Fight Aging! Newsletter, June 26th 2017

Investigators have successfully repaired severe limb fractures in laboratory animals with an innovative technique that cues bone to regrow its own tissue. If found to be safe and effective in humans, the pioneering method of combining ultrasound, stem cell and gene therapies could eventually replace grafting as a way to mend severely broken bones. "We are just at the beginning of a revolution in orthopedics. We're combining an engineering approach with a biological approach to advance regenerative engineering, which we believe is the future of medicine."

The new technique could provide a much-needed alternative to bone grafts. In their experiment, the investigators constructed a matrix of collagen, a protein the body uses to build bones, and implanted it in the gap between the two sides of a fractured leg bone in laboratory animals. This matrix recruited the fractured leg's stem cells into the gap over two weeks. To initiate the bone repair process, the team delivered a bone-inducing gene directly into the stem cells, using an ultrasound pulse and microbubbles that facilitated the entry of the gene into the cells. Eight weeks after the surgery, the bone gap was closed and the leg fracture was healed in all the laboratory animals that received the treatment. Tests showed that the bone grown in the gap was as strong as that produced by surgical bone grafts.

We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area.

Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

If humanity were to appoint a general in our war against aging, Aubrey de Grey would likely earn the honor. The British author and biomedical gerontologist has been on the front line for years, researching ways to free the world of age-related disease and, ultimately, extend human life indefinitely. From the SENS Research Foundation Research Center (SRF-RC) in Mountain View, CA, foundation scientists conduct proof-of-concept research with the goal of addressing the problems caused by aging. They focus on repairing damage to the body at the molecular level, and their work is helping advance the field of rejuvenation biotechnology.

SRF-RC teams are currently focusing on two equally complex-sounding research projects, one centered on allotopic expression (a way to bypass the harmful effects of age-caused mitochondrial mutations) and the other on telomerase-independent telomere elongation (a little-researched process by which some cancer cells overcome mortality). Either project could lead to major breakthroughs in anti-aging treatments, but as de Grey explains, the path to immortality doesn't just run through the science lab. While the research being conducted at the SRF-RC is far from simple, de Grey claims DNA mutations and cancer cells aren't the biggest hurdles to anti-aging breakthroughs: "The most difficult aspect of fighting age-related diseases is raising the money to actually fund the research." The nature of most science research is exploratory. Researchers don't know that what they're working on is going to yield the results they expect, and even if it does, turning basic research into income is no easy task. To support their work, most have to rely on funding from outside sources, such as government grants, educational institutions, or private companies.

"It's still an incredibly hard sell," de Grey claims. "We have very limited resources. We only have about 4 million a year to spend, and so we spent it very judiciously." That money isn't going to just the two in-house projects, either. The SENS Research Foundation funds anti-aging research at institutions across the globe and provides grants and internships for students, so raising money to support those endeavors is key to continued success in its fight against aging. The benefits of ending the problem of aging would be tremendous. Not only would we be living longer, we'd be living healthier for longer.

Essential to raising money for anti-aging research is ensuring that those with the funds understands why it's worth the investment - a not-so-easy task given current misconceptions about aging. In 2015, eight major aging-focused organizations, released a report detailing what they call the many "notable gaps" that exist between expert perspectives on aging and the public's perception of the process. If the public isn't well informed on aging, it's even less knowledgeable about anti-aging. Fifty-eight percent of respondents in a 2013 Pew Research study said they had never even heard of radical life extension before. When asked if they would undergo treatments that would allow them to live to the age of 120 or older, the majority of those surveyed said they would not, and 51 percent thought such treatments would be "bad for society."

"There is still a huge amount of resistance to the logic that aging is bad for you and that it's a medical problem that needs to be addressed," explains de Grey. "It's really, really extraordinary to me that it's so hard to get this through to people, but that is the way it is. Aging is not mysterious. We understand it pretty well. It's not even a phenomenon of biology. It's more a phenomenon of physics. Any machine with moving parts is going to damage itself ... and the result is inevitably going to be that eventually the machine fails. It's the same for the human body as it is for a car, for example, and if we think about it that way, it becomes pretty easy to actually see what to do about it."

