Keep checking back as this post is often updated! Updates usually added at the end.

MAKE SURE YOU CHECK OUT UPDATE #9 – It is amazing!  #13 is pretty cool too!

See Update #23  For Another study that proves all of what you are about to read is correct- aging  was recently reversed in old rats by 50 to 75% using the ideas discussed herein!

Update #24 7/19/2021     Update #25  8/5/2021  Aging reversed by 2 years in humans with GH, DHEA, and metformin


Feeling lazy?>>>  watch these podcasts   >>>.

or  >>>>>>>>>  but make sure you look over this blog post  afterwards  has a lot more interesting explanations of additional very interesting things  like progeria and Werner’s syndrome , rapidly aging salmon, etc.



Before we get started let me just whet your appetite about what is contained in the rest of this article. The results of the most important study on aging EVER, that will be the most important study of aging for all time- have just been released! Steve Horvath’s :

Universal DNA methylation age across mammalian tissues

The study proves conclusively that aging is selected for by evolution and is programmed. A result that contradicts all major mainstream theories of aging that have been proposed since the early 1900’s. It turns out August Weisman got the right answer in 1882 but with the wrong reasoning.

The new study also reveals the true cause of aging at the cellular level- the programmed loss of cellular differentiation.

Recently, a preprint of a journal article that is expected to be published in Nature, was released that completely breaks open the cause of aging in mammals of almost all species. The paper shows that this aging is highly conserved by evolution and ends the debate about whether aging is caused by accidental DNA damage  or is programmed. The answer?- aging evolved, is highly conserved, and  is programmed- no doubt about it!

The paper was lead-authored by  Steve Horvath and co-authored with a long list of collaborators. It is currently titled>> Universal DNA methylation age across mammalian tissues.

I think it is the most important study concerning aging and always will be. And I have been studying aging for 35+ years and have seen almost everything! Horvath’s travels through the methylation of the DNA of so many animals is certainly as important as  and probably more so than Darwin’s 5 years on the HMS Beagle.

Here is the link  to the preprint>>

In the last sentence of his abstract Horvath bravely states-

“Collectively, these new observations support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species – a notion that was long-debated without the benefit of this new and compelling evidence.”




All those evolution professors  are going to have to go back to the drawing board because in their view of evolution it is impossible for aging to have evolved  and be selected for because it is bad for the spread of your selfish genes! Virtually all evolutionary biologists believe that aging being selected for by  evolution is impossible!

Actually, I propose in another article with a link at the end of this one, that the selfish gene theory of evolution is mostly correct  but it is only half the story. There is a missing half  of the theory of evolution that has yet to be revealed. I take a stab at it, and succeed,  in the article linked to at the end. Okay  back to aging…

Here is a summary of what Horvath et al  found:

Horvath and this team looked at the DNA of a large number of mammals  and determined what were the genes that experienced  major changes of DNA methylation (both increases and decreases) at older ages.  Increased methylation at the beginning of a gene would basically shut it down, removal of methylation from the beginning of a gene would allow that gene to be expressed at older ages

They looked at the DNA methylation changes with age in 59 different tissue types from 128 mammalian species to see what they all had in common.

They found a highly conserved aging program driven by DNA methylation changes that for the most part  shut down genes that produced transcription factors  by adding methyl groups to the promoter area of the genes. They found 36 genes that were affected  /shut down  by DNA methylation and almost all of them were transcription factors that are involved in the differentiation of cells during development  that have homeobox domains. They found very few (12) genes that experienced loss of methylation which was a surprise to me based on my predictions in my 1998 paper . I expected it to be the other way around because the entire genome loses a lot of methylation during aging. So, these instances of hypermethylation must be very  special to buck the overall trend in the global DNA demethylation with age, apparently most DNA methylation is uninvolved with direct aging control.

Overall, they found 3,617  cases of hypermethylated  cytosines in the DNA associated with aging and only  12 hypomethylated cytosines!  This blew my mind.

Well, those 12 hypomethylated  sites must be next to some very interesting genes! They analyzed these hypomethylated genes and found the #1 gene that was most hypomethylated in liver and #2  across all tissue types was the LARP1 gene.  This gene being more expressed at older ages  must be doing something very naughty!  Let us take a look at the LARP 1 gene’ function as described by Wikipedia>>>>

Well, what do you know?? LARP1 has a unique region that binds to RNA transcripts! My guess is that this is the protein that is involved in truncating the Lamin A protein in normally aging cells, and likely it is truncating the WRN protein in normally aging cells (truncated WRN protein being found in normally aging and senescent cells has yet to be shown true-but I predict someday this will be found to be occurring).

(If this LARP1 info is boring to you now  you can skip over it to get to the good stuff and come back later!)


From Wikipedia, the free encyclopedia

La-related protein 1 (LARP1) is a 150 kDa protein that in humans is encoded by the LARP1 gene.[5][6][7] LARP1 is a novel target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, a circuitry often hyperactivated in cancer which regulates cell growth and proliferation primarily through the regulation of protein synthesis.[8]


LARP1 is the largest of a 7-member family of LARP proteins (others are: LARP1B, LARP3 (aka genuine La or SSB), LARP4A, LARP4B, LARP6 and LARP7).[9] All LARP proteins, including human LARPs, contain 2 conserved regions. The first conserved region shares homology with La proteins (called the La motif, see SSB) whereas the second conserved region (called the LA- motif) is restricted to LARP proteins. LARP1 and 1B also contain a conserved “DM15 region” within their C-terminus.[10] This region is unique and has been shown to be required for RNA-binding. Mouse Larp1 is expressed in dorsal root ganglia and spinal cord, as well as in developing organs characterized by epithelialmesenchymal interactions.[6] Human LARP1 is present at low levels in normal, non-embryonic cells but is highly expressed in epithelial cancers (such as ovarian, colorectal, prostate, non-small cell lung, hepatocellular and cervical cancers).[11][12][13][14] Some studies have shown that high levels of LARP1 protein correlate with worse prognosis in cancer patients.[15][16]

LARP1 binds to and regulates the translation of terminal oligopyrimidine motif (TOP mRNAs) and can directly interact with the 5′ cap of mRNAs.[17][18] It has also been shown to interact with the 3′ end and coding regions (CDS) of other genes.[17] LARP1 protein colocalizes with stress granules and P-bodies,[19] which function in RNA storage and degradation. It has been suggested that LARP1 functions in P-bodies to attenuate the abundance of conserved RasMAPK mRNAs. The cluster of LARP1 homologs may function to control the expression of key developmental regulators.[19]

Several studies have demonstrated that LARP1 deficiency selectively affects the recruitment of TOP mRNAs to polysomes In some cancer cells, LARP1 deficiency reduces proliferation and activates apoptotic cell death.[13] Even though a decrease abundance of proteins encoded by TOP mRNAs has been reported in LARP1 silenced cells, some researchers believe that this can be explained simply by the reduced number of TOP mRNA transcripts in LARP1-deficient cells.

It turns out I predicted most of this  long ago in 1998.

In 1998, I published a paper titled “The Evolution of Aging: A New Approach to an Old Problem of Biology”

in Medical Hypotheses Sep 1998.

This paper was the result of almost 10 years of non-stop 7 day a week, feverish research at the Northwestern Medical School library where I read everything I could find about aging. At the end of 10 years, I was like the first paleontologist who had uncovered the complete skeleton of a dinosaur but the bones were strewn about. I had identified almost all the relevant factors related to aging. It was time to put the bones together to see what the dinosaur looked like. Just like that first paleontologist’s attempt, my assembled dinosaur (aging theory)   was mostly correct, but there were some bones placed in the wrong position.

Many good predictions came out of the paper which proved to be true such as:

-Aging is driven by the loss of DNA methylation of cytosines (actually driven by cytosine’s gain or loss of methylation  (CH3’s))  also known as epigenetics. The next paper confirming this prediction did not come out until 2012 >  Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock. Cell 2012 Jan 20;148(1-2):46-57.  (written by some guy at Stanford- who did not mention my 1998 paper )

-Alzheimer’s and dementia would be found to be driven by the increase in Luteinizing Hormone that occurs  after age 50 in both men and women. Confirmed in 2005 at the NIH>> Evidence for the role of gonadotropin hormones in the development of Alzheimer disease. Cell Mol Life Sci. 2005 Feb;62(3):293-8.

-Luteinizing Hormone and Follicle Stimulating Hormone would be found to play a central role in aging . Confirmed >> Data mining of human plasma proteins generates a multitude of highly predictive aging clocks that reflect different aspects of aging. October 2020  Aging Cell 19(1):e13256 ( the #1 and #2  proteins that increase the most in the aging cell are related to LH(#1) and FSH (#2).)

-The Hierarchy of programmed aging control was predicted to be

Hormone Changes>> Loss of Methylation >>> Expression of genes that cause aging.

This study proves this to be true, but to a lesser extent than I expected. What I did not expect was another hierarchy revealed by this study

Hormone Changes >>>>  Gain of Methylation >>>  Suppression of genes required for cellular differentiation.

-And one more  little thing predicted in my 98 paper , that aging EVOLVED  and is PROGRAMMED and is controlled by the same things that control development.

-The first 2 sentences in the abstract of my paper claimed that aging evolved and aging and development were intimately linked. This new study proves it to be 100% true.

The evolution of aging: a new approach to an old problem of biology”

Bowles, JT Medical Hypotheses Sep 98


“Most gerontologists believe aging did not evolve, is accidental, and is unrelated to development.

The opposite viewpoint is most likely correct.”

The problems with the paper were caused by my trying to put all the aging puzzle pieces together without enough information. For example, I imagined that the protein that is defective in Werner’s Syndrome (truncated) was generating excessive free radicals during the DNA unwinding process that catalyzed the demethylation of cytosines in the DNA. I thought this allowed pro-aging genes to be expressed, filling the body with destructive proteins. I received endless  ridicule and derision from mainstream aging theorists who believed that the evolution of pro-aging genes was impossible. The new study shows that there are pro-aging genes, just not as many as I had imagined. It turns out that  a lot of the programmed aging is caused by the suppression of genes that make transcription factors involved in maintaining cellular differentiation during and after development.

In reality what was happening was that the WRN helicase consists of 6 identical subunits which come together to form a helicase. The job of a helicase is to unwind and rewind the DNA. The single subunit also has another job as a transcription factor that binds to and silences some genes in stem cells to allow them to retain their differentiation and remain stem cells. But this was not known when I wrote the paper, so I gave it my best guess.

See>> A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging  Science. 2015 Jun 5; 348(6239): 1160–1163.


Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of  and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.

“Finally, we asked whether heterochromatin disorganization could be a common hallmark for physiological human stem cell aging. For this purpose, we compared the levels of heterochromatin marks in primary dental pulp MSCs derived from six young (7–26 year old) and six old (58–72 year old) individuals (fig. S10I, and Table S4) (20). A marked downregulation of WRN protein associated with a decrease in H3K9me3, HP1α, SUV39H1, and LAP2β levels in MSCs derived from old individuals (Fig. 4E). Therefore, specific heterochromatin changes may underlie both pathological as well as physiological mesenchymal stem cell aging.

In summary, we have found that WRN protein, besides its role in DNA repair, functions to safeguard heterochromatin stability (fig. S11). Our results unveil that the progressive heterochromatin disorganization observed in WRN deficient MSCs underlies cellular aging, but more extensive studies are needed to examine its role during physiological aging.”

Werner’s Syndrome is a rapid aging disease that kicks in around puberty and leads to  thoroughly aged people by the age of 45 or so>>

Werner’s Syndrome is caused by the WRN protein being improperly truncated so that it is too short  to do its job properly of preserving the differentiation status  of human stem cells.

Werner’s Syndrome is very much the same as normal aging. These patients have all the classic signs of aging , but they also have some extra-rare forms of disease which is what has led scientists to try and claim that this was not real aging. WS patients are afflicted with quite a few rare cancers  as well as the normal aging processes.

I believe that the excess of rare cancers  and other oddities associated with WS  are caused not by the single truncated protein which causes all the features of normal aging, but rather by the improper functioning for the DNA helicase made by the 6 identical but defective WRN helicase subunits. Because proper functioning of DNA helicases are required for proper DNA maintenance and repair, it is not surprising that defective helicases would be associated with various odd forms of cancer.

(To my knowledge, truncated WRN protein being found in normally aging and senescent cells has yet to be discovered-but I predict someday this will be forthcoming).

The truncated differentiation/helicase protein found in Werner’s Syndrome is similar in concept to the disease called progeria which attacks  young children from the time they are born and turns them into very old  decrepit individuals by the age of 12 or so where they usually die of heart disease or atherosclerosis. Progeria  is also caused by a truncated protein , the Lamin A protein which is a protein that is found in the nuclear envelope inside the cell. I proposed that progeria recapitulates many of the aging symptoms seen as more pronounced  in normally aging males.

The truncated Lamin A protein causes the envelope that surrounds the DNA in the nucleus to be misshapen>>

Normal Nucleus         Progeria Nucleus

What is not that well known is that the progeria Lamin A protein has a 2nd function of binding to the DNA to act as a transcription factor that silences various genes  so that various cells maintain their differentiation with the proper gene expression profile (for example so that a skin cell remains a skin cell by keeping a certain set of genes silenced).

See UPDATE #12 at the end of this article- it turns out that Lamin A is NOT expressed in induced pluripotent stem cells (undifferentiated cells)  and the nucleus of these undifferentiated cells looks a lot like the nucleus in progeria cells! This gives further weight to the idea that aging is caused by loss of cellular differentiation.

Efficient Induction of Pluripotent Stem Cells from Granulosa Cells by Oct4 and Sox2 | Stem Cells and Development

Well, it turns out that this truncated Lamin A protein is not unique to progeria kids but is also seen in normal aging at older ages in normal adults! It is found in senescent cells in normal humans- there are a number of  studies on this  for example>>>

PLoS One. 2007; 2(12): e1269.

Published online 2007 Dec 5.

The Mutant Form of Lamin A that Causes Hutchinson-Gilford Progeria Is a Biomarker of Cellular Aging in Human Skin

Abstract Hutchinson-Gilford progeria syndrome is a rare disorder characterized by accelerated aging and early death, frequently from stroke or coronary artery disease. 90% of HGPS cases carry the LMNA G608G (GGC>GGT) mutation within exon 11 of LMNA, activating a splice donor site that results in production of a dominant negative form of lamin A protein, denoted progerin. Screening 150 skin biopsies from unaffected individuals (newborn to 97 years) showed that a similar splicing event occurs in vivo at a low level in the skin at all ages. While progerin mRNA remains low, the protein accumulates in the skin with age in a subset of dermal fibroblasts and in a few terminally differentiated keratinocytes. Progerin-positive fibroblasts localize near the basement membrane and in the papillary dermis of young adult skin; however, their numbers increase, and their distribution reaches the deep reticular dermis in elderly skin. Our findings demonstrate that progerin expression is a biomarker of normal cellular aging and may potentially be linked to terminal differentiation and senescence in elderly individuals. “

So, in both cases of Werner’s Syndrome  and progeria we find a truncated protein that is used for differentiating cells is defective and unable to properly do its job of maintaining the differentiated state of the cell.

So, what could have been predicted from these facts?

That aging is caused by nothing more than cells losing their differentiation or becoming de- differentiated as I state in the title of this article. In reality, this should have been predicted long ago after studying Werner’s Syndrome and progeria. This prediction could have easily been made in 2014  and probably earlier after studies came out showing that Lamin A protein was involved in maintaining cellular differentiation in stem cells.

