammatory, supplements, drugs, herbs, and fish oil. Eat like crap if you want it will only help. 

 

Very good to know, hope everyone sees this. Sounds pretty important. I'll try to get in on the next deal, let me know when.



#286 stefan_001

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Posted 03 February 2018 - 05:42 PM

removed

 


Edited by stefan_001, 03 February 2018 - 05:53 PM.


#287 stefan_001

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Posted 03 February 2018 - 05:52 PM

 

Also according to the paper if Anti-inflammatories were used the peptide was not effective and and more inflamed the cells were the better it worked. So when you take it you should be off all Anti-inflammatory, supplements, drugs, herbs, and fish oil. Eat like crap if you want it will only help. 

 

Very good to know, hope everyone sees this. Sounds pretty important. I'll try to get in on the next deal, let me know when.

 

 

The broad range SASP inhibitors Cortisol and Rapamycin and an antibody against IL-1α reduce the effectiveness of FOXO4 DRI in lowering senescent cell viability, while the general inflammation enhancer Lipopolysaccharide (LPS) improves it. Together these two panels demonstrate FOXO4 DRI specifically targets those senescent cells which express high SASP as is the case in many senescence-related diseases in vivo



#288 stefan_001

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Posted 03 February 2018 - 06:39 PM

@meatsauce when do you close the registration for the next batch? I would be interested as well. Also very curious to hear more results. Thanks already.


Edited by stefan_001, 03 February 2018 - 06:42 PM.


#289 meatsauce

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Posted 03 February 2018 - 08:23 PM

@meatsauce when do you close the registration for the next batch? I would be interested as well. Also very curious to hear more results. Thanks already.

Im going to place the order soon so If you want to get in PM me!



#290 Nate-2004

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Posted 04 February 2018 - 02:33 PM

 

 

Also according to the paper if Anti-inflammatories were used the peptide was not effective and and more inflamed the cells were the better it worked. So when you take it you should be off all Anti-inflammatory, supplements, drugs, herbs, and fish oil. Eat like crap if you want it will only help. 

 

Very good to know, hope everyone sees this. Sounds pretty important. I'll try to get in on the next deal, let me know when.

 

 

The broad range SASP inhibitors Cortisol and Rapamycin and an antibody against IL-1α reduce the effectiveness of FOXO4 DRI in lowering senescent cell viability, while the general inflammation enhancer Lipopolysaccharide (LPS) improves it. Together these two panels demonstrate FOXO4 DRI specifically targets those senescent cells which express high SASP as is the case in many senescence-related diseases in vivo

 

So, obviously avoid anything anti-inflammatory that day. I wonder what I would eat during a day of dosing.

 

By the way, how is everybody dosing this or are you all doing this differently? I think that info got lost in the mess of this thread.



#291 RenewYou

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Posted 04 February 2018 - 03:08 PM

 

 

 

Also according to the paper if Anti-inflammatories were used the peptide was not effective and and more inflamed the cells were the better it worked. So when you take it you should be off all Anti-inflammatory, supplements, drugs, herbs, and fish oil. Eat like crap if you want it will only help. 

 

Very good to know, hope everyone sees this. Sounds pretty important. I'll try to get in on the next deal, let me know when.

 

 

The broad range SASP inhibitors Cortisol and Rapamycin and an antibody against IL-1α reduce the effectiveness of FOXO4 DRI in lowering senescent cell viability, while the general inflammation enhancer Lipopolysaccharide (LPS) improves it. Together these two panels demonstrate FOXO4 DRI specifically targets those senescent cells which express high SASP as is the case in many senescence-related diseases in vivo

 

So, obviously avoid anything anti-inflammatory that day. I wonder what I would eat during a day of dosing.

 

By the way, how is everybody dosing this or are you all doing this differently? I think that info got lost in the mess of this thread.

 

 


I would avoid ALL anti-inflammatories for a full 2 weeks prior. & 2 weeks following, Including fish oils, tumeric etc etc

 

Read pre-op instructions for anti-inflammatories - or for something like PRP

 

 



#292 Nate-2004

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Posted 04 February 2018 - 03:23 PM

Why such a long period prior and post?



#293 RenewYou

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Posted 04 February 2018 - 03:30 PM

Vacillates between 1 & 2 weeks depending on who you speak with. The anti-inflammatory effect remains in system / blood many days after ingesting. 2 is on the very 'safe' sde

 

You can stop a day before as well & see how it goes 



#294 jdlancaster519

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Posted 04 February 2018 - 06:10 PM

When will the first results be coming in?



#295 recon

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Posted 04 February 2018 - 06:45 PM

I’m wondering about the lack of experience posts. Usually these threads will be filled with experience posts almost immediately, even on benign substances.

#296 meatsauce

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Posted 04 February 2018 - 07:03 PM

I’m wondering about the lack of experience posts. Usually these threads will be filled with experience posts almost immediately, even on benign substances.

I'm the only one who has tried it so fat. The few other people who got the first part of the order Are beginning soon. There will be more reports once this second batch goes out. 


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#297 Vantika

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Posted 04 February 2018 - 08:05 PM

When will the first results be coming in?

 

I’m wondering about the lack of experience posts. Usually these threads will be filled with experience posts almost immediately, even on benign substances.