Among age-related diseases, cardiovascular disease has an impressive prevalence, considering that the remaining lifetime risk for cardiovascular disease is about 50% at the age of 40. Consistently, the pathophysiologic modifications that are observed in aging hearts and arteries interact with alterations that characterize atherosclerosis progression, concurring to the development of age-associated heart failure. This latter is due to a combined diastolic and systolic dysfunction, caused by cardiac hypertrophy, replacement fibrosis, and myocardial ischemia, even in the absence of atherosclerotic coronary disease.

Morphometric data acquired in the early 1990s suggested that the number of left ventricle cardiomyocytes declines progressively with aging. Consistently, investigators have documented that although cardiomyocyte turnover occurs postnatally, the rate of cardiomyocyte renewal declines as age advances. Intriguingly, while the same investigators have recently suggested that the total number of cardiomyocytes residing in the left ventricle does not change with aging, evidence of myocyte death has been shown to occur both in male primates and in humans. In these latter, cardiac troponin T levels increase with aging and can predict cardiovascular events and death in the general population. This finding is thought to be the consequence of the age-related reduction of expression or activity of proteins that are involved in cardioprotection, a condition that eventually leads to an increased susceptibility of cardiac myocytes to injury.

To understand the mechanisms leading to heart failure, we and other authors hypothesized that the reduced cardiomyocyte turnover observed in aging was a consequence of the reduced cardiac growth reserve. Several independent groups have shown that undifferentiated, primitive cells reside in mammalian hearts and are involved in cardioprotection against heart failure, possibly generating new myocytes. Conversely, different lines of evidence obtained in animal models of heart failure and in humans indicate that senescent and dysfunctional cardiac resident stem/progenitor cells (CS/PC) accumulate as a consequence of cardiac pathology. Furthermore, with organism aging, senescent primitive and differentiated cells accumulate in mammalian hearts.

Although the concept of cellular senescence was introduced more than 50 years ago, the debate around this programmed cellular behavior is still ongoing. Specifically, in relatively recent years, it has been shown that cell senescence may exert positive effects, by promoting tissue healing after injury and protecting young organisms from cancer. However, in line with the antagonistic pleiotropy theory of aging, these beneficial effects exerted by cell senescence in young animals may be also responsible for the occurrence of functional impairment and age-related pathologies. Consistently, "rejuvenation" strategies aimed at reducing the frequency of senescent cells in the organism or designed to modulate those pathways whose activation status is altered in cell senescence can restore cardiac function in aged and failing hearts. Finally, we should emphasize that, while it has been postulated that reactive oxygen species (ROS) play a primary role in the development of cell senescence, the molecular mechanisms responsible for the development and evolution of cellular senescence are still a matter of intense research.

For many years it was believed that ROS were produced in an unregulated manner as a byproduct of cellular metabolism. Moreover, their ability to cause damage to macromolecules was thought to be responsible for organism aging (also known as the mitochondrial free radical theory of aging, MFRTA). Consistently, several pieces of evidence have shown an age-dependent decrease in mitochondrial integrity, and a parallel increase in the level of oxidized DNA (including mitochondrial DNA). These alterations have led to the formulation of "the vicious cycle hypothesis of mitochondrial ROS generation," according to which the mitochondrial production of ROS would damage mitochondrial DNA (mtDNA) and lead to mitochondrial dysfunction, thus increasing ROS generation. However, discordant results have been obtained in more recent years, which have either supported or refuted the increased production of mitochondrial ROS with aging.