See> Gerontology. 2014;60(3):197-203. Epigenetic involvement in Hutchinson-Gilford progeria syndrome: a mini-review

Take a skin cell for example, as it loses  the factors that are suppressing genes that are not involved with being a skin cell, the cell starts adopting a more and more unusual (undifferentiated) phenotype.

If the process were to continue long enough the skin cell would be unrecognizable eventually. In some ways  you could say the skin cell is returning to its undifferentiated, earlier (younger) state, but in an unhealthy way that ends up killing the bearer of these undifferentiated cells throughout the body. Counterintuitively, detrimental aging appears to actually be caused  by cells becoming younger in a way, less differentitated, more like an embryonic stem cell!

I theorized  in my 1998 paper,  that more primitive organisms , early in evolution, probably reproduced in this manner- a quote-

“At this point in evolution, reproduction likely occurred through parthenogenesis and possibly the complete dissociation of the multi-celled organism into a myriad of single cell, clonal  spores; in an unrestricted environment, this would provide a great reproductive advantage.”

And it turns out that there are still animals on earth that can reproduce this way..take the immortal jellyfish for example:

From National Geographic Magazine-

How the Jellyfish Becomes “Immortal”

“Turritopsis typically reproduces the old-fashioned way, by the meeting of free-floating sperm and eggs. And most of the time they die the old-fashioned way too. But when starvation, physical damage, or other crises arise, “instead of sure death, [Turritopsis] transforms all of its existing cells into a younger state,” said study author Maria Pia Miglietta, a researcher at Pennsylvania State University.The jellyfish turns itself into a bloblike cyst, which then develops into a polyp colony, essentially the first stage in jellyfish life.The jellyfish’s cells are often completely transformed in the process. Muscle cells can become nerve cells or even sperm or eggs.Through asexual reproduction, the resulting polyp colony can spawn hundreds of genetically identical jellyfish—near perfect copies of the original adult.”

It appears that our human development/ aging program of increasing differentiation then decreasing differentiation probably evolved from this ancient form of a reproduction system. Instead of a human  dissolving into 30 trillion identical clonal spores to reproduce (which one would expect to happen if the selfish gene theory of evolution was the only way evolution worked), we instead lose our cellular differentiation in a way that harms and eventually kills.  We might go so far to say  that we age by getting younger from a differentiation point of view!-talk about an unexpected conclusion!

If this concept is correct then we can expect that with the addition of a number of healthy transcription factors back to an older cell that it could be made younger, and this has indeed been proven to be the case. All that was needed were the four transcription factors known as Yamanaka factors to reverse aging in the cell dramatically. The first experiment with Yamanaka factors  took an adult cell and reprogrammed it all the way back to an embryonic state. They later  just subjected an adult cell to transient expression of the Yamanaka factors and were able to make the cell significantly younger, but not return all the way to embryo status.

See> Cell. 2016 Dec 15; 167(7): 1719–1733.e12.In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming

“Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.”


See Update #23 with a link to a new Horvath study in press where he determined that this technique of transient expression of Yamanaka Factors that was applied to old rats   reversed the aging in their cells by 54% to 75% depending on the tissue type, and had a major rejuvenating effect on them!

Okay, so here is another a little prediction that could be made:

If aging is caused by the loss of differentiation in your cells, then one would expect to see genes that produce transcription factors  shut down. Also, thinking back to Werner’s Syndrome and progeria  we would also expect to see some sort of pro-aging related protein unleashed that leads to truncated differentiation proteins like Lamin A  and WRN.

What kind of gene product would we be looking for that truncates differentiation proteins? The easiest way to truncate proteins would not be at the protein level, but rather at the mRNA level. The way proteins are produced is that the genes in our DNA  are read and copied to a very similar molecule called mRNA which is almost identical to  DNA with the exception of using the base pair Uracil in place of Thymine in the GCAT  alphabet of your DNA. The only difference  between Uracil and Thymine is a single methyl group (CH3) which is found attached to the 5’ carbon in thymine but only an H is attached to the 5’ carbon in uracil.

Prediction: There should exist some sort of protein that truncates mRNA transcripts at inappropriate places that increase with age to cause impairment of various differentiation proteins  like WRN and Lamin A. This would be a lot  easier that cutting the proteins after they have already been made. In fact, I did make this prediction to a pair of researchers  who were able to rejuvenate old mice by removing half their blood plasma and replacing it with saline and albumin. I suggested they look for an aging-promoting RNA-ase  that ran around truncating mRNA transcripts in inappropriate places-I never heard back from them.

Well as mentioned before, LARP1  seems to fit the description of this hypothetical protein! It has a very unique sequence that is specific for binding to RNA transcripts. It is found at high levels in cancers. Werner’s Syndrome victims suffer from normal cancers at a high rate as well- is LARP1  cleaving the WRN protein which leads to cancer?

So, the bottom-line conclusions we can draw from this amazing new study are these:

  1. Because the large set of genes shut down by methylation during the aging process (as well as the upregulated LARP1 gene( a true aging gene) ) are primarily the same across all mammalian species it very, very, strongly suggests that aging evolved and is highly conserved. This is in complete contradiction to modern mainstream evolutionary theory which proclaims that aging could never have evolved because it is bad for  the individual and reduces the spread of the individual’s genes. For most modern aging theorists, they think an evolved aging program is something akin to a perpetual motion machine, completely impossible. In fact, this was once the quote by Aubrey De Grey in his sophomoric paper about how programmed aging was impossible.

See> Calorie restriction, post-reproductive life span, and programmed aging: a plea for rigor. Grey AD,  Ann N Y Acad Sci. 2007 Nov;1119:296-305.

Please Notice  I did not cross out the hormonal/neuroendocrine theory of aging…that one is still valid  and will be found to be the upstream controller of the programmed loss of cellular differentiation primarily through the large/dramatic  post age 50 increases in LH, FSH, and hCG with the simultaneous dramatic decline in night time melatonin peaks, dhea, pregnenolone, and progesterone.

Interestingly, melatonin has been found to do all sorts of amazing things, like reversing recent onset menopause (probably due to melatonin’s ability to suppress LH and FSH), preventing the progression of Alzheimer’s, increasing dramatically during caloric restriction, acting as birth control in women at 75 mg per night, and even extending the lives of mice by 20%. I can easily imagine that melatonin somehow has a central role in maintaining  the methylated status  of the circadian rhythm and Alzheimer’s genes that become hypomethylated during aging (Horvath found these  in the small group of genes that get activated with aging along with LARP1).

Fig. 8. Comparison of MNR brain recording of monozygotic twins, both of them were 
suffering from the Alzheimer's disease. The patient on the left (NN) was given melatonin 
(6 mg/day) for the period of 36 months, whereas patient on the right (ZZ) 
was given a placebo. Note the bitemporal atrophy and an enlargement of ventricules 
in the non-treated patient on the right (ZZ). (Adapted from Brusco et al. 1998).

A quick Pub Med search of the terms  “melatonin AND DNA AND methylation” gives you 96 studies , most of which show that melatonin is intricately involved with DNA methylation, and the decline of melatonin with age might be the reason for the global hypomethylated status of DNA in the elderly. Studies with titles such as>> Melatonin and sirtuins: A “not-so unexpected” relationship., or Neuroendocrine aging precedes perimenopause and is regulated by DNA methylation  Melatonin-induced demethylation of antioxidant genes increases antioxidant capacity through RORalpha in cumulus cells of prepubertal lambs, Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation, Melatonin restores the pluripotency of long-term-cultured embryonic stem cells through melatonin receptor-dependent m6A RNA regulation (of Yamanaka factors) are not uncommon.

Likewise, a few studies have recently shown that DHEA also affects  DNA methylation and DNA methyltransferase activity>> Epigenetic Age Reversal by Cell-Extrinsic and Cell-Intrinsic Means. A DNA Methylation Signature of Addiction in T Cells and Its Reversal With DHEA Intervention. Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus.

There are numerous studies showing that progesterone, testosterone, and estrogen have dramatic effects on DNA methylation.

Similarly, given that the LH and FSH subunits are found to be the #1 and #2 proteins that increase inside a cell during aging, it is not too far a leap to suggest that these proteins might have a central role in the methylation of  the 36 genes that become hypermethylated with aging. LH and FSH  not only increase dramatically during aging after the age of female menopause (in both men and women-up to 1,000%+!), they also have a primary role in initiating pre-pubertal/pubertal  development in children as well when they increase.

I also did not cross out the telomere theory of aging. However, this theory becomes just a subset of the loss of cellular differentiation theory of aging in that telomeres when they are long, fold back over on the coding DNA and suppress various genes, probably aging genes. As the telomere shortens, these genes are then expressed. This is called the telomere position effect. (Maybe the genes that , when expressed, lead to the methylation/suppression  of those 36 transcription factor genes might be found here-( However  I kind of doubt it because mice have been shown to have no increase in aging symptoms after their telomerase gene is knocked out and they have continually shortening telomeres. The only aging symptoms that show up in the experimental mice occur  in the 4th generation when they start showing hair-graying,  alopecia, and infertility-see DePinho)). (Update! Stay turns out that TET enzymes are the likely suspects involved with the methylation of the 36 genes-to be discussed later).

Also, Horvath noted that some other aging genes that were hypomethylated and thus shut down were a group of genes involved with the circadian rhythm. This suggests to me a connection to melatonin and other hormones that vary throughout the day. He noted another set of genes involved with causing Alzheimer’s disease that also lose methylation and are more highly expressed. (This might explain why melatonin seems so effective at stopping the progression of Alzheimer’s).

So of course this study raises the important question-What is the purpose of the evolution of programmed aging and how could it evolve?

A brief article about how evolution can select FOR aging>>

A Unifying Theory of The Evolution of Sex and Aging Via Predator Selection

Or a more in depth book on the topic>>>>

Update 1. The recent study where 50% of the blood plasma of mice was replaced with saline and albumin which led to a dramatic rejuvenation of the mice earlier was suggested herein to possibly be caused by a reduction of the LARP1 protein. However, what if LARP1 protein does not circulate in the blood but is only found inside the cell? What else could be being removed from the blood that stops the aging process and allows rejuvenation to happen? How about a 50% reduction in the circulating gonadotropins LH, FSH, and hCG ? These are the pro-aging hormones that increase with age by hundreds of percent and even up to 1,000% in women and men after age 50.
Image result for fsh lh aging levels

Update 2. It is interesting to note how babies often look so much alike  due to their not being fully “differentiated”. They are much more unique and differentiated as children and adults. But then think of the elderly; don’t they seem to be very similar looking? Is this an example of a gain then a loss of cellular differentiation manifesting itself in physical appearance?

Image result for babiues that look the same

Image result for very old peopleImage result for very old peopleImage result for very old people
Image result for baby to adult pictures


Update 3- It appears this aging system is kind of a case of antagonistic pleiotropy (AP). How? It is a new kind of AP where something that was good for your distant ancestors (dissolution of the organism into millions of billions  of single cell clones that can each grow into a new adult) from an evolutionary perspective, evolves into something that is bad for the more modern descendants of the ancestral species. The ancient, dramatically prolific  system of reproduction has evolved into something that now kills an individual at a programmed time.

Update 4– If progeria and Werner’s Syndrome are both manifestations of a malfunctioning  development/differentiation program we can make two very important observations:

  1. The genes silenced by the Lamin a protein that is defective in progeria, are most likely the same genes that control the developmental changes  where an infant develops into a prepubescent juvenile. (Keep in mind that progeria  begins at birth) and
  2. The genes silenced by the WRN protein that is defective in Werner’s Syndrome, are most likely the genes that control the developmental changes where a prepubescent juvenile develops into a fully sexually developed/differentiated fertile adult. (Remember that Werner’s Syndrome does not kick in until puberty begins).

Update 5- For decades if not a century, evolutionary biologists and gerontologists, in order to maintain the illusion that aging is not programmed and was not selected for have had to hide certain facts that just screamed out  “aging is programmed!”.   The two diseases that are the main topic of this article, Werner’s Syndrome, and progeria have been referred to over and over  as “not real examples of aging” because they are caused by genetic mutations and have a few differences when compared to regular aging . This is especially true in the case of Werner’s disease where a 50 year old  woman will look almost identical to a normally aging 85 or 90 year old woman! Because Werner’s Syndrome patients have a higher incidence of some rare cancers – scientists of the past have relied on this canard to declare Werner’s Syndrome is not a case of accelerated programmed aging. With the programmed loss of cellular differentiation theory of aging we can finally do away with this pretense.

Another glaring fact that screams “aging is programmed” is the existence of semelparous aging. The most famous example consists of the rapid aging and death of the Pacific Salmon immediately after breeding around the age of three years old. When the Pacific Salmon is castrated , it can live 7 or more years. How was this explained using the other theories of aging of the past? Simply by saying semelparous aging is not a real form of aging and can be ignored! It had long been hypothesized that the rapid aging and death of the Pacific Salmon was caused by its huge exertion of energy and large amounts of resources spent in traveling the long journey from the ocean to its riparian birthplace to reproduce. Some suggested that the Salmon just died of exhaustion. Others made a slightly less simple case and suggested that the hormonal changes occurring during the great trip of the Salmon led to very high levels of the stress hormone cortisol. Supposedly that was what was killing them, although the cortisol increase comes long before they reproduce.

Well, recently Craig Atwood did a study of changes in the pro-aging hormones  LH and FSH various species including the semelparous Salmon. Although apparently he could not find any data on changes in LH levels in Salmon he did find that FSH levels skyrocket 4,500% post reproduction as compared to even the high level reached in humans after age 50 of about a 500% increase.

I would like to point out to Craig that there is a 1998 study on hormone changes in Salmon that shows LH levels skyrocket as well.  Biol Reproduction 1998 Mar;58(3):814-20. B. Borg et. al.


So the bottom line here is we see that semelparous species no longer have to be put in a special category and hidden away and ignored as not related to normal aging. Rather they now provide a somewhat typical case of programmed aging being driven by the post reproductive dramatic increases in FSH and LH  seen also in humans and most other animals from fish to mammals to birds, etc. The salmon are only unusual in the speed at which they age and the height to which their LH and FSH levels can reach.