 

I got most of the first batch, and still have it in my freezer.  I'm still preparing to take it, trying to ramp up SASP to maximize the number of senescent cells killed, which in my case involves temporarily abandoning my otherwise strict ketogenic diet, increasing my omega-6:omega-3 ratio (I normally consume a minimal amount of omega-6 fatty acids), washing out various anti-inflammatory supplements, and figuring out what else I can do to maximize the effect.
 
As for results/experiences, what are you expecting?  I'm in my mid 30s and am in generally good health, so I expect no perceptible results.  Senescent cells accumulate throughout the lifespan at an exponential rate (I mean exponentially literally, in accordance with its mathematical meaning, not as some general, flippant figure of speech). Thus, at my age I have relatively few of them -- and consequently very little pathology caused by them.
 
My expectation here is to kill a subset of them in some or most of my organs/tissues, thereby paying down some of the "interest" on this continuously compounding "debt" (the metaphor is mathematically accurate, in that both undergo exponential growth).  Ultimately, if all goes well, I should slow my rate of aging to varying degrees in various tissues, roughly in proportion to the respective extent of senescent cell clearance within each.
 
EDIT: To clarify the above, senescent cells eventually convert (many of) their neighbors to senescence, through paracrine signalling. This is why they accumulate exponentially (i.e. as an exponential function of their current population levels) and why clearing some of them out should reduce their rate of accumulation.  Look at the graph of an exponential function if this isn't clear.  Periodic clearance should keep the rate of population growth low, thereby keeping things from (exponentially) spiraling out of control, into the rapidly accelerating progression of aging that naturally happens.
 
Furthermore, given that I'm feeling a bit shitty right now from all of these pro-inflammatory dietary changes I've recently made (not from the peptide, which, again, I haven't even started yet), I expect any effects from the peptide itself that otherwise would be perceptible will instead be masked -- when I do take it -- by my transition from an anti-inflammatory diet/supplement regimen to a pro-inflammatory diet/supplement regimen and then finally back to an anti-inflammatory diet/supplement regimen after I've finished dosing.
 
So don't expect much (if any) useful information from me. I'm going for maximal effect for myself, not maximal information for everybody else. :)

Edited by Vantika, 04 February 2018 - 08:39 PM.

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#298 jdlancaster519

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Posted 04 February 2018 - 08:30 PM

 

When will the first results be coming in?

 

I’m wondering about the lack of experience posts. Usually these threads will be filled with experience posts almost immediately, even on benign substances.

 

I got most of the first batch, and still have it in my freezer.  I'm still preparing to take it, trying to ramp up SASP to maximize the number of senescent cells killed, which in my case involves temporarily abandoning my otherwise strict ketogenic diet, increasing my omega-6:omega-3 ratio (I normally consume a minimal amount of omega-6 fatty acids), washing out various anti-inflammatory supplements, and figuring out what else I can do to maximize the effect.
 
As for results/experiences, what are you expecting?  I'm in my mid 30s and am in generally good health, so I expect no perceptible results. ..
 
Furthermore, given that I'm feeling a bit shitty right now from all of these pro-inflammatory dietary changes I've recently made (...I expect any effects from the peptide itself that otherwise would be perceptible will instead be masked -- when I do take it -- by my transition ... back to an anti-inflammatory diet/supplement regimen after I've finished dosing.
 
So don't expect much (if any) useful information from me. I'm going for maximal effect for myself, not maximal information for everybody else. :)

 

My hope was for objective data such as blood work and break them down into categories such as regimen and age. It would take a long time to get enough data points for any significant statistical analysis (especially since there doesn't seem to be a single protocol that is being followed), but it seems a shame to take the risk of an unproven treatment without collecting the data. As well as unperceived benefits, there could also be unperceived negatives. The negatives (such as increase in bp, cholesterol, strange enzyme levels, etc.) may be easier to see in a young healthy persons bloodwork... this is all speculation of course, but it couldn't hurt to do a before and after bloodwork at any age.


Edited by Michael, 29 July 2018 - 09:57 PM.
trim quotes

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#299 jdlancaster519

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Posted 07 February 2018 - 08:10 PM

I was reading some papers today and saw  Quantitative identification of senescent cells in aging and disease by Biran et al. 2017. They compared the amount of senescent cells in young (2 months) and old (24 months) mice as a small part of their paper. The relevant data : "quantification showed the percentage of senescent cells in the various young and old mouse tissues with age (respectively, 1.4 ± 0.35% and 13.8 ± 4.34% in subcutaneous adipose-tissue-derived stromal cells, 0.19 ± 0.06% and 3.53 ± 1.59% in spleen cells, 0.3 ± 0.06% and 3.3 ± 0.76% in small intestinal cells, 0.16 ± 0.03% and 1.48% ±1.1% in lymph node cells, and 5.9 ± 0.86% and 6.7 ± 0.6% in lung cells)".

I found the great difference by tissue type to be interesting. 


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#300 The Capybara

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Posted 07 February 2018 - 09:33 PM

This brings up a point I had made a ways back.
It seems that if FOXO4-dri pans out for people, as it did for rodents, one should at least be aware of tumor lysis syndrome.
Along the same logic, I'm a little concerned about the lack of reported side effects, albeit on the start of a very limited human trial here on Longecity. I'd think that effectiveness of this compound as well as proper dosage, might be positively correlated with certain side effects related to a cellular die off.
Mice seem far far less susceptible to tumor lysis syndrome than humans, which would argue against using the rodent studies as an argument against this concern.


Edited by The Capybara, 07 February 2018 - 09:37 PM.

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