These seemingly contradictory results can be reconciled if we consider that ROS have a dual nature. In fact, on top of their ability to damage in non-specific fashion biological molecules, ROS can exert useful and beneficial effects, by regulating signaling pathways. According to current models, ROS generation is highly regulated, and therefore oxidative stress would arise from the loss of this architecture. Importantly, redox signaling is a crucial regulator of stem cell quiescence, self-renewal, and differentiation. Conversely, loss of controlled redox signaling (oxidative stress) can obliterate stem cell function and promote cell senescence of stem and differentiated cells, two conditions that have been associated with the progressive loss of tissue renewal and reparative reserve that characterize aging.

Cardiac stem cells are not immune from these pathological processes, becoming dysfunctional and unable to effectively repair cardiac damage with organism aging and pathology. mTOR signaling, which associates with cardiac stem cell senescence, may affect redox signaling at multiple levels, overloading the endoplasmic reticulum, and inhibiting both lysosomal function and autophagy. Therefore, innovative interventions aimed at restoring proper redox signaling in primitive cells have the potential to reverse or attenuate age-associated stem cell dysfunction.

Since SENS Research Foundation's founding in 2009, we've worked toward bringing our vision of a world free of age-related disease from concept to reality. In challenging ourselves on this front, we have likewise challenged you, our supporters. We've asked a lot of all of you, and not only have you accepted this challenge, you have delivered. The rejuvenation biotechnology community that has emerged over the past several years owes its existence to each and every one of you. You have become our most vocal advocates. Over 2000 of you have become our funders.

We asked you to help us change how the world researches and treats age- related disease. You did. Through the efforts of our donors, collaborators, and our advisory board, world-renowned institutions are pursuing age- related disease research specifically focused on the damage-repair paradigm. We asked you to help us move from basic research to translational research and clinical trials. You did. In 2016 we launched Project|21, our five-year plan to help move rejuvenation biotechnologies from concept to human clinical trials. Project|21 is now backed by a number of generous and forward-thinking individuals.

You asked us to follow through. We did. In lending your support, you place not only resources in our hands, but trust. We know that a world-changing nonprofit cannot operate on the power of vision alone; and we are here not just to inspire, but to deliver results. The purpose of this report is to demonstrate concrete examples of those results to you. With your help, we have taken great steps toward the establishment of a robust rejuvenation biotechnology industry and the realization of our vision. And every step we are able to take is proof of the power of your community.

Death-Resistant Cells: Toward Neutralizing the SASP

Buck Institute researchers led by Dr. Judith Campisi had shown that the presence of senescent cells alongside cancer cells can stimulate those cells to both multiply more rapidly and to spread to other parts of the body - the metastasis process, which ultimately makes most cancers so deadly. Repeating these studies in cell culture while inhibiting the senescence-associated secretory phenotype (SASP) with apigenin almost completely nullified the proliferation-stimulating and pro-metastatic effects of senescent cells on breast cancer cells. Drugs based on parts of apigenin's structure could dampen some of the harmful effects the SASP in senescent cells. Removing these cells is the ultimate solution to these problems, and in the last year several groups have made rapid progress toward this goal. In the meantime, these studies using apigenin may demonstrate important principles from which senescent-cell-focused rejuvenation biotechnologies may be derived.

Target Prioritization of Tissue Crosslinking

Our arteries slowly stiffen with age, in substantial part because of random crosslinking of the structural proteins collagen and elastin. Developing rejuvenation biotechnologies to break these crosslinks is key to restoring youthful arterial function. To tease out the effects and relative importance of all of these different sources of crosslinking in aging tissues, the Babraham Institute team has been studying the crosslinking process in the tissues of aging mice. This has required the development and validation of new experimental methods and assays, which are now ready for use. The team has evaluated multiple tissues for crosslink presence. Importantly, some of the crosslinks that have been reported by others to accumulate in aging tissues were not detected. While further studies are needed to confirm it decisively, these results suggest that several crosslinks now believed to accumulate in aging tissues may actually be experimental artifacts.