Update 6-

Now this one really gets into the weeds of this theory-probably not suitable for the casual reader:

Anyway in my 1998 paper  I described evidence that I had found that suggested  FSH  is the hormone that drives the age changes  seen in progeria  (what I called male aging). And now  I also call it asexual development /aging >>>>from the infant into the prepubescent juvenile>> basically the hormone that drives the development of the asexual body from an infant into a prepubuscent juvenile.
Why FSH? I once searched  many cancers  vs  various hormones  and found involvement of LH  with most cancers  but never found FSH being involved with any cancers…
I also searched high and low for a year+  and could not find any cases of cancer in progeria patients…I went deep into the med school basement  on this quest and looked up every case of progeria that could be found.
There was one case of BPH  and possibly  some associated cancer of the prostate but they were not sure.. That one “possible”  was all I could find , and I looked a long time! I did find a case of hypertension occurring in an abnormally long-lived progeria victim who lived into his 20’s back in the 1940’s and was an Esperanto teacher. Hypertension is another disease that seems to occur at a higher rate in males than in females. Back to cancer>
I say there is NO CANCER in progeria and no cancer from FSH.
The chart below suggests that FSH may play the major role in driving the development of infant to prepubescent juvenile- asexual development.
Also above, we see a bigger role of LH  in driving the development associated with development of the virtually asexual juvenile into a sexual adult. Most of the sexual development occurs in the female who is the one given all the complicated reproduction equipment. (Males undergo very little sexual development as far as organs of the body go-and cannot reproduce. Males just develop a tiny little sperm which is basically like a switch that turns on female reproduction).  I associated LH with “female aging” and now I would add sexual development/aging. Werner’s syndrome kicks in at puberty-I have always believed  LH was involved in driving the aging changes seen in Werner’s Syndrome in normal aging.
After menopause driven by FSH and LH increases…..and melatonin decreases… these development hormones  continue to drive the development/aging program which eventually ends in the unhealthy loss of cellular differentiation and death.
Update 7- You might wonder how my 1998 paper on aging was able to make so many seemingly outrageous predictions that ended up being confirmed. Was it just luck? How did I put together all the facts about aging in a coherent way that was relatively correct?
The fact is I kind of cheated! What I did was while trying to solve the Rubik’s cube of the many facts about aging, I was simultaneously trying to imagine a logical way that evolution could have proceeded from an immortal  clonally reproducing single photosynthetic cell to lead to the outcome that we see today in the large number of facts about aging and reproduction in today’s organisms. I was basically working two Rubik’s cubes at once that were connected to each other. I worked them simultaneously long enough so that when I solved one, it  had to have solved the other-or else back to the drawing board. I worked on this for years. Once both were simultaneously solved and made sense, to me that was the confirmation I needed to know I was on the right track.  One of the ideas I worked with in the sequence of evolution was the jump from single cell life to multiple-celled organisms. All the theorizing and facts told me that the first multi-cellular organisms likely evolved before the evolution of sex (or at least  sophisticated  forms of sex with more than one mating type). I imagined that the most logical way for asexual multi-celled organisms to reproduce  would be to turn all their cells into single-cell clones and to dissociate into thousands or millions of spores that would be dispersed far and wide. All this evolution was driven purely by selfish gene mechanics of genes doing nothing more than trying to make more and more copies of themselves. This, I imagined would be the best and most efficient way for multi-celled organisms to spread their genes at a rapid rate as the selfish gene theory would favor. So this form of development and then de- development>reproduction could be called Primordial asexual reproduction. And we do indeed see this type of reproduction still exists today in the example provided previously in the immortal jellyfish  which when environmentally stressed, instead of reproducing sexually, deprograms all its cells back to the single cell embryo state and then just dissociates into a huge number of spores. So this form of development and then deprogramming, I believe, was the precursor to what we now see in humans as the development system controlled by lamin A proteins which causes the infant human to develop into a prepubescent juvenile. I then imagined the next step- the evolution of sexual reproduction which was a response to the initial evolution of predation. In this case I imagined a form of metamorphosis where the multi-celled organism changes into a mating phenotype that required major mixing of their DNA with another mating phenotype in order to reproduce. You might imagine a caterpillar turning into a butterfly. It was this second development program that I expected evolved  into the development program we see that causes the development of sexually-reproducing phenotypes we see in humans as they develop from prepubescent juveniles into sexually reproducing male or female adults. In its original state this sexual development program likely also led to the de-programming of cells and mass  dispersal of single cell embryos , except this new form of embryo produced by sexual reproduction would have a much higher diversity of genetic variation as mutations in various individuals could come together much more quickly in a single individual through the process of meiotic recombination. This new form of sexual reproduction allowed for a much faster pace for prey species to evolve defenses to predation than waiting for multiple mutations to all occur simultaneously in a single clone.  So we see in the immortal jellyfish example that in times of low environmental stress where predation as opposed to starvation  is the biggest danger, the jellyfish reproduces  sexually. I believe both of these two types of reproduction systems  were both changed by evolution into programmed development/aging systems in humans and other animals as another response to the evolution of evolving predation. You will see in my other article referenced herein, that aging also speeds up the pace of evolution in prey animals by protecting phenotypic diversity as well as  the genetic diversity of the gene pool by preventing any one individual from contributing too many genes to the gene pool. In the end it seems the aging program that kills the oldest living humans is probably the asexual development/aging program that is malfunctioning in progeria. However the sexual development/aging program which is malfunctioning in Werner’s Syndrome likely kills many humans as well at much younger ages, from say age 50 to age 70 even though its primary goal is likely stopping human females from reproducing. Keep in mind that females are the bottleneck to population growth, males are not. It only takes one male to keep the group reproducing.

It is interesting to consider these two abstracts  concerning menopause, hormones and methylation>


Within an evolutionary framework, aging and reproduction are intrinsically linked. Although both laboratory and epidemiological studies have observed associations between the timing of reproductive senescence and longevity, it is not yet known whether differences in the age of menopause are reflected in biomarkers of aging. Using our recently developed biomarker of aging, the “epigenetic clock,” we examined whether age at menopause is associated with epigenetic age of blood, saliva, and buccal epithelium. This is a definitive study that shows an association between age of menopause and biological aging (measured using the epigenetic clock). Our results also indicate menopause may accelerate the epigenetic aging process in blood and that age at menopause and epigenetic age acceleration share a common genetic signature.

Neuroendocrine aging precedes perimenopause and is regulated by DNA methylation

Perimenopause marks initiation of female reproductive senescence. Age of onset is only 47% heritable suggesting that additional factors other than inheritance regulate this endocrine aging transition. To elucidate these factors, we characterized transcriptional and epigenomic changes across endocrine aging using a rat model that recapitulates characteristics of the human perimenopause. RNA-seq analysis revealed that hypothalamic aging precedes onset of perimenopause. In the hypothalamus, global DNA methylation declined with both age and reproductive senescence. Genome-wide epigentic analysis revealed changes in DNA methylation in genes required for hormone signaling, glutamate signaling, and melatonin and circadian pathways. Specific epigenetic changes in these signaling pathways provide insight into the origin of perimenopause-associated neurological symptoms such as insomnia. Treatment with 5-aza-2′-deoxycytidine, a DNA-methyltransferase-1 inhibitor, accelerated transition to reproductive senescence/ whereas supplementation with methionine, a S-adenosylmethionine precursor, delayed onset of perimenopause and endocrine aging. Collectively, these data provide evidence for a critical period of female neuroendocrine aging in brain that precedes ovarian failure and that DNA methylation regulates the transition duration of perimenopause to menopause.

Update 8-

Once evolutionary biologists realize that there is something more going on to drive evolution than the selfish gene, once they realize there are evolutionary forces that also limit the spread of an individual’s genes for the good of the species which I describe in my article  “Sex & Aging , How Evolution Selects For Them Almost Everywhere All the Time” many enduring mysteries of evolution can be easily explained. For example, here is a large portion of the chapter on homosexuality in my book “What Darwin Could Not See- The Missing Half of the Theory”:

CHAPTER 6:  The Sixth Puzzle Piece-Homosexuality In Animals & Humans

One would think that anyone defending the primacy of the selfish gene as the major driving force behind all of evolution would have some sort of reasonable explanation for how something as widespread as human homosexuality could evolve.

Homosexuality is a condition where the possessor of the homosexual trait will, if left to nature only, will choose to never have sex with the opposite sex and thus not have any offspring and not pass on a single gene to the gene pool! Yet human homosexuality exists and has persisted throughout history.

Certainly, this must be harder for selfish gene promoters to explain than sex. At least with sex, the reproducer gets to pass on half of his or her genes. Here the selfish gene-ist has to explain how it evolved that someone passes on NO GENES WHATSOEVER!  Given the very tough problem to solve here, the topic of homosexuality is just ignored for the most part, by evolutionary biologists.

Richard Dawkins, as courageous as he is, at least gives an explanation a try in a 2015 YouTube video. I share the link with you below. If you want a good laugh give it a watch. I am not laughing at Dawkins himself just at him trying to perform the impossible task of explaining homosexuality from the selfish gene point of view.

Darwin Day 2015 Questions: #4 How does evolution explain homosexuality?

Richard Dawkins Foundation for Reason & Science


After viewing this I think Dawkins might seem to focus more on male than female homosexuality and was so bold as to suggest that bottle feeding babies (male I presume) might make them more inclined to be homosexual. I am guessing he thinks sucking on a rubber nipple trains the young lad to want to suck on other protuberances?

Biologists in general tend to also discuss the evolutionary puzzle of homosexuality as mainly a human condition. Applying it to just humans makes the fact conveniently unique and a special category that can be ignored. They do it all the time. Menopause and suicide are also promoted by most biologists as being exceptions that apply to just humans.

Well it turns out that humans aren’t the only ones where homosexuality is common.

There are some estimates that up to 1500 species have been documented to have homosexual individuals in their numbers! If you just do a quick perusal of the Wikipedia entry for homosexuality in animals, you will get all sorts of examples.

So apparently, homosexuality cannot be dumped into the unique human exception category and thus can no longer be ignored by biologists. It must be addressed; trying to address it from the perspective of the preeminence of the selfish gene just leads us into another blind alley with no way out.

What the Wikipedia entry fails to describe are the conditions affecting the pregnant mother of future homosexual offspring.

A number of studies in rodents have shown that if you stress the pregnant mother at certain times during her pregnancy she will tend to give birth to homosexual males and promiscuous females and a smaller number of homosexual females.

What is this telling us from the perspective of the BIG PICTURE? What causes stress? Too many close encounters with predators. This fits quite easily into the BIG PICURE of most unexplained biological phenomenon as being defenses to evolving predation.

How is having homosexual offspring a defense to predation? Having homosexual offspring is a form of birth control for mothers who are considered by evolution to be unfit in the presence of predation. The stress from predator encounters if extreme enough can kill the mothers and their unborn babies. If the stress is less extreme it can lead to homosexual offspring that need to be nursed for a relatively significant period of time. Nursing prevents the mother from becoming fertile for mating. So, in a sense having homosexual offspring is just nature’s form of birth control for mothers perceived as temporarily “unfit” due to stress.

Let us consider the case of female offspring of stressed mothers being more promiscuous than the female offspring of non-stressed mothers. This also jibes well with the BIG PICTURE as promiscuous females who have offspring from multiple males rather than bonding with a single one will add more diversity to the gene pool than if she just mated with a single male for life. As we will see later diversity in the gene pool is the defense to evolving predation that evolution seeks with all these mysterious adaptations.

Now we get to humans; is there any evidence that stressing pregnant female humans can cause their male offspring to be born as homosexual? I wrote about this topic in my December 2000 paper published in Medical Hypotheses titled “Sex, Kings, & Serial Killers and other Group Selected Traits”

Here is the excerpt:

Homosexuality:  Birth Control for “Unfit” Mothers?

 Prevailing evolutionary theory cannot explain the conundrum of homosexuality. Current theory requires defining homosexuality as an evolutionary accident as homosexual offspring would not be expected to reproduce. Is evolution so sloppy that the sexual preferences of 10% to 20% of the human population (78) is simply a random mistake of nature? And why does it also occur throughout the animal kingdom from sheep (79) on down to rats (80)? If one accepts group selection as a reality, the purpose of homosexuality has a simple explanation.

Various studies show that when stressed at a certain time during gestation, rats give birth to males that exhibit female behavior and females that are more masculine (81). (The literature is relatively conclusive on this for males, but the data on females is somewhat ambiguous. Some female offspring of stressed rats also show more promiscuous mating behavior).  Stress increases cortisol levels in rats, and the Prior Paper referred to studies showing that cortisol appears to oppositely affect the sex hormones in human females and males which we will assume extends to rats.

If stress induces high maternal cortisol levels during gestation and the cortisol reaches the developing embryo, endogenous embryonic sex hormones may be altered. Testosterone and estradiol levels in male and female embryos respectively may be decreased. Decreased embryonic sex hormones likely affect the development of the brain’s sexuality. It has been shown that the prenatal stress-induced feminization of male rats is prevented by perinatal androgen treatment (82).

Studies have shown that human females, male transsexuals, and homosexuals share similarities in certain brain structures which differ with heterosexual males (83, 84). Also, it is believed that testosterone derived DHT is required during fetal brain development to create a “male brain” (85). Likewise, we might assume that estradiol exposure creates a “female brain” by feminizing some brain structures. If a stress-induced maternal cortisol surge suppresses the embryo’s testosterone or estradiol, then homosexual offspring, of either sex could result. Interestingly, some researchers found that in a large group of homosexuals interviewed in Germany, many more were born during the war years of 1941 to 1947 than before or after this stressful period with the birth peak occurring in 1944-1945 (86).

Why would evolution create such a system? If a pregnant female is stressed in the wild, it may imply close encounters with predators or maladaptation to her group. Evolution, through group selection, has likely selected for groups that remove or inhibit the spread of her “less fit” genes. While a spontaneous miscarriage or stressed-induced cannibalization of her young (which is common in rodents) is a simple solution, it would leave the female ready to reproduce again. A more clever and effective solution is to give her effectively sterile offspring which she will raise, and which will keep her from reproducing much longer than if she were childless. Also, if group survival required the homosexual children to reproduce, homosexual females could be forced to have sex by dominant heterosexual males. Homosexual males, however, who could not be forced, are evolutionarily irrelevant anyway as long as a single heterosexual male existed.

The only attempt at an evolutionary explanation of homosexuality that the author could find was one that proposed that a homosexual male child would be generated if it was prenatally stressed. The stressor was assumed to be the mother’s living in a crowded environment. The homosexual male, as an adult would not reproduce so that in times of famine there would be fewer grandchildren, and thus an increased likelihood of the grandchildren’s survival (87). One does not have to work long to find counter arguments to this reasoning, but it is a creative attempt to overcome the conundrum of homosexuality and borders on using group selection as an argument. It is only referenced here to show the difficulties that exist in trying to explain homosexuality without the unabashed acceptance of some form of group selection.

(Recent note-which we will later find this to be not group but an even  higher-level form of selection called species selection which I have refined and coined the name “Predator Selection”).

One must wonder about the seemingly high levels of human homosexuality. Were so many mothers severely stressed by predators or wars during pregnancy? Not likely. However, a source of artificial stress has been unleashed this century on humans in epidemic proportions: cigarette smoking.  Nicotine from smoking induces a significant increase in cortisol levels. If a pregnant female has the genetic predisposition to bear homosexual children when stressed, and she smokes during early pregnancy, the nicotine-induced cortisol increase may be sufficient to induce homosexuality in her offspring. This speculation could easily be confirmed or refuted with a simple epidemiological study.

  1. Sell R. Wells J. Wypij D. The prevalence of homosexual behavior and attraction in the United States, the United Kingdom, and France: results of national population-based samples. Archives of Sexual Behavior 24(3). 1995. 235-248.
  2. Perkins A. Fitzgerald J. Moss G. A comparison of LH secretion and brain estradiol receptors in heterosexual and homosexual rams and female sheep. Hormones & Behavior 29(1). 1995. 31-41.
  3. Ferguson T. Alternative sexualities in evolution. Evolutionary Theory 11(1). 1995. 55-64
  4. Ohkawa T. Sexual differentiation of social play and copulatory behavior in prenatally stressed male and female offspring of the rat: the influence of simultaneous treatment by tyrosine during exposure to prenatal stress. Nippon Naibunpi Gakkai Zasshi-Folia Endocrinolgica Japonica. 63(7):823-35, 1987 Jul.
  5. Dorner G. Gotz F. Docke W. Prevention of demasculization and feminization of the brain in prenatally stressed male rats by perinatal androgen treatment. Experimental & Clinical Endocrinology. 81(1):88-90 1983 Jan.
  6. Swaab D. Gooren L. Hofman M. Gender and sexual orientation in relation to hypothalamic structures. Hormone Research. 38 Suppl 2:51-61, 1992.
  7. Zhou J, Hofman M. Gooren L. Swaab D. A sex difference in the human brain and its relation to transsexuality. Nature. 378(6552):68-70, 1995 Nov.
  8. Connolly P. Choate J. Resko J. Effects of endogenous androgen on brain androgen receptors of the fetal rhesus monkey. Neuroendocrinology. 59(3):27 1994 Mar.
  9. Dorner G. Prenatal stress as possible aetiogenetic factor of homosexuality in human males. Endokrinologie.75(3):365-8, 1980 Jun.
  10. 81.

-END Excerpt from my 1998 paper-

(A natural born homosexual?)

While searching for the old studies that showed a sharp rise in the birth of homosexuals in Germany during the WWII years (this supposedly also happened in England as well amongst the pregnant women who hid in London’s subway tunnels during the German bombing campaigns) I found an article about a new book by Dr. Dick Swaab, a well-known neuroscientist who is best known for his research and discoveries in the field of brain anatomy and physiology, in particular the impact that various hormonal and biochemical factors in the womb have on brain development. The book is called “We Are Our Brains” and he puts forth the controversial “new” idea that homosexuality occurs in the brains of fetuses in the womb of stressed mothers. He also notes, like I did in my 2000 paper, that smoking by pregnant mothers can lead to homosexual offspring because nicotine stimulates the release of the stress hormone cortisol and in effect acts as a predator encounter as perceived by evolution. He does add some new evidence that stress in mothers causes homosexuality by noting, as one would expect, that amphetamines (also fake stress) and various other substances lead to an excess of homosexual offspring.

So, that’s about it for homosexuality. If we have learned another fact about evolution, we can say that homosexuality fits in to the big picture as follows:

-Homosexuality is birth control for mothers perceived as stressed by evolution and thus possibly having less than optimal genes for the particular environment. Having an effectively “sterile” child reduces her potential contribution to the gene pool by preventing her from getting pregnant while nursing, and investing resources into the sterile child which also reduces her total potential reproductive output.

One more Thing-Dr. Dick Swaab, for some reason is getting death threats from some gay people who don’t like homosexuality being portrayed as a pathology! I guess they want to think it is a choice. But if you ask most gay people they are happy to say they were born that way. So please don’t send me any death threats thank you.

One other thing I have seemed to notice is that when you look at large groups of either homosexual males, or homosexual females, the males still tend to maintain their evolved desire to stand out and draw attention to themselves while groups of lesbians seem to act more like the camouflaged females of other species, who desire and have evolved to avoid attention. You can also do a crude test of this idea by searching google-images for group of gay men   and then for group of lesbians and you will see the difference. Most of the pictures are along the lines that follow: (see pictures in the book).


Update 9-  This is a good one!

While doing research for my 1998 paper- The Evolution of Aging a New Approach to an Old Problem of Biology

I studied all accelerated aging diseases of progeria, the segmental aging diseases caused by various mitochondrial defects, ataxia telangiectasia (AT), Cockayne syndrome (CS) , xeroderma pigmentosum (XP), and Werner’s syndrome and compared them with respect to what aging symptoms they had at an accelerated rate…and made a table to compare them! (see table bellow). You could call this the periodic table of accelerated aging symptoms.

I had designated Werner’s syndrome as  the dominant aging system that coopted and controlled all the other aging systems-you will see why when you study the table- but basically because WS shows all the symptoms of normal human aging while other rapid aging diseases just show a segment..

It turns out, I believe, given the connection between development and aging we now understand thanks to Horvath,  that the normal Werner’s syndrome protein apparently is also the coordinator and master regulator of all other development genes and transcription factors. The WRN protein does not do all the work itself, it has apparently coopted downstream transcription/differentiation factors and tells them when to be turned on and turned off. WRN is the master regulator of development.

Somehow WRN controls how and when Lamin A protein shuts down various genes in its purview, as well as the normal  proteins that are defective in the mitochondrial diseases , AT, CS, and XP. WRN tells them all what to do and when.

It also now reveals , what I believe , is the master plan of how development is regulated and orchestrated as you will soon see- I had an inkling about it when I made the table but I never articulated it. Well hold onto your hats…here it comes>>

It is pretty clear to me that progeria and Werner’s syndrome are both caused by defective development caused by defective transcription factors as earlier stated.
How about the rest of these diseases? More defective transcription factors?
Now concentrate on just the aging symptoms unique to each aging system . You will notice something amazing.
Now notice that aging system #4 (progeria )relates to somatic development of the child to a pre teen  and AS #6 relates to sexual development of the adult
Now look at As #5a (mitochondrial aging) -relates to development of the brain and muscles
Aging system #5b relates to development of the immune system! (Keep in mind the skin is the largest immune system organ in the body and the immune system and tissue remodeling systems are two sides of the same coin. Also cancer is a result of an underactive immune system.)
And finally aging system #6 relates to the development of the sexually reproducing adult. But actually controls the whole show through the other transcription factors.
Is there evidence that aging systems #5a and #5b are also involved in maintaining differentiation of cells? Well yes there is-you can do the research, but through Pub Med searching I have found that mitochondria are somehow involved with differentiation as well as the wild type  CS and AT proteins. I am still working on XP.
I present the periodic table of accelerated aging disease symptoms>>

 Check out my table again.. I just noticed  something  
Aging System #4 relates to somatic development 
AS #5A relate to brain and muscle development 
#5b immune system development 
#6 sexual development
Aging System #4 Senescent Gene Expression: FSH/DHT driven, seen in men at higher rate. (co-opts #3) (and #1?)
Aging System #5A Somatic atrophy: Mitochondrial Apoptosis, LH/hCG driven, seen in women at a higher rate
(co-opts #2)
Aging System #5B Somatic atrophy: nDNA Fragmentation Apoptosis, LH/hCG driven, seen in women at a higher rate
Aging System # 6 Sex tissue atrophy:
estrogen/DHT driven, seen in women at higher rate (co-opts #4, #5,  (and #1))
Progeria only. Defective Lamin A protein-truncated
Mitochondrial Myopathy (MM), NARP (N), CPEO (CP), MELAS (ME), MERRF (MR) , KSS (K), Dystonia (D), Leigh’s Syndrome (LS)
Ataxia Telangiectasia (AT), Xeroderma Pigmentosum (XP),  Cockayne Syndrome (CS). Various Defective proteins.
Werner’s Syndrome. (WS),
Bloom’s Syndrome (BS), Defective DNA helicase protein WRN- truncated
Original to #4 alone (likely defects of development)
Coxa Valga & necrosis of head of femur
Dysplastic osteoporosis (growing bones)
Symptoms of #4 co-opted by  #6
Symptoms of #6 co-opted from  #4
Gray Hair
Gray Hair-WS
Calcification of Heart Valves
Calc.  of Heart Valves-WS
Laryngeal Atrophy
Laryngeal Atrophy-WS
Loss of subcutaneous   tissue
Loss of subcut.  tissue-WS
Hypermelanosis of Skin
Hypermelanosis of Skin-WS
Hypogonadism (defect of development?)
Hypogonadism -AT, XP
Hypogonadism -WS, BS
Symptoms of #5A also seen in #5B and co-opted by #6
Symptoms of #5B also seen in #5A and co-opted by #6
Symptoms of #6  co-opted from  #5A and #5B
Muscle Wasting-MM, N
Muscle Wasting-AT
Muscle Wasting-WS
Neuronal Degeneration/Brain Atrophy-CP, ME, MR, K
Neuronal Degeneration/Brain Atrophy -AT, XT
Neuronal Degeneration, Brain Atrophy -WS
Basal Ganglion Calcification – D, LS
Basal Ganglion Calcification – CS
Basal Ganglion Calcification -WS
Diabetes-BS, WS
Alzheimer’s Disease-mitochondrial induced
Alzheimer’s Disease-XP
Alzheimer’s Disease-WS
Symptoms of #5B co-opted by #6
Symptoms of #6 co-opted from  #5B
Poor Healing -XP
Poor Healing -WS
Skin Ulcers -XP
Skin Ulcers -WS
Thymic Atrophy-AT
Thymic Atrophy-BS, WS
Scaly Skin-XP
Scaly Skin-WS
Somatic Cancers-XP,AT
Somatic  Cancers- BS, WS
Lipofuscin Accumulation-CS,XP
Lipofuscin Accumulation-WS
Peripheral Osteoporosis-CS
Peripheral Osteoporosis-WS (growth plate closure) maybe  unique to #6?
Symptoms unique to #6
Breast, Uterine, and Ovarian atrophy and cancer-WS
Prostate atrophy-WS, hyperplasia-WS, and cancer-WS

An interesting coincidence is that from this table we see there appears to be 4 unique development/aging programs for 4 different groups of tissue types.  Does this somehow relate to the fact that there are 4 Yamanaka factors that can reverse aging?  I will be looking into this.

So that’s it for update #9!


Update #10:

One thing I am wondering about…

If Yamanaka factors can turn a somatic cell into a pluripotent stem cell by removing most of  the transcription factors on the DNA back to time 0……..
aging is caused by the gradual loss of transcription factors and loss of cellular differentiation as the Horvath study shows….
then how do Yamanaka factors  just transiently expressed    make the cell younger again??
If the Yamanaka factors only remove transcription factors  they should actually make the cell older in the sense that the cell would be less differentiated.
The only solution or answer I can imagine is that the Yamanaka factors  aren’t that smart or efficient
….  they don’t just yank transcription factors off of the DNA of an old cell and viola  it becomes a pluripotent stem cell again….
I think it is possible that somehow the Yamanaka factors  have to cause the cell to refollow its development /aging program exactly in reverse to get back to the pluripotent when you give an old cell a little jolt of Yamanaka factors  the cell responds by initially adding transcription factors to re differentiate the cell back to a younger (less aged) state  before it starts to remove the transcription factors again to get back to the pluripotent state….
One might describe the development/aging program as the world’s first Blockchain.

The discovery of Yamanaka factors that can age somatic cells in reverse  should not have come as much of a surprise. Why? They should have been predicted the day it was announced that Dolly the sheep was cloned. In her case an adult somatic nucleus was inserted into an ovum, zapped with a little electricity and the nucleus reprogramed itself  all the way back to a single cell undifferentiated embryo. What is being done with Yamanka factors is the same thing that has been  going on inside cloned embryos since the 1990’s. The real question here is what took so long to discover the Yamanaka factors?

Update #11

This update discussion has been deleted I am still working on it but I provide an interesting  abstract to consider

J Gerontol A Biol Sci Med Sci

.2019 Aug 16;74(9):1391-1395.

Centenarians Overexpress Pluripotency-Related Genes

Marta Inglés 1 2José Viña 2


Human mesenchymal cells can become pluripotent by the addition of Yamanaka factors OCT3/4, SOX2, c-MYC, KLF4. We have recently reported that centenarians overexpress BCL-xL, which has been shown to improve pluripotency; thus, we aimed to determine the expression of pluripotency-related genes in centenarians. We recruited 22 young, 32 octogenarian, and 47 centenarian individuals and determined the mRNA expression of Yamanaka factors and other stemness-related cell surface marker genes (VIM, BMP4, NCAM, BMPR2) in peripheral blood mononuclear cells by reverse transcription polymerase chain reaction. We found that centenarians overexpress OCT3/4, SOX2, c-MYC, VIM, BMP4, NCAM, and BMPR2, when compared with octogenarians (p < .05). We further tested the functional role of BCL-xL in centenarians’ ability to express pluripotency-related genes: lymphocytes from octogenarians transduced with BCL-xL overexpressed SOX2, c-MYC, and KLF4. We conclude that centenarians overexpress Yamanaka Factors and other stemness-related cell surface marker genes, which may contribute to their successful aging.


Update #12 Turns out lamin A is missing in undifferentiated embryonic stem cells and is defective in the rapid aging disease of progeria :

Efficient induction of pluripotent stem cells from granulosa cells by Oct4 and Sox2 

Jian Mao 1Qian ZhangXiaoying YeKai LiuLin Liu


Various types of somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells. Somatic stem cells exhibit enhanced reprogramming efficiency by fewer factors, in contrast to fully differentiated cells. Nuclear Lamin A is highly expressed in differentiated cells, and stem cells are characterized by the absence of Lamin A. Granulosa cells (GCs) and cumulus cells in the ovarian follicles effectively and firstly generated cloned mice by somatic cell nuclear transfer, and these cells lack Lamin A expression. We tested the hypothesis that GCs could be effectively used to generate iPS cells with fewer factors. We show that iPS cells are generated from GCs at high efficiency even with only two factors, Oct4 and Sox2, like the iPS cells generated using four Yamanaka factors. These iPS cells show pluripotency in vitro and in vivo, as evidenced by high expression of pluripotency-associated genes, Oct4, Nanog, and SSEA-1, differentiation into three embryonic germ layers by embryoid body formation and teratoma tests, as well as high efficient generation of chimeras. Moreover, the exogenous genes are effectively silenced in these iPS cells. These data provide additional evidence in supporting the notion that reduced expression of LaminA and stem cells can improve the reprogramming efficiency to pluripotency.

Update #13

I wrote in my 1998 paper >  ”  Cancer, in a broad sense,  may simply be a cell returning to its earlier, primitive, immortalized, state. It should not be very surprising  that a mortal life form that evolved from a previously immortalized life form could spontaneously become immortalized through loss of some type of control. However, if the mortal life form had evolved from mortal ancestors, spontaneous immortalization would seem to be quite a miracle indeed.”  see comment in full context  at the end of this update .

Well, well, well>>  I have been reading all the abstracts  in Pub Med that contain the term “Yamanaka Factors” and what have I found?  The cancer cells are basically just malfunctioning de-differentiated embryonic stem cells.  And yes it seems very likely they are simply a reemergence of our oldest ancestors…Single cell life  that lived before the age of oxygen. Both cancer cells and embryonic stem cells can divide indefinitely (immortal).  Both of the them do not use oxygen for energy  even when oxygen is present but rather switch to an anaerobic form of glycolysis for energy ! What follows are the interesting abstracts that show how this view of cancer being a reversion of cells to their most primitive state seems to be correct:


The role of pluripotency factors to drive stemness in gastrointestinal cancer


A better molecular understanding of gastrointestinal cancers arising either from the stomach, the pancreas, the intestine, or the liver has led to the identification of a variety of potential new molecular therapeutic targets. However, in most cases surgery remains the only curative option. The intratumoral cellular heterogeneity of cancer stem cells, bulk tumor cells, and stromal cells further limits straightforward targeting approaches. Accumulating evidence reveals an intimate link between embryonic development, stem cells, and cancer formation. In line, a growing number of oncofetal proteins are found to play common roles within these processes. Cancer stem cells share features with true stem cells by having the capacity to self-renew in a de-differentiated state, to generate heterogeneous types of differentiated progeny, and to give rise to the bulk tumor. Further, various studies identified genes in cancer stem cells, which were previously shown to regulate the pluripotency circuitry, particularly the so-called “Yamanaka-Factors” (OCT4, KLF4, SOX2, and c-MYC). However, the true stemness potential of cancer stem cells and the role and expression pattern of such pluripotency genes in various tumor cell types remain to be explored. Here, we summarize recent findings and discuss the potential mechanisms involved, and link them to clinical significance with a particular focus on gastrointestinal cancers.