Engineering New Mitochondrial Genes to Restore Mitochondrial Function

Free radicals derived from our energy-producing mitochondria can mutate the organelle's DNA, leading to deletions of large stretches of the mitochondrial genome. These deletion mutations prevent the mitochondria from building various pieces of the electron transport chain (ETC), with which mitochondria generate most cellular energy. The accumulation of deletion-mutation-containing cells is a significant consequence of aging. A potential rejuvenation biotechnology to recover ETC function is the allotopic expression of functional mitochondrial genes: placing "backup copies" of all of the protein-coding genes of the mitochondria in the "safe harbor" of the nucleus, thereby giving the mitochondrion all of the proteins it needs to continue producing energy normally even when the original mitochondrial copies have been mutated.

This year, the SRF MitoSENS team reported a tremendous success: for what they believe is the first time, they have used allotopic expression to rescue the complete loss of a mitochondrially-encoded protein in a mammalian cell. A publication announced their success in the fall of 2016. The results show that their targeted and recoded ATP8 protein can be expressed from the nucleus, turned into protein in both normal and mutant cells, and efficiently targeted to the mitochondria. Furthermore, they can demonstrate functional rescue of cells. Under conditions where mutant cells die for lack of ability to produce energy, the cells with engineered allotopically-expressed proteins were able to survive and replicate. In addition to ATP8, the SRF MitoSENS team has further demonstrated expression and targeting of a second re-engineered protein, ATP6. It is proof-of-concept that ATP8 is not a special case.

Identification of the Genetic Basis of ALT in Cancer

Telomeres shorten every time a cell divides, and thus all cancers have to find a way to keep their telomeres long enough to prevent senescence or death. Most cancers use an enzyme called telomerase for this purpose, but about 10-15% of cancers use a telomerase-independent mechanism known as Alternative Lengthening of Telomeres (ALT). The ALT mechanism remains largely a mystery, and therefore the OncoSENS team at SENS Research Foundation is working hard to find new ways to attack ALT cancers. First, the team has developed and established two separate high-throughput assays measuring different ALT-specific biomarkers. These assays will finally enable cancer researchers to screen hundreds of thousands of compounds across multiple drug libraries, or even test every single one of the more than 20,000 genes in the human genome, for ways to shut down ALT cancers. In addition to their biomarker work, the team is also pursuing more targeted methods to kill ALT cancer cells.

Glucosepane Crosslinks and Routes to Cleavage

One major cause of crosslink accumulation in aging is Advanced Glycation Endproducts (AGE), and one AGE in particular, called glucosepane, is currently thought to be the single largest contributor to tissue AGE crosslinking. The Yale AGE team is studying the role of AGEs in aging, and developing novel tools and strategies for reversing AGE-mediated protein damage and develop new antibodies and reagents to enable rejuvenation research. Our pilot lab at Cambridge University found that all of the commercially-available antibodies for the major AGE-related molecules are actually highly unreliable. This is a serious impediment. The Yale glucosepane team is now tackling this problem via the novel chemistry and methods they have developed. In the last twelve months, the Yale team has made exciting progress in their work. Most notably, they have developed the first synthetic route to produce glucosepane. Their novel synthetic strategy is the first ever to provide high yields of pure samples of glucosepane, putting them (and soon other scientists) for the first time in a position to explore mechanisms through which crosslinks can be broken.

In collaboration with a colleague at Yale, they have also developed a high-throughput assay for screening proprietary libraries of organic catalysts for agents capable of breaking synthetic glucosepane. One of these libraries has already been taken forward for proof of concept, which led to the identification of several leads for catalysts that could be capable of breaking glucosepane. Beyond that, the Yale group has successfully generated proteins containing their synthetic glucosepane that can be used to identify antibodies that label glucosepane-containing proteins. These antibodies will enable the immunochemical detection of glucosepane crosslinks for a wide range of applications.