The oncogene c-Jun impedes somatic cell reprogramming

Oncogenic transcription factors are known to mediate the conversion of somatic cells to tumour or induced pluripotent stem cells (iPSCs).


. 2014 Mar;15(3):244-53.

Dedifferentiation and reprogramming: origins of cancer stem cells


Regenerative medicine aims to replace the lost or damaged cells in the human body through a new source of healthy transplanted cells or by endogenous repair. Although human embryonic stem cells were first thought to be the ideal source for cell therapy and tissue repair in humans, the discovery by Yamanaka and colleagues revolutionized the field. Almost any differentiated cell can be sent back in time to a pluripotency state by expressing the appropriate transcription factors. The process of somatic reprogramming using Yamanaka factors, many of which are oncogenes, offers a glimpse into how cancer stem cells may originate. In this review we discuss the similarities between tumor dedifferentiation and somatic cell reprogramming and how this may pose a risk to the application of this new technology in regenerative medicine.

J Cell Sci

. 2013 Aug 15;126(Pt 16):3638-48.

The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation


Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs

Expert Rev Anticancer Ther

. 2021 Apr 8.

 Pluripotency inducing Yamanaka factors: role in stemness and chemoresistance of liver cancer


Introduction: Liver cancer is a major cause of mortality and is characterized by the transformation of cells into an uncontrolled mass of tumor cells with many genetic and epigenetic changes, which lead to the development of tumors. A small subpopulation of cell population known as Cancer Stem Cells (CSCs) is responsible for cancer stemness and chemoresistance. Yamanaka factors [octamer-binding transcription factor 4 (OCT4), SRY (sex-determining region Y)-box 2 (SOX2), kruppel like factor 4 (KLF4), and Myelocytomatosis (MYC); OSKM] are responsible for cancer cell stemness, chemoresistance, and recurrence.

Biochem Biophys Res Commun

. 2019 Sep 17;517(2):324-329.

Silencing of the transcription factors Oct4, Sox2, Klf4, c-Myc or Nanog has different effect on teratoma growth


Induced pluripotent stem cells (iPSC) have a great potential, but their clinical application depends on finding strategies to abolish their tumorigenic potential. The use of Oct4, Sox2, Klf4, c-Myc and Nanog to generate iPSC demonstrated the already known importance of these genes to maintain stemness. Therefore, the presence of these genes is responsible for iPSC-derived teratomas. Similar to iPSC, P19 teratocarcinoma cell line also has characteristics of embryonic carcinoma cells and the ability to differentiate into many cell types. We separately silenced the transcription factors Oct4, Sox2, Klf4, c-Myc and Nanog in P19 cells and measured the impact of this silencing in vivo. All silenced cells generated tumors when injected in immunosuppressed mice, but silencing of Oct4, Sox2 and Klf4 generated mainly teratomas with mesoderm tissue. Our results suggest that downregulation of these transcription factors is not enough to avoid the formation of teratomas, but their silencing affect their differentiation potential.


. 2019 Aug;38(34):6226-6239.

Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity


Pancreatic ductal adenocarcinoma (PDAC) arises through accumulation of multiple genetic alterations. However, cancer cells also acquire and depend on cancer-specific epigenetic changes. To conclusively demonstrate the crucial relevance of the epigenetic programme for the tumourigenicity of the cancer cells, we used cellular reprogramming technology to reverse these epigenetic changes. We reprogrammed human PDAC cultures using three different techniques – (1) lentivirally via induction of Yamanaka Factors (OSKM), (2) the pluripotency-associated gene OCT4 and the microRNA mir-302, or (3) using episomal vectors as a safer alternative without genomic integration. We found that induction with episomal vectors was the most efficient method to reprogram primary human PDAC cultures as well as primary human fibroblasts that served as positive controls. Successful reprogramming was evidenced by immunostaining, alkaline phosphatase staining, and real-time PCR. Intriguingly, reprogramming of primary human PDAC cultures drastically reduced their in vivo tumourigenicity, which appeared to be driven by the cells’ enhanced differentiation and loss of stemness upon transplantation. Our study demonstrates that reprogrammed primary PDAC cultures are functionally distinct from parental PDAC cells resulting in drastically reduced tumourigenicity in vitro and in vivo. Thus, epigenetic alterations account at least in part for the tumourigenicity and aggressiveness of pancreatic cancer, supporting the notion that epigenetic modulators could be a suitable approach to improve the dismal outcome of patients with pancreatic cancer.

Methods Mol Biol

. 2019;1916:249-261.

Reprogramming of Human Melanocytes and Melanoma Cells with Yamanaka Factors


The expression of Yamanaka factors (Oct3/4, Klf-4, Sox-2, c-Myc) can reprogram cancer cells to a pluripotent stage. This may cause the removal of their epigenetic memory and result in altered tumorigenicity. Various studies in the literature have shown that cancer cell reprogramming is a potential tool to study disease progression or discover novel therapeutic or diagnostic markers in cancer research. In this chapter, we aim to introduce the cancer cell reprogramming protocol in detail by using human melanocytes and melanoma cell lines, and Sendai viral vectors encoding Yamanaka factors have been used to reprogram cells. Representative results are discussed and important notes have been summarized in order to point out important steps during cancer cell reprogramming.

J Biomed Sci

. 2018 Jul 19;25(1):57.

Incomplete cellular reprogramming of colorectal cancer cells elicits an epithelial/mesenchymal hybrid phenotype


Background: Induced pluripotency in cancer cells by ectopic expression of pluripotency-regulating factors may be used for disease modeling of cancers. MicroRNAs (miRNAs) are negative regulators of gene expression that play important role in reprogramming somatic cells. However, studies on the miRNA expression profile and the expression patterns of the mesenchymal-epithelial transition (MET)/epithelial-mesenchymal transition (EMT) genes in induced pluripotent cancer (iPC) cells are lacking.

Methods: iPC clones were generated from two colorectal cancer (CRC) cell lines by retroviral transduction of the Yamanaka factors. The iPC clones obtained were characterized by morphology, expression of pluripotency markers and the ability to undergo in vitro tri-lineage differentiation. Genome-wide miRNA profiles of the iPC cells were obtained by microarray analysis and bioinformatics interrogation. Gene expression was done by real-time RT-PCR and immuno-staining; MET/EMT protein levels were determined by western blot analysis.

Results: The CRC-iPC cells showed embryonic stem cell-like features and tri-lineage differentiation abilities. The spontaneously-differentiated post-iPC cells obtained were highly similar to the parental CRC cells. However, down-regulated pluripotency gene expression and failure to form teratoma indicated that the CRC-iPC cells had only attained partial pluripotency. The CRC-iPC cells shared similarities in the genome-wide miRNA expression profiles of both cancer and pluripotent embryonic stem cells. One hundred and two differentially-expressed miRNAs were identified in the CRC-iPC cells, which were predicted by bioinformatics analysis be closely involved in regulating cellular pluripotency and the expression of the MET/EMT genes, possibly via the phosphatidylinositol-3 kinases-protein kinase B (PI3K-Akt) and transforming growth factor beta (TGF-β) signaling pathways. Irregular and inconsistent expression patterns of the EMT vimentin and Snai1 and MET E-cadherin and occludin proteins were observed in the four CRC-iPC clones analyzed, which suggested an epithelial/mesenchymal hybrid phenotype in the partially reprogrammed CRC cells. MET/EMT gene expression was also generally reversed on re-differentiation, also suggesting epigenetic regulation.

Conclusions: Our data support the elite model for cancer cell-reprogramming in which only a selected subset of cancer may be fully reprogrammed; partial cancer cell reprogramming may also elicit an epithelial-mesenchymal mixed phenotype, and highlight opportunities and challenges in cancer cell-reprogramming.

Biochim Biophys Acta Rev Cancer

. 2018 Jan;1869(1):1-10.

Deubiquitylating enzymes as cancer stem cell therapeutics


The focus of basic and applied research on core stem cell transcription factors has paved the way to initial delineation of their characteristics, their regulatory mechanisms, and the applicability of their regulatory proteins for protein-induced pluripotent stem cells (protein-IPSC) generation and in further clinical settings. Striking parallels have been observed between cancer stem cells (CSCs) and stem cells. For the maintenance of stem cells and CSC pluripotency and differentiation, post translational modifications (i.e., ubiquitylation and deubiquitylation) are tightly regulated, as these modifications result in a variety of stem cell fates. The identification of deubiquitylating enzymes (DUBs) involved in the regulation of core stem cell transcription factors and CSC-related proteins might contribute to providing novel insights into the implications of DUB regulatory mechanisms for governing cellular reprogramming and carcinogenesis. Moreover, we propose the novel possibility of applying DUBs coupled with core transcription factors to improve protein-iPSC generation efficiency. Additionally, this review article further illustrates the potential of applying DUB inhibitors as a novel therapeutic intervention for targeting CSCs. Thus, defining DUBs as core pharmacological targets implies that future endeavors to develop their inhibitors may revolutionize our ability to regulate stem cell maintenance and differentiation, somatic cell reprogramming, and cancer stem cells.

Biochim Biophys Acta Mol Cell Res

. 2017 Jul;1864(7):1359-1369.

Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences


Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called “Yamanaka factors” (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis.

Iran J Basic Med Sci

. 2016 Oct;19(10):1131-1135.

Linc-ROR and its spliced variants 2 and 4 are significantly up-regulated in esophageal squamous cell carcinoma


Objectives: Similar characteristics of molecular pathways between cellular reprogramming events and tumorigenesis have been accentuated in recent years. Reprogramming-related transcription factors, also known as Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC), are also well-known oncogenes promoting cancer initiation, progression, and cellular transformation into cancer stem cells. Long non-coding RNAs (lncRNAs) are a major class of RNA molecules with emerging roles in stem cell pluripotency, cellular reprogramming, cellular transformation, and tumorigenesis. The long intergenic non-coding RNA ROR (lincRNA-ROR, linc-ROR) acts as a regulator of cellular reprograming through sponging miR-145 that normally negatively regulates the expression of the stemness factors NANOG, OCT4, and SOX2.

Stem Cells

. 2016 Nov;34(11):2613-2624.

Positive Feedback Loop of OCT4 and c-JUN Expedites Cancer Stemness in Liver Cancer


The network of stemness genes and oncogenes in human patient-specific reprogrammed cancer stem cells (CSCs) remains elusive, especially in liver cancer. HepG2-derived induced pluripotent stem cell-like cells (HepG2-iPS-like cells) were generated by introducing Yamanaka factors and the knockdown vector shTP53. They exhibited features of stemness and a higher tumorigenesis after xenograft transplantation compared with HepG2 cells. The cancerous mass of severe combined immunodeficiency (SCID) mice derived from one colony was dissected and cultured to establish reprogrammed HepG2-derived CSC-like cells (designated rG2-DC-1C). A single colony exhibited 42% occurrence of tumors with higher proliferation capacities. rG2-DC-1C showed continuous expression of the OCT4 stemness gene and of representative tumor markers, potentiated chemoresistance characteristics, and invasion activities. The sphere-colony formation ability and the invasion activity of rG2-DC-1C were also higher than those of HepG2 cells. Moreover, the expression of the OCT4 gene and the c-JUN oncogene, but not of c-MYC, was significantly elevated in rG2-DC-1C, whereas no c-JUN expression was observed in HepG2 cells. The positive-feedback regulation via OCT4-mediated transactivation of the c-JUN promoter and the c-JUN-mediated transactivation of the OCT4 promoter were crucial for promoting cancer development and maintaining cancer stemness in rG2-DC-1C. Increased expression of OCT4 and c-JUN was detected in the early stage of human liver cancer. Therefore, the positive feedback regulation of OCT4 and c-JUN, resulting in the continuous expression of oncogenes such as c-JUN, seems to play a critical role in the determination of the cell fate decision from iPS cells to CSCs in liver cancer. Stem Cells 2016;34:2613-2624.

Stem Cell Reports

. 2016 Jul 12;7(1):1-10.

MiR-31/SDHA Axis Regulates Reprogramming Efficiency through Mitochondrial Metabolism


Metabolism is remodeled when somatic cells are reprogrammed into induced pluripotent stem cells (iPSCs), but the majority of iPSCs are not fully reprogrammed. In a shift essential for reprogramming, iPSCs use less mitochondrial respiration but increased anaerobic glycolysis for bioenergetics. We found that microRNA 31 (miR-31) suppressed succinate dehydrogenase complex subunit A (SDHA) expression, vital for mitochondrial electron transport chain (ETC) complex II. MiR-31 overexpression in partially reprogrammed iPSCs lowered SDHA expression levels and oxygen consumption rates to that of fully reprogrammed iPSCs, but did not increase the proportion of fully reprogrammed TRA1-60(+) cells in colonies unless miR-31 was co-transduced with Yamanaka factors, which resulted in a 2.7-fold increase in full reprogramming. Thus switching from mitochondrial respiration to glycolytic metabolism through regulation of the miR-31/SDHA axis is critical for lowering the reprogramming threshold. This is supportive of multi-stage reprogramming whereby metabolic remodeling is fundamental.

Cancer cells exhibit aerobic glycolysis. This means that cancer cells derive most of their energy from glycolysis that is glucose is converted to lactate for energy followed by lactate fermentation, even when oxygen is available. This is termed the Warburg effect.

Full context extract from my 1998 paper>>>

“So, if mitochondria existed as separate organisms prior to their merging with the drifting Archaea, then it might be expected  that they had evolved their own separate aging system. Once the two life forms merged and the larger, combined, life form  was completely dependent on the mitochondrial energy source, whenever enough mitochondria in the cell had died, the cell itself would also die. If the mitochondrial imposed death occurred before death caused by telomeric shortening, two aging systems could exist in the same organism, one dominant and one vestigial. Mitochondrial aging will be referred to as Aging System #2 or (AS#2).

The next step in evolution would likely have been the vast colonization of the oceans by these photosynthetic Archaea. (We will now refer to them as algae). With the sun providing unlimited energy and the ocean an unrestricted habitat, evolution would select for maximal reproductive potential and therefore maximal life spans. The first two aging systems, therefore were likely deactivated. The symbiotic mitochondria could simply evolve longer life spans, and the telomeric aging system could be deactivated by the creation of telomerase which  rebuilds the ends of the chromosomes after each round of replication. Also, to counter the effect of the sun’s deadly mutating  UV, Gamma, and X rays ( referred to herein as solar radiation)  a DNA repair system had to evolve that could excise damaged base pairs and replace them with the proper ones. Additionally, to protect against the free radicals generated by the oxygen produced from photosynthesis and solar radiation, an antioxidant protective system had to evolve as well. After a billion years of this selection pressure it could be expected that the algae evolved into non-aging, rapidly-reproducing  organisms with perfect DNA repair and free radical defense systems. Is there any evidence that single cell organisms were once immortal? Many cell-types with the proper manipulations can become immortalized cancer strains and reproduce indefinitely as a culture. Cancer, in a broad sense,  may simply be a cell returning to its earlier, primitive, immortalized, state. It should not be very surprising  that a mortal life form that evolved from a previously immortalized life form could spontaneously become immortalized through loss of some type of control. However, if the mortal life form had evolved from mortal ancestors, spontaneous immortalization would seem to be quite a miracle indeed.

Update #14-  A new Horvath article has just been published in turns out as expected  transient expression of Yamanaka factors can reverse the aging process!!
Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells


Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.
Update #15-

In my 1998 paper I predicted that antioxidants would be found to  catalyze the methylation of cytosines, and free radicals would be found to catalyze the demethylation of cytosines…here is the exact language>>>

This was in the abstract>>  “Free radicals catalyze the demethylation of 5mC while antioxidants catalyze the remethylation of cytosine by altering the activity of DNA methyltransferases. Hormones act as either surrogate free radicals by stimulating the cAMP pathway  which alters free radical levels within cells, or as surrogate antioxidants through cGMP pathway stimulation. Access to  DNA containing 5mC-inhibited developmental and aging genes and restriction sites  is allowed by  DNA helicase strand separation. Tightly wound  DNA does not allow this access. ”

In the concluding remarks>>>

  • “A major manner in which genetic signaling occurs is through alteration of the methylation status of  5mC in  5mC demethylation is catalyzed by free radicals and methylation by antioxidants.”
  • “The main assumptions that this theory relies upon are that free radicals catalyze the demethylation of 5mC while antioxidants catalyze the methylation of 5mC by influencing  DNA methyltransferase activity. This theory was constructed prior to the author’s knowledge that there existed  any evidence that supported the above assumptions. After studies were located that confirmed the major assumptions of this theory, the likelihood of it being correct increased significantly.”

So my examination of the 200+  journal articles in Pub Med that contain the term “Yamanaka factors ” yields some very interesting results… It turns out that it appears that the general rule is that adding antioxidants to the Yamanaka factors greatly increases their differentiation into specific cell lines (presumably through increasing general cytosine methylation and shutting down of various genes.). It also appears to be generally true that adding free radicals to the mix  greatly increases the loss of differentiation of the cells and allows them to become pluripotent (blank) stem cells  (presumably by increasing loss of cytosine methylation) .

Now, if these phenomena are not occurring via DNA methylation/demethylation, then the other idea that might hold water is that antioxidants catalyze the binding of transcription factors to their binding sites while free radicals catalyze the removal of transcription factors.  Another way to think of it is that in essence is that methyl groups are the simplest form of transcription factors and the antioxidants and free radicals are catalyzing all the reactions.

Well what do you know?  This all seems to be revealed to likely be true in the following relevant abstracts of Yamanaka factor articles. There is one odd abstract however where it refers to a TET protein that is involved with demethylating cytosines and antioxidants  increase its activity of demethylation! Here is a definition of TET protein’s. The TET enzymes are a family of ten-eleven translocation (TET) methylcytosine dioxygenases. They are instrumental in DNA demethylation of 5-Methylcytosine.  

This little clue gives me an idea as to what to look for to find the methyltransferase that is activated during aging to methylate the 36 development-related genes uncovered by Horvath. There might exist a special methyltransferase that is activated by free radicals to methylate those 36 genes. This would make sense if the pro-aging cAMP stimulating hormones LH, FSH, and hCG are in actuality as I predicted in the 1998 paper – free radical generating hormones.

Okay  here are the interesting abstracts>>>

Ascorbic acid promotes the direct conversion of mouse fibroblasts into beating cardiomyocytes


Recent advances in the direct conversion of fibroblasts to cardiomyocytes suggest this process as a novel promising approach for cardiac cell-based therapies. Here, by screening the effects of 10 candidate small molecules along with transient overexpression of Yamanaka factors, we show ascorbic acid (AA), also known as vitamin C, enhances reprogramming of mouse fibroblasts into beating cardiomyocytes. Immunostaining and gene expression analyses for pluripotency and cardiac lineage markers confirmed beating patches were derived from non-cardiac lineage cells without passing through a pluripotent intermediate. Further analysis revealed that AA also increased the size of the beating areas and the number of cardiac progenitors. Immunostaining for cardiac markers, as well as electrophysiological analysis confirmed the functionality of directly converted cardiomyocytes. These results illustrate the importance of AA in direct conversion of fibroblasts to cardiomyocytes and may open new insights into future biomedical applications for induced cardiomyocytes.

Optimal ROS Signaling Is Critical for Nuclear Reprogramming

Free PMC article


Efficient nuclear reprogramming of somatic cells to pluripotency requires activation of innate immunity. Because innate immune activation triggers reactive oxygen species (ROS) signaling, we sought to determine whether there was a role of ROS signaling in nuclear reprogramming. We examined ROS production during the reprogramming of doxycycline (dox)-inducible mouse embryonic fibroblasts (MEFs) carrying the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM]) into induced pluripotent stem cells (iPSCs). ROS generation was substantially increased with the @@@@@@READ THIS- I believe ROS catalyze the demethylation of 5mC!

onset of reprogramming. Depletion of ROS via antioxidants or Nox inhibitors substantially decreased reprogramming efficiency. Similarly, both knockdown and knockout of p22(phox)-a critical subunit of the Nox (1-4) complex-decreased reprogramming efficiency. However, excessive ROS generation using genetic and pharmacological approaches also impaired reprogramming. Overall, our data indicate that ROS signaling is activated early with nuclear reprogramming, and optimal levels of ROS signaling are essential to induce pluripotency.

Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors

Free PMC article


The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source-peripheral blood-has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34(+) cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-)/CD19(-) cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Cell 1993 Oct 22;75(2):241-51.

Bcl-2 functions in an antioxidant pathway to prevent apoptosis


Bcl-2 inhibits most types of apoptotic cell death, implying a common mechanism of lethality. Bcl-2 is localized to intracellular sites of oxygen free radical generation including mitochondria, endoplasmic reticula, and nuclear membranes. Antioxidants that scavenge peroxides, N-acetylcysteine and glutathione peroxidase, countered apoptotic death, while manganese superoxide dismutase did not. Bcl-2 protected cells from H2O2- and menadione-induced oxidative deaths. Bcl-2 did not prevent the cyanide-resistant oxidative burst generated by menadione. Two model systems of apoptosis showed no increment in cyanide-resistant respiration, and generation of endogenous peroxides continued at an inherent rate that was unaltered by Bcl-2. Following an apoptotic signal, cells sustained progressive lipid peroxidation. Overexpression of Bcl-2 functioned to suppress lipid peroxidation completely. We propose a model in which Bcl-2 regulates an antioxidant pathway at sites of free radical generation.

JMJD3 acts in tandem with KLF4 to facilitate reprogramming to pluripotency

Free PMC article


The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3’s catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.

Nuclear S-Nitrosylation Defines an Optimal Zone for Inducing Pluripotency


Background: We found that cell-autonomous innate immune signaling causes global changes in the expression of epigenetic modifiers to facilitate nuclear reprogramming to pluripotency. A role of S-nitrosylation by inducible nitric oxide (NO) synthase, an important effector of innate immunity, has been previously described in the transdifferentiation of fibroblasts to endothelial cells. Accordingly, we hypothesized that S-nitrosylation might also have a role in nuclear reprogramming to pluripotency.

Methods: We used murine embryonic fibroblasts containing a doxycycline-inducible cassette encoding the Yamanaka factors (Oct4Sox2Klf4, and c-Myc), and genetic or pharmacological inhibition of inducible NO synthase together with the Tandem Mass Tag approach, chromatin immunoprecipitation-quantitative polymerase chain reaction, site-directed mutagenesis, and micrococcal nuclease assay to determine the role of S-nitrosylation during nuclear reprogramming to pluripotency.

Results: We show that an optimal zone of innate immune activation, as defined by maximal yield of induced pluripotent stem cells, is determined by the degree of activation of nuclear factor κ-light-chain-enhancer of activated B cells; NO generation; S-nitrosylation of nuclear proteins; and DNA accessibility as reflected by histone markings and increased mononucleosome generation in a micrococcal nuclease assay. Genetic or pharmacological inhibition of inducible NO synthase reduces DNA accessibility and suppresses induced pluripotent stem cell generation. (free radicals catalyze demethylation) The effect of NO on DNA accessibility is mediated in part by S-nitrosylation of nuclear proteins, including MTA3 (Metastasis Associated 1 Family Member 3), a subunit of NuRD (Nucleosome Remodeling Deacetylase) complex. S-Nitrosylation of MTA3 is associated with decreased NuRD activity. Overexpression of mutant MTA3, in which the 2 cysteine residues are replaced by alanine residues, impairs the generation of induced pluripotent stem cells.

Conclusions: This is the first report showing that DNA accessibility and induced pluripotent stem cell yield depend on the extent of cell-autonomous innate immune activation and NO generation. This “Goldilocks zone” for inflammatory signaling and epigenetic plasticity may have broader implications for cell fate and phenotypic fluidity.

This one doesn’t fit>>>>
. 2019 Feb 12;5:11.

Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

Free PMC article


The relationship between active DNA demethylation induced by overexpressing Tet1 and passive DNA demethylation induced by suppressing Dnmt1 remains unclear. Here, we found that DNMT1 preferentially methylated, but TET1 preferentially demethylated, hemi-methylated CpG sites. These phenomena resulted in a significant overlap in the targets of these two types of DNA demethylation and the counteractions of Dnmt1 and Tet1 during somatic cell reprogramming. Since the hemi-methylated CpG sites generated during cell proliferation were enriched at core pluripotency loci, DNA demethylation induced by Tet1 or sh-RNA against Dnmt1 (sh-Dnmt1) was enriched in these loci, which, in combination with Yamanaka factors, led to the up-regulation of these genes and promoted somatic cell reprogramming. In addition, since sh-Dnmt1 induces DNA demethylation by impairing the further methylation of hemi-methylated CpG sites generated during cell proliferation, while Tet1 induced DNA demethylation by demethylating these hemi-methylated CpG sites, Tet1-induced DNA demethylation, compared with sh-Dnmt1-induced DNA demethylation, exhibited a higher ability to open the chromatin structure and up-regulate gene expression. Thus, Tet1-induced but not sh-Dnmt1-induced DNA demethylation led to the up-regulation of an additional set of genes that can promote the epithelial-mesenchymal transition and impair reprogramming. ????????? When vitamin C was used to further increase the demethylation ability ?????????of TET1 during reprogramming, Tet1 induced a larger up-regulation of these genes and significantly impaired reprogramming. Therefore, the current studies provide additional information regarding DNA demethylation during somatic cell reprogramming.

Stem Cell Res. 2016 Sep;17(2):296-305.

Robust reprogramming of Ataxia-Telangiectasia patient and carrier erythroid cells to induced pluripotent stem cells

Free article


Biallelic mutations in ATM result in the neurodegenerative syndrome Ataxia-Telangiectasia, while ATM haploinsufficiency increases the risk of cancer and other diseases. Previous studies revealed low reprogramming efficiency from A-T and carrier fibroblasts, a barrier to iPS cell-based modeling and regeneration. Here, we tested the feasibility of employing circulating erythroid cells, a compartment no or minimally affected in A-T, for the generation of A-T and carrier iPS cells. Our results indicate that episomal expression of Yamanaka factors plus BCL-xL in erythroid cells results in highly efficient iPS cell production in feeder-free, xeno-free conditions. Moreover, A-T iPS cells generated with this protocol maintain long-term replicative potential, stable karyotypes, re-elongated telomeres and capability to differentiate along the neural lineage in vitro and to form teratomas in vivo. Finally, we find that haploinsufficiency for ATM does not limit reprogramming from human erythroid cells or in vivo teratoma formation in the mouse.

Update #16-  in progress

After writing update #15 it quickly became obvious that we need to focus on the activity of the odd TET enzymes that in the presence of antioxidants maintain the unmethylated status of various unmethylated genes. Do they maintain the methylation status of Horvath’s 36 development/aging genes the become hypermethylated (probably gradually) during aging? Does the decline in the antioxidant status of older individuals lead to the turning off of these TET enzymes which allows the methylation of various genes? Why does the antioxidant status of individuals decline with aging? Let’s take another look at the hormones that change the most with aging.

We see  a huge decline in the hormones melatonin, DHEA, pregnenolone, estrogen, progesterone, and DHEA. What do all these hormones have in common? They all are either antioxidants (some very strong) or at least stimulate antioxidant activity. We also see a huge rise in a a number of (I propose) free radical stimulating hormones  like FSH, LH, and hCG. Here is some info  about these hormones:

Melatonin as an antioxidant: under promises but over delivers

Melatonin is uncommonly effective in reducing oxidative stress under a remarkably large number of circumstances. It achieves this action via a variety of means: direct detoxification of reactive oxygen and reactive nitrogen species and indirectly by stimulating antioxidant enzymes while suppressing the activity of pro-oxidant enzymes. In addition to these well-described actions, melatonin also reportedly chelates transition metals, which are involved in the Fenton/Haber-Weiss reactions; in doing so, melatonin reduces the formation of the devastatingly toxic hydroxyl radical resulting in the reduction of oxidative stress. Melatonin’s ubiquitous but unequal intracellular distribution, including its high concentrations in mitochondria, likely aid in its capacity to resist oxidative stress and cellular apoptosis. There is credible evidence to suggest that melatonin should be classified as a mitochondria-targeted antioxidant. Melatonin’s capacity to prevent oxidative damage and the associated physiological debilitation is well documented in numerous experimental ischemia/reperfusion (hypoxia/reoxygenation) studies especially in the brain (stroke) and in the heart (heart attack). Melatonin, via its antiradical mechanisms, also reduces the toxicity of noxious prescription drugs and of methamphetamine, a drug of abuse. Experimental findings also indicate that melatonin renders treatment-resistant cancers sensitive to various therapeutic agents and may be useful, due to its multiple antioxidant actions, in especially delaying and perhaps treating a variety of age-related diseases and dehumanizing conditions. Melatonin has been effectively used to combat oxidative stress, inflammation and cellular apoptosis and to restore tissue function in a number of human trials; its efficacy supports its more extensive use in a wider variety of human studies. The uncommonly high-safety profile of melatonin also bolsters this conclusion. It is the current feeling of the authors that, in view of the widely diverse beneficial functions that have been reported for melatonin, these may be merely epiphenomena of the more fundamental, yet-to-be identified basic action(s) of this ancient molecule.

2020; 11: 360.

Pregnenolone Inhibits Osteoclast Differentiation and Protects Against Lipopolysaccharide-Induced Inflammatory Bone Destruction and Ovariectomy-Induced Bone Loss
Using reactive oxygen species (ROS) detection assays, we found that Preg exhibits anti-oxidant properties inhibiting the generation of intracellular ROS following RANKL stimulation. Consistent with these in vitro results, we confirmed that Preg protected mice against local Lipopolysaccharide (LPS)-induced inflammatory bone destruction in vivo by suppressing osteoclast formation. Furthermore, we did not find any observable effect of Preg on osteoblastogenesis and mineralization in vitro. Finally Preg was administered to ovariectomy (OVX)-induced bone loss and demonstrated that Preg prevented systemic OVX-induced osteoporosis. Collectively, our observations provide strong evidence for the use of Preg as anti-osteoclastogenic and anti-resorptive agent for the potential treatment of osteolytic bone conditions.
Antioxidant effects of dehydroepiandrosterone (DHEA) and 7alpha-hydroxy-dehydroepiandrosterone in the rat colon, intestine and liver

This study examined in healthy male Wistar rats the in vivo antioxidant effect of dehydroepiandrosterone (DHEA) and 7alpha-hydroxy-DHEA administered by intraperitoneal injections (50 mg/kg body weight) for 2 or 7 days. Markers of oxidative damage to lipids (thiobarbituric acid-reacting substances, TBARS) and to proteins (protein carbonyls) were assessed in colon, small intestine, and liver homogenates. DHEA and 7alpha-hydroxy-DHEA caused a decrease in body weight. DHEA treatment significantly increased liver, colon, and small intestine cell weights. After 7 days, DHEA exerted an antioxidant effect in all organs studied. In the colon, oxidative damage protection was accompanied by a goblet cell proliferation and increase in acidic mucus production. After 2 days, the antioxidant effect of 7alpha-hydroxy-DHEA was mainly observed in the liver. Nonprotein sulfhydryl groups (mostly glutathione levels) were altered by DHEA in the liver whereas they remained unchanged after 7alpha-hydroxy-DHEA treatment. The results indicate that in healthy animals, DHEA exerts a protective effect, particularly in the colon, by reducing the tissue susceptibility to oxidation of both lipids and proteins. This effect was not limited to a specific tissue, whereas the metabolite 7alpha-hydroxy-DHEA exerted its antioxidant effect towards the two markers of oxidative damage earlier than DHEA, and mainly in the liver.