Tissue-Engineered Thymus

The thymus gland is responsible for the development of a class of immune cells called T-cells. As part of the degenerative aging process, the thymus shrinks in size. This process of thymic atrophy prevents the body from maturing new T-cells, progressively weakening the immune system's ability to fight off never-before-encountered infections. Engineering new, healthy thymic tissue would help to restore the vigorous immune response of youth. SENS Research Foundation has therefore funded a Wake Forest Institute for Regenerative Medicine (WFIRM) group to apply tissue engineering techniques to the creation of functional thymic tissue to fortify or replace the aging thymus. Engineering new tissues requires a "scaffold" in which to embed cells to give them structure and functional cues, and the WFIRM group has tested different scaffolding systems: decellularized donor scaffolds and hydrogels.

In the decellularized scaffold paradigm, an organ of the type that is needed is taken from a donor, but is then stripped of its original cells and DNA, leaving behind a protein structure with low potential for immunological rejection that can be repopulated with cells taken from the new organ recipient. The WFIRM group initially began work in this paradigm using mouse organs, but they found that once decellularized, mouse thymuses lacked the rigidity to serve in that role. They accordingly moved on to the pig thymus - a species that not only worked well as an experimental system, but has some clinical potential as well. The pig is closer to humans both immunologically and in terms of size.

Catalytic Antibodies Targeting Transthyretin Amyloid

As part of the degenerative aging process, proteins that normally remain dissolved in bodily fluids become damaged, and adopt a misfolded form called amyloid. Amyloid composed of the transporter protein transthyretin (TTR) deposits in the heart and other organs with age, beginning to impair heart function. With SRF funding, the University of Texas-Houston Medical School (UTHMS) extracellular aggregate team is working to develop novel catalytic antibodies ("catabodies") that would recognize and cleave TTR amyloid deposited in the heart and other tissues. Catabodies have the potential to be safer and more effective than conventional antibody-based immunotherapies: their catalytic activity minimizes the amount of antibody required to clear deposits from tissues, and the fact that they don't form stable complexes with their targets or engage immune cells is expected to minimize the inflammatory side-effects seen with other experimental antibody therapies.

Work has resulted in the identification of two powerful TTR-cleaving catabodies. When tested for their ability to degrade misfolded wild-type TTR, these candidates were able to hydrolyze both soluble aggregates and deposit-like particulates, while having no effect on either TTR in its healthy, normal conformation or on a selection of fourteen other physiologically important proteins. Concentrations required to disintegrate 80% of a sample of amyloid were many hundreds of times lower than those routinely achieved in the blood using other infused antibodies. The establishment of stable cell lines will enable larger-scale production, as the team works to develop these candidates into functional rejuvenation biotechnologies.

Rejuvenation of the Systemic Environment

There might be a misunderstanding of what was really going on in parabiosis. When animals are connected, they are not just given reciprocal blood transfusions, but are surgically joined together. So in addition to receiving young blood, the old animals also have their old blood filtered through the young animals' livers and kidneys, and diluted with the young pairmate's own blood. Might the effects of parabiosis mostly come from the removal of toxic or suppressive factors from the old animals' sluggish circulation instead of from the delivery of active rejuvenating factors?

To test this possibility - and to accelerate identification and testing of potential pro- and anti-rejuvenation factors in the exchanged blood - SENS Research Foundation funded Dr. Conboy and the UC Berkeley systemic environment team's development of a novel technological platform. Using a mixture of off-the-shelf and custom 3-D printed parts, this platform enables the group to easily and safely extract blood from small animals and transfuse it quickly and directly into another animal, without the reciprocal exchange of its blood or the passage of its blood through the pairmate's system. It thus separates the effects of the young animals' metabolic and excretory systems from the pure effects of their blood.

The team then used the new system to repeat key parabiosis experiments from Dr. Conboy's and others' labs. As compared with the impact of full-on parabiosis, the effects of isolated young blood on old muscles' ability to repair an injury were still substantial: the stem cells recovered significant regenerative powers, and less residual fibrosis remained after the wound was resolved. But by contrast, previously-reported benefits of parabiosis in the brain and the liver were either not present, or were far more modest. Another critical finding was the confirmation of suppressive factors in the old animals' blood, which inhibited neurogenesis and other regenerative responses of young animals transfused with it. While this clearer picture of the basis of the "parabiosis effect" indicates a lower likelihood of isolating true pro-rejuvenation factors in the blood of young mice, we are nonetheless closer to being able to filter out factors responsible for suppressing the regenerative potential of an older body.

Commercial Development

Two of the companies SENS Research Foundation has supported are moving to raise funding to move their research from the lab to clinical trials. Ichor Therapeutics announced a Series A offering to bring its Lysoclear product for age-related macular degeneration and Stargardt's macular degeneration through Phase I clinical trials. In 2014, Ichor Therapeutics completed a material and technology transfer agreement for rights to concepts and research pioneered by SENS Research Foundation. Lysoclear, which Ichor announced in 2017, is a recombinant enzyme product based on extending SRF's prior work that selectively localizes to the lysosomes of retinal pigment epithelium cells where A2E accumulates, and destroys it. Ongoing studies suggest that Lysoclear is safe and effective at targeting A2E, the main toxin driving these diseases, eliminating up to 10% with each dose. This product would be the first clinical candidate based on concepts and research pioneered by SENS Research Foundation.

Oisin Biotechnologies is focused on the genetic elimination of unwanted cells, but without involving the immune system. Oisin reports significant progress in showing that their vector works, efficiently transducing cells and delivering a DNA construct which can kill targeted cells on command. Oisin closed a 500K oversubscribed convertible debt round in mid-December and is working towards a substantial Series A in the next few months that would take it towards a Phase 1 clinical trial.

Traditional population projections categorize "old age" as a simple cutoff at age 65. But as life expectancies have increased, so too have the years that people remain healthy, active, and productive. In the last decade, researchers have published a large body of research showing that the very boundary of "old age" should shift with changes in life expectancy, and have introduced new measures of aging that are based on population characteristics, giving a more comprehensive view of population aging. The study combines these new measures with UN probabilistic population projections to produce a new set of age structure projections for four countries: China, Germany, Iran, and the USA.

One of the measures used in the paper looks at life expectancy as well as years lived to adjust the definition of old age. Probabilistic projections produce a range of thousands of potential scenarios, so that they can show a range of possibilities of aging outcomes. For China, Germany, and the USA, the study showed that population aging would peak and begin declining by 2040 in Germany and by 2070 in China, well before the end of the century. Iran, which had an extremely rapid fall in fertility rate in the last 20 years, has an unstable age distribution and the results for the country were highly uncertain. Population aging - when the median age rises in a country because of increasing life expectancy and lower fertility rates - is a concern for countries because of the perception that population aging leads to declining numbers of working age people and additional social burdens.

Probabilistic population forecasts were motivated by Keyfitz. Keyfitz wrote: "Demographers can no more be held responsible for the inaccuracy in forecasting population 20 years ahead than geologists, meteorologists, or economists when they fail to announce earthquakes, cold winters, or depressions 20 years ahead. What we can be held responsible for is warning one another and our public what the error of our estimates is likely to be."

There is now an extensive literature of probabilistic forecasting. All the probabilistic measures of aging produced by the United Nations assume that the threshold of old age is a fixed chronological age, regardless of time, place, education, or other characteristics of people. Researchers have questioned this assumption: "To the extent that our concern with age is what it signifies about the degree of deterioration and dependence, it would seem sensible to consider the measurement of age not in terms of years elapsed since birth but rather in terms of the number of years remaining until death." It is suggested the old age threshold be defined on the basis of some plausible remaining life expectancy rather than any specific chronological age. We call the ages, that are obtained when life expectancy is the characteristic that is held constant, prospective ages and measures that use them prospective measures of population aging.