Hormones and oxidative stress: an overview
Spotlight on a New Heme Oxygenase Pathway: Testosterone-Induced Shifts in Cardiac Oxidant/Antioxidant Status
 Received: 15 July 2019 / Revised: 2 August 2019 / Accepted: 3 August 2019 / Published: 7 August 2019
A low testosterone level contributes to the development of oxidative damages; however, the cardiovascular effects of exogenous hormone therapy are not well elucidated. The aim of our work is to study the association of the testosterone level, antioxidant/oxidant system, and anti-inflammatory status related to the heme oxygenase (HO) system. To determine the effects of testosterone, 10-week-old, and 24-month-old sham-operated and castrated male Wistar rats were used. One part of the castrated animals was daily treated with 2.5 mg/kg cyproterone acetate, while the hormone replacement therapy was performed via an i.m. injection of a dose of 8.0 mg testosterone undecanoate/kg/once a week. The plasma testosterone level, the activity of HO and myeloperoxidase (MPO) enzymes; the concentrations of the HO-1, tumor necrosis alpha (TNF-α), and cyclic guanosine monophosphate (cGMP), as well as the total level of glutathione (GSH + GSSG) were determined from the cardiac left ventricle. In accordance with the testosterone values, the aging process and castration resulted in a decrease in antioxidant HO activity, HO-1 and cGMP concentrations and in the level of GSH + GSSG, whereas the inflammatory TNF-α and MPO activity significantly increased. Testosterone therapy was able to restore the physiological values. Our results clearly show that testosterone replacement therapy increases the antioxidant status and mitigates the inflammatory parameters via the modulation of the HO system. (like estrogen, T has both pro and anti oxidant effects.)
Aging and Luteinizing Hormone Effects on Reactive Oxygen Species Production and DNA Damage in Rat Leydig Cells

We observed previously that after long-term suppression of luteinizing hormone (LH) and thus of Leydig cell steroidogenesis, restimulation of the Leydig cells by LH resulted in significantly higher testosterone production than by age-matched cells from control rats. These studies suggest that stimulation over time may elicit harmful effects on the steroidogenic machinery, perhaps through alteration of the intracellular oxidant-to-antioxidant balance. Herein we compared the effects of LH stimulation on stress response genes, formation of intracellular reactive oxygen species (ROS), and ROS-induced damage to ROS-susceptible macromolecules (DNA) in young and in aged cells. Microarray analysis indicated that LH stimulation resulted in significant increases in expression of genes associated with stress response and antiapoptotic pathways. Short-term LH treatment of primary Leydig cells isolated from young rats resulted in transiently increased ROS levels compared to controls. Aged Leydig cells also showed increased ROS soon after LH stimulation. However, in contrast to the young cells, ROS production peaked later and the time to recovery was increased. In both young and aged cells, treatment with LH resulted in increased levels of DNA damage but significantly more so in the aged cells. DNA damage levels in response to LH and the levels of intracellular ROS were highly correlated. Taken together, these results indicate that LH stimulation causes increased ROS production by young and aged Leydig cells and that while DNA damage occurs in cells of both ages, there is greater damage in the aged cells.


Human chorionic gonadotropin (hCG) may be a marker of systemic oxidative stress in normotensive and preeclamptic term pregnancies

In vitro studies on placental function have revealed interactions between levels of secretion of human chorionic gonadotropin (hCG) by trophoblastic cells and oxidative stress generated by hydrogen peroxide (H2O2). Here, we have examined the relationship between maternal levels of hCG and H2O2 in vivo in term pregnancies with and without preeclampsia. We measured serum levels of hCG and H2O2 in twenty preeclamptic and twenty normotensive term pregnant women (controls), using an enzymatic immunoassay and an electrochemical method, respectively. Higher levels of serum hCG and H2O2 were observed in patients with preeclampsia in comparison to controls. A significant positive correlation between serum hCG concentration and H2O2 production was found. Our results show that: (1) systemic hCG levels are correlated with an oxidative stress state in term pregnant women with preeclampsia and (2) circulating hCG may be a monitoring tool of oxidative stress during pregnancy.

FSH is an odd case and I have yet to completely unravel its mystery. Given that it is a cAMP stimulating hormone like LH and hCG I would expect it to be primarily a free radical generating hormone. However FSH also has antioxidant like qualities. One case is that like just antioxidants, FSH can protect the developing ovum from free radical damage. But this hormone increases dramatically after age 50 in both men and women. I have also found that FSH is not associated with any cancers when I did a Pub Med search on this question for my 1998 paper. However, all the other cAMP stimulating hormones were in some way associated with various cancers. I believe somehow FSH is associated with the develpoment/aging  program that is accelerated in progeria as progeria kids never get cancer. Somehow FSH must be associated with Lamin A proteins- this would be a good area for some studies. Maybe FSH is like estrogen and progesterone havng both free radical and antioxidant potential.

FSH protects mouse granulosa cells from oxidative damage by repressing mitophagy

Oxidative stress has been implicated in triggering granulosa cell (GC) death during follicular atresia. Recent studies suggested that follicle-stimulating hormone (FSH) has a pivotal role in protecting GCs from oxidative injury, although the exact mechanism remains largely unknown. Here, we report that FSH promotes GC survival by inhibiting oxidative stress-induced mitophagy. The loss of GC viability caused by oxidative stress was significantly reduced after FSH treatment, which was correlated with impaired activation of mitophagy upon oxidative stress. Compared with FSH treatment, blocking mitophagy displayed approximate preventive effect on oxidative stress-induced GC death, but FSH did not further restore viability of cells pretreated with mitophagy inhibitor. Importantly, FSH suppressed the induction of serine/threonine kinase PINK1 during oxidative stress. This inhibited the mitochondrial translocation of the E3 ligase Parkin, which is required for the subsequent clearance of mitochondria, and ultimately cell death via mitophagy. In addition, knocking down PINK1 using RNAi confirmed the role of the FSH-PINK1-Parkin-mitophagy pathway in regulating GC survival under oxidative conditions. These findings introduce a novel physiological function of FSH in protecting GCs against oxidative damage by targeting PINK1-Parkin-mediated mitophagy.

My 1998 paper predicted that hormones that stimulate the cAMP pathway  could be considered free radical hormones and hormones that stimulated the cGMP pathway  could be considered antioxidant hormones :

cGMP Signaling Increases Antioxidant Gene Expression by Activating Forkhead Box O3A in the Colon Epithelium
Mitochondrial Biogenesis and Mitochondrial Reactive Oxygen Species (ROS): A Complex Relationship Regulated by the cAMP/PKA Signaling Pathway


Abstracts concerning TET enzymes>>>

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study- Published 2016

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

This Looks Interesting-
Tet1 Deficiency Leads to Premature
Reproductive Aging by Reducing Spermatogonia
Stem Cells and Germ Cell Differentiation

Ten-eleven translocation (Tet) enzymes are involved in DNA demethylation, important in regulating
embryo development, stem cell pluripotency and tumorigenesis. Alterations of DNA methylation
with age have been shown in various somatic cell types. We investigated whether Tet1 and Tet2 regulate aging. We showed that Tet1-deficient mice undergo a progressive reduction of spermatogonia
stem cells and spermatogenesis and thus accelerated infertility with age. Tet1 deficiency decreases
5hmC levels in spermatogonia and downregulates a subset of genes important for cell cycle, germ
cell differentiation, meiosis and reproduction, such as Ccna1 and Spo11, resulting in premature reproductive aging. Moreover, Tet1 and 5hmC both regulate signaling pathways key for stem cell development, including Wnt and PI3K-Akt, autophagy and stress response genes. In contrast, effect of Tet2
deficiency on male reproductive aging is minor. Hence, Tet1 maintains spermatogonia stem cells
with age, revealing an important role of Tet1 in regulating stem cell aging.

Effect of aging on 5-hydroxymethylcytosine in the mouse hippocampus


Aging is believed to affect epigenetic marking of brain DNA with 5-methylcytosine (5mC) and possibly via the 5mC to 5-hydroxymethylcytosine (5hmC) conversion by TET (ten-eleven translocation) enzymes. We investigated the impact of aging on hippocampal DNA 5-hydroxymethylation including in the sequence of aging-susceptible 5-lipoxygenase (5-LOX) gene.


Hippocampal samples were obtained from C57BL6 mice. Cellular 5hmC localization was determined by immunofluorescence. The global 5mC and 5hmC contents were measured with the corresponding ELISA. The 5-LOX 5hmC content was measured using a glucosyltransferase/enzymatic restriction digest assay. TET mRNA was measured using qRT-PCR.


Global hippocampal 5hmC content increased during aging as did the 5hmC content in the 5-LOX gene. This occurred without alterations of TET1–3 mRNAs and without changes in the content of 8-hydroxy-2-deoxy-guanosine, a marker of non-enzymatic DNA oxidation.


The aging-associated increase of hippocampal 5hmC content (global and 5-LOX) appears to be unrelated to oxidative stress. It may be driven by an altered activity but not by the increased expression of the three TET enzymes. Global 5hmC content was increased during aging in the absence of 5mC decrease, suggesting that 5hmC could act as an epigenetic marker and not only as an intermediary in DNA demethylation. Further research is needed to elucidate the functional implications of the impact of aging on hippocampal cytosine hydroxymethylation.

Additional abstracts of interest>>>

During caloric restriction we find that antioxidant hormones increase dramatically while free radical hormones decline dramatically-this leads to an altered (increased) antioxidant status in the nucleoplasm. This in turn would lead to the TET enzymes working better at keeping  (the 36?) anti-aging genes demethylated. This should lead to rejuvenating the animal undergoing CR at at least slowing the aging process. This allows the aging system to be malleable  depending on the hormonal milieu which is dictated by environmental conditions. It even allows for reversal of aging symptoms which would be beneficial during a famine for making sure that if a single mating pair of individuals survived a long famine (or drought) they would be young enough to reproduce and reconstitute the group…SO now we can see why melatonin given to women who have recently entered menopause can experience menopause reversal.

In my 1998 paper I studied the hormone changes that occur during caloric restriction and came up with this from the paper

Melatonin: the famine and drought hormone.

During famine conditions or CR one would expect that in addition to the increase in cGMP activity, that an increase in cGMP stimulating hormones would be seen. Also, one would expect a decline in cAMP stimulating hormones. In a study of human males undergoing 5 days of fasting (136)  the following hormone level changes were seen, (for hormones not measured in  this study other references are noted):

cAMP stimulating hormones:

TSH declined by 67%-as expected

LH decreased by 33%-as expected

FSH decreased by 33%-as expected

cortisol increased by 110%-unexpected

estrogen -increased by 10%-unexpected

cGMP stimulating hormones

Melatonin increased +/-100% in rats  (137)-as expected

GH increased 200%-400% in men  (138) -as expected

DHEA-S increased 100%-expected

Testosterone-decreased 50%- unexpected

T3 and T4 were relatively unaffected, and prolactin declined 25% but is not listed because it is an “ambidextrous” hormone stimulating both cAMP and cGMP depending on which receptors it influences.

The above results reasonably conform to expectations based on the prior hypothesis regarding cAMP and cGMP stimulating hormones.  However, by examining  the exceptions additional important insights can be gained. First, the cortisol increase of 110% is definitely not expected as it is a cAMP hormone and  the hormone is widely known to be implicated in accelerating the diseases of aging in persons where it is chronically elevated. What is also known about cortisol is that it has been implicated in triggering apoptosis is various cell types including thymocytes of the thymus gland (139). If the early stages of CR require a large scale induction of apoptosis in various cells, it is likely that the increased cortisol  is involved. The other contradiction about the large cortisol increase is that when it occurs  during CR one must assume that it does not lead to the deleterious accelerated age changes that are normally associated with high cortisol levels as CR’d animals live much longer than controls. One study explains the contradiction: during CR, although the baseline levels of cortisol are elevated, increases in peak cortisol levels from stress are shown to be lower in CR’d animals than in ad lib fed animals (140). The idea that evolution has designed stress so that at times it kills and at other times it does not suggests that stress is also an aging system. This idea will be explored shortly.

The other exceptions include a 10% increase in estrogen and a 50% decrease in testosterone. If one remembers that inhibition of reproduction  would be a primary goal of the CR response, then a drop in the male reproductive hormone is not illogical even though it is a cGMP hormone. The corresponding increase in DHEA of 100% which in absolute terms is of equal magnitude to the testosterone decline might be seen as CR’s version of testosterone that does not induce sexuality in the male. Finally, if the only male aging symptoms associated with AS#6 include prostatic atrophy (assuming no malfunctions in apoptosis) then the estrogen increase of 10% can also be seen as an anti-reproductive hormone change. An estrogen increase however, would not be expected to occur in the female during CR, and studies show that this is likely true (141).

CR leads to quite a complicated array of hormone changes, but can it all be simplified? A simple Medline search of melatonin  against each of the individual hormones mentioned above provides the answer. Melatonin administration has been shown to suppress LH (142), FSH (143), and testosterone(144) while increasing DHEA(145),  GH(146), and in some cases cortisol(147)  levels in either rats, mice or humans. In females, 300 mg. of  melatonin was shown to suppress estrogen (E2) levels (148). More definitive studies do need to be made in this area, however, as most studies are short term in nature while melatonin induced hormone changes seem to  take much longer to occur in humans.  Melatonin’s effect on prolactin, however, was not clear and is generally suggestive of increasing  levels in humans but this might only be a short term effect  due to the short term nature of  human melatonin studies. Melatonin, did however, reduce prolactin levels in the rat pituitary (149).  TSH was also shown to be suppressed in the rat by  melatonin (150)  In most cases of hormone changes induced by CR, melatonin administration induced the same effect. What is also interesting, a reduction in body temperature in animals is seen during CR and posited by some to be the potential candidate as the active life-extending mechanism in CR. As one would expect, melatonin administration leads to reduced body temperature as well (151a). It is interesting to note that water deprivation, as would be expected, has also been shown to increase melatonin levels in rodents (151b).”


Update #18 A List of Aging Researchers

Michael Lemonick

Dr. Michael Fossel

Stephen Spindler

Richard Miller

Arlan Richardson

Update #19

I always was curious about this study and now I thin we have shed some light on it. BHT certainly looks like a molecule that could colocalize with DNA.

Effects of the antioxidant butylated hydroxytoluene (BHT) on mortality in BALB/c mice

Butylated hydroxytoluene (BHT) was given in the feed to determine its effect on life span in genetically well-defined, barrier-derived BALB/c mice. Both sexes received 0.75% BHT for three different treatment periods: (A) 8 to 11 weeks of age; (B) for life, beginning at 11 weeks; (C) for life, beginning at 8 weeks of age. The control group (D) was untreated. All BHT treatment groups had mean survival times which exceeded that of controls. The order of survival was B greater than C greater than A greater than D (Males: 890, 832, 726, 684 days; Females: 875, 798, 759, 701 days). Most of the increases in mean survival time were related to a reduction in early deaths (350–600 days) in BHT-treated mice. The reason for the life-lengthening effect on BHT was not identified, but it may relate to alterations in specific disease incidences.

Molecular structure of BHT. | Download Scientific Diagram

Update #20

Vitamin D3 is also a hormone with antioxidant effects that declines with age. Why does it decline? Elderly skin makes less than half the amount of Vitamin D3 as young skin does from the same amount of sunlight.

Table 2: Vitamin D levels between male and female of different age groups

Vitamin D is a membrane antioxidant: thus Vitamin D3 (cholecalciferol) and its active metabolite 1,25-dihydroxycholecalciferol and also Vitamin D2 (ergocalciferol) and 7-dehydrocholesterol (pro-Vitamin D3) all inhibited iron-dependent liposomal lipid peroxidation.(Vitamin D3 levels are also positively correlated linked to longer telomeres) . This leads us to another purported source of aging- iron accumulation which apparently leads to a more oxidized ( free radicalized) cellular environment which is conducive to catalyzing demethylation of cytosines and loss of transcription factors as well as deactivating TET enzymes.

Male Fischer 344 rats fed ad libitum or dietary restricted (maintained on 60% of ad libitum food intake) were sacrificed at 6, 12 and 24 months of age. Portions of kidney, liver and brain were removed for total iron content analysis and oxidative stress assessment. Total iron content was measured directly and lipid peroxidation (LPO) was assayed as an index of oxidative stress. Tissue total iron content was shown to increase significantly with age in animals fed ad libitum (AL). At 24 months, these animals showed comparable iron content increases in the liver and kidney, but were significantly greater than measurements found in brain. This age-related iron accumulation, however, was found to be markedly suppressed by dietary restriction (DR) in all tissues. Similarly, LPO measurements increased in an age-related, tissue-specific fashion. At 24 months of age, measurements of LPO in AL rats brain and liver exceeded measurements in kidney. Again, we found DR to markedly suppress age-related LPO in all tissues. Reported here are our findings on the ability of DR to modulate iron status at the tissue level. Consistent with the proposed anti-oxidative mechanism of DR, these findings further suggest that the modulation of tissue total iron content is an important component of that mechanism.
(Thinking about the fact that calorically restricted rats  do not load iron into their tissues with age suggests to me that iron accumulation  is also part of the aging program and is no accident!). And yes iron accumulates in normal aging humans as well>>>
In healthy individuals brain iron levels increase with age (Bartzokis et al., 1994) and abnormally high brain iron levels are observed in age-related degenerative diseases.)

Here is an interesting abstract>>>

The Aging of Iron Man

Brain iron is tightly regulated by a multitude of proteins to ensure homeostasis. Iron dyshomeostasis has become a molecular signature associated with aging which is accompanied by progressive decline in cognitive processes. A common theme in neurodegenerative diseases where age is the major risk factor, iron dyshomeostasis coincides with neuroinflammation, abnormal protein aggregation, neurodegeneration, and neurobehavioral deficits. There is a great need to determine the mechanisms governing perturbations in iron metabolism, in particular to distinguish between physiological and pathological aging to generate fruitful therapeutic targets for neurodegenerative diseases.  Based on the evidence discussed here, we suggest a synergistic use of iron-chelators and anti-inflammatories as putative anti-brain aging therapies to counteract pathological aging in neurodegenerative diseases.

Update #21

After pondering the idea that the accumulation of iron in the elderly participates in driving the aging process by increasing the oxidation potential of the cellular environment, I then wondered if deficiencies in other minerals that are very common in the elderly also helped drive the aging process by reducing the antioxidant potential in the cell…There are two critical factors that are very often deficient in the elderly due to increasing difficulties in absorbing these minerals from the diet as one ages. It is estimated  that 80% to 95% of the elderly are magnesium deficient. Most are zinc deficient as well.

Magnesium in Aging, Health and Diseases
Abstract: Several changes of magnesium (Mg) metabolism have been reported with aging, including
diminished Mg intake, impaired intestinal Mg absorption and renal Mg wasting. Mild Mg deficits
are generally asymptomatic and clinical signs are usually non-specific or absent. Asthenia, sleep
disorders, hyperemotionality, and cognitive disorders are common in the elderly with mild Mg
deficit, and may be often confused with age-related symptoms. Chronic Mg deficits increase the
production of free radicals which have been implicated in the development of several chronic age

related disorders. Numerous human diseases have been associated with Mg deficits, including
cardiovascular diseases, hypertension and stroke, cardio-metabolic syndrome and type 2 diabetes
mellitus, airways constrictive syndromes and asthma, depression, stress-related conditions and
psychiatric disorders, Alzheimer’s disease (AD) and other dementia syndromes, muscular diseases
(muscle pain, chronic fatigue, and fibromyalgia), bone fragility, and cancer. Dietary Mg and/or Mg
consumed in drinking water (generally more bioavailable than Mg contained in food) or in alternative
Mg supplements should be taken into consideration in the correction of Mg deficits. Maintaining
an optimal Mg balance all through life may help in the prevention of oxidative stress and chronic
conditions associated with aging. This needs to be demonstrated by future studies.

Zinc, aging, and immunosenescence: an overview

Zinc plays an essential role in many biochemical pathways and participates in several cell functions, including the immune response. This review describes the role of zinc in human health, aging, and immunosenescence. Zinc deficiency is frequent in the elderly and leads to changes similar to those that occur in oxidative inflammatory aging (oxi-inflamm-aging) and immunosenescence. The possible benefits of zinc supplementation to enhance immune function are discussed.

 Magnesium increases the activity of an important glutathione enzyme called glutathione peroxidase (GPx). This enzyme accelerates how quickly glutathione neutralizes free radicals. Because free radicals are often considered to be “Public Enemy Number One” when it comes to cellular health, longevity and quality of life, you want antioxidants to neutralize them and protect against their damaging effects as quickly as possible. Supplementation with magnesium wasn’t just relegated to glutathione; it also increased the activity of two other extremely important antioxidants that reside in our bodies—catalase and superoxide dismutase.

Zinc is an Antioxidant and Anti-Inflammatory Agent: Its Role in Human Health

Zinc supplementation trials in the elderly showed that the incidence of infections was decreased by approximately 66% in the zinc group. Zinc supplementation also decreased oxidative stress biomarkers and decreased inflammatory cytokines in the elderly. In our studies in the experimental model of zinc deficiency in humans, we showed that zinc deficiency per se increased the generation of IL-1β and its mRNA in human mononuclear cells following LPS stimulation. Zinc supplementation upregulated A20, a zinc transcription factor, which inhibited the activation of NF-κB, resulting in decreased generation of inflammatory cytokines. Oxidative stress and chronic inflammation are important contributing factors for several chronic diseases attributed to aging, such as atherosclerosis and related cardiac disorders, cancer, neurodegeneration, immunologic disorders and the aging process itself. Zinc is very effective in decreasing reactive oxygen species (ROS). In this review, the mechanism of zinc actions on oxidative stress and generation of inflammatory cytokines and its impact on health in humans will be presented.

Update #22

We are looking for up to 30 people to participate in a demonstration project where they will get their DNA methylation age tested and then embark on a 3  to 4 month protocol involving  taking high dose melatonin (up to 400 mg/night) ,  DHEA, pregnenolone, and Vitamin D3 and then retesting their DNA methylation age to see if, as predicted, the DNA methylation age will be significantly lowered. If interested email me at

Update #23

Just in case you think this is all a lot of theory with no proof, take a look at another Horvath  recent study that is in press where he has reversed aging in old rats by 50 to 75%! By using the transient expression of Yamanaka Factors as designed by Dr. Harold Katcher.


Reversing age: dual species measurement of epigenetic age with a single clock



Young blood plasma is known to confer beneficial effects on various organs in mice. However, it was not known whether young plasma rejuvenates cells and tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly-accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n=593 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain-, liver-, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n=850 human tissue samples to the training data. We employed these six clocks to investigate the rejuvenation effects of a plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. Cellular senescence, which is not associated with epigenetic aging, was also considerably reduced in vital organs. Overall, this study demonstrates that a plasma-derived treatment markedly reverses aging according to epigenetic clocks and benchmark biomarkers of aging.


Update #24 Great News! 

Another antioxidant that declines dramatically with age (some say 90%) ,  but at least 50% has been identified >>  Alpha -Keto-Glutarate . Which just so happens, when combined with Vitamin C, keeps the TET enzymes working that keep your 36  anti aging genes turned on by demethylating them. Studies also show that this antioxidant extends the lifespan of mice  by about 15% and increases health span as well. Also, a related antioxidant the increases  by 1,000% during caloric restriction Butylated Hydroxy Butyrate has been shown to have many health improving and anti aging effects!

Image result for alpha ketoglutarate chemical structure      Image result for 3-Hydroxybutyrate



Update #25 More  Great News! 

Aging Cell. 2019 Dec; 18(6): e13028.

Reversal of epigenetic aging and immunosenescent trends in humans


Epigenetic “clocks” can now surpass chronological age in accuracy for estimating biological age. Here, we use four such age estimators to show that epigenetic aging can be reversed in humans. Using a protocol intended to regenerate the thymus ( DHEA, Growth Hormone, and metformin adminstration), we observed protective immunological changes, improved risk indices for many age‐related diseases, and a mean epigenetic age approximately 1.5 years less than baseline after 1 year of treatment (−2.5‐year change compared to no treatment at the end of the study). The rate of epigenetic aging reversal relative to chronological age accelerated from −1.6 year/year from 0–9 month to −6.5 year/year from 9–12 month. The GrimAge predictor of human morbidity and mortality showed a 2‐year decrease in epigenetic vs. chronological age that persisted six months after discontinuing treatment. This is to our knowledge the first report of an increase, based on an epigenetic age estimator, in predicted human lifespan by means of a currently accessible aging intervention.





  1. interesting article and brilliant analysis!!…quite paradoxical to other schools ( even lifeextensionists)….my questionists is do u have hint on “how to reactivate the 36 genes that are shut down by methylation and how to suppress that LARP1 gene to allow us to return back to a more differentiated version of ourselves”. I’m just curious to what youthink, since your high intuitions predicted this close since 1998 without sophisticated data..

  2. How could we possible know that it is hormone changes driving aging, and not aging driving hormone changes? I do not say rebalancing hormones to a more youthful state could not be beneficial, but it seems a stretch to say they are the cause of aging. To do that we’d need to see what the signal is that causes them to go awry. It could just be a feedback mechanism that gradually fails.

    1. Good question- and valid question..but after 35+ years of research on the question..I have found that it is the hornones that drive the aging process
      It is a bit slow to see in instead why dont you take a look at the atlantic salmon..who rapidly age and die in 3 days after spawning

      check out their hormone changes you will see LH shooting up 10,000 percent

      also check out the Pacific Salmon who also show the rapid aging and death after spawening and only live 3 years..
      you cut out their sex hormone producing glands and they live 7 years.

      1. I understand what you are saying, but I’d still like to know WHY hormone levels change so much. In the various Salmon cases you could either argue that the massive hormone boost is required to make the arduous journey back up rivers. Or perhaps it is intentionally to kill the salmon and make way for their young. It is plausible that a similar, albeit slower signal could be occurring in humans. But what is driving it? How does the body know when to turn on the kill switch?

        Regarding LH and FSH, is there a recommended range that older folks should aim for?

        1. HI there..good points/questions. One way I look at it is rather than ask if aging is programmed..Ask if menopause appears programmed. You would agree that that is true right? Well menopause just so happens to be the evolutionary equivalent of death . Once you cannot reproduce from either menopause death or aging, evolution does not care about you and you can have no more effect on the gene pool.How is menopause triggered? By a huge drop in melatonin levels. Melatonin suppresses LH and FSH. LH and FSH are very important hormones that control women’s reproductive cycle ona monthly basis. At menopause these hormoens do not go down. they shoot sky high..! And the same thing happens in men. If a women is just eentereing menopause, melatonin can reverse the process. For how long I don’t know that would make a great study. But what I can say is taking high doses of melatonin at night (75 mg + for women and maybe 120 mg + for men -due to weight differences) will help keep LH and FSH suppressed.

          1. This seems similar to Walter Pierpaoli’s research decades ago on melatonin as the master regulator of the hormonal aspects of programmed aging, of the master aging clock.

          2. Hi there yeah pretty similar but this takes it a lot furhter !! HAHA
            Funny , my uncle (godfather) George Solomon MD the famous psychiatrist
            was good friends with Walter Pierpoli and he told me to send my
            1998 paper to him…Never got any response back
            he probably didn’t understand it!

  3. Jeff

    Does Melatonin poser taste a bit chalky flavor?
    Just confirming I got what they claimed it was

    How do you measure it (eg 10mg, 20mg, 75mg, 100mg etc) given the “standard” dose in pills 3mg is tiny

    Do you use a sensitive electronic scale?

    1. HI there

      yeah melatonin does not have much taste where did you get yours from….

      If you bought bulk powder from peter at he gives you little measuring spoons…

      1. Jeff, how much powdered melatonin should one take in MG? I know there are no set rules here, but for a 50 year old man, what dose in mg would you suggest?

        1. hi there

          All I can say is I have tried many differnt doses over the years and I have finally settled on what I think is about 400 mg of powder under my tongue every night before bed…abut as much as I can handle wihtout puking

          if you do it…
          but that is going to probsably make you sleep likle crazy for up to 3 months before you get used to it and there will likely be short term sexual side effects..kibd of like propecia …

        2. Hey if you want to participate in a mini study we can get your DNA methylation age tested now before you try it and then 3 to 4 months later….are you game?? I would do it but Ive been taking too much melatonin for too long…

          1. Jeff, I may be interested in the melatonin study but may have some confounding factors. I am 67 and take bio-identical estrogen/testosterone and progesterone. No DHEA or pregnenolone. I have been taking melatonin at night but only 1.5 mg dose.

  4. my theory again around microbiome and microb! I read some where that the virus and microb, bacteria should be in body all intact and they just try to curb diseased cells! I wonder if this idea help with senescence cells removal from body so take out a lot of inflammatory source from body!?

    1. HI there yeah Harold Katcher is getting a patent on some plant orthologs of yamanaka factors to give you a blast of anti aging
      effects with no need for blood transfusion..It should be out soon
      You might get some good effects by just reversign the hormoes changes that occurred with aging…

      hgh dose melatonin which is a great antioxidant but also suppresses LH and FSH…pregnenolone dhea progesterone

  5. I have a question. Do we need to stop Vitamin D3 supplementation if we happen to be on vacation where we spend 2-3 hours at the beach with just a bathing suit?
    I vacation in Spain for 2-3 weeks in the summer and spend it at the beach almost every day. Can we over do it if we are also supplementing with D3? or can we still supplement while also spending time in the Sun.

    1. HI there

      If you are in the summer sun you don’t have to take as much D3 as your skin will make about 20,000 with 1/2 hour of sunbathing but it is good to take d3 before you get out in the many people say it prevents sunburn. Interestingly they found that Israeli lifeguards had about a 7x higher incidence of kidney stones than the normal population which means they were generating excess calcium in their blood form the d3 being made in their skin and this caused kidney stones because they weren’t taking vitamin k2!!

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