New measures of population aging are useful because tomorrow's older people will not be like today's. They may well have longer life expectancies, better cognition, better education, and fewer severe disabilities. In most OECD countries, the labor force participation of people 65+ years old is increasing as are the ages at which people can receive a normal national pension. Since changes in the characteristics of people are ignored in the conventional measures of aging, they become more outdated with the passage of time. The use of prospective ages is one way to create measures of aging that are more in line with observable changes.

We chose a remaining life expectancy of 15 years. That was the life expectancy at age 65 in many low mortality countries around 1970. We show estimated and forecasted old age thresholds based on a remaining life expectancy of 15 years for China, Germany, Iran and the US for the years 2013 through 2098. In 2013, the old age threshold was 66 in China and 72 in Germany. By 2098, the old age threshold is forecasted to increase to 79 in Germany and 77 in China. Most of the population aging that we measure in China, Germany, and the US occurs between now and around 2040. The probabilistic forecasts show that it is highly likely that the prospective median age of the population of Germany and the US will be lower in 2098 than it is today. Even in China there is around a 50 percent chance that the prospective median age will be lower at the end of the century than it is today. It is possible that people's concern about the future is related to the number of additional years they expect to live. Lower prospective median ages in 2098 than now indicate that the people at the median age will have even more years of additional life ahead of them than people at the median age have currently, despite that median age being higher.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excess hepatic fat (steatosis) in individuals who drink little or no alcohol. NAFLD is more prevalent in older populations. The mechanisms underlying this condition are not understood nor is why its prevalence increases with ageing. It has been speculated that ageing processes may promote NAFLD via different mechanisms, including adipose tissue dysfunction, impaired autophagy, and oxidative stress.

Cellular senescence is a state of irreversible cell-cycle arrest, which can be induced by a variety of stressors, including telomere dysfunction and genotoxic and oxidative stress. Senescent cells frequently have increased secretion of a broad repertoire of proinflammatory factors, collectively known as the senescence-associated secretory phenotype, which can induce tissue dysfunction in a paracrine manner. Senescent cells have mitochondrial dysfunction, with decreased oxidative phosphorylation and concomitantly increased generation of reactive oxygen species (ROS), caused at least partly by failing mitophagy.

A significant fraction of hepatocytes develop a senescent phenotype during the life course of mice and with age-related liver disease in humans. However, the relationship between cellular senescence and liver fat accumulation remains unclear. Here we hypothesized that cellular senescence results in impaired fat metabolism and that removal of senescent cells may diminish liver steatosis.

We found a close relationship between senescence markers and fat accumulation in hepatocytes of mice fed ad libitum (AL), dietary restricted (DR) or following dietary crossover and in a small cohort of NAFLD patients. Furthermore, clearance of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice and a senolytic cocktail of dasatinib plus quercetin reduced overall hepatic steatosis in ageing, obese, and diabetic mice. In contrast, hepatocyte-specific induction of senescence by a local DNA repair defect resulted in liver steatosis. Finally, we found that induction of senescence in mouse fibroblasts and hepatocytes resulted in decreased ability to metabolize fat. Our findings suggest that interventions targeting senescent cells may be developed into therapies to reduce steatosis during NAFLD.

We have made huge strides in the last decade or so and we know a great deal about the processes and damages aging causes, but sadly this does not mean we know enough. There is much more to be learned in order to develop effective interventions and therapies to address these processes, and this is where basic science comes in. The countdown to accessible therapies against aging will not start unless each mechanism of aging is well understood. If all of them were understood right now, you would still need to wait for another 17 years to get a full range of therapies against aging. If you add 17 years to your current age and don't like the resulting number and its relation to the onset of age-related diseases, then ask yourself this, is supporting basic research on aging now in your interest? You may not be very excited about life extension in mice, but remember, no results in mice equals no translation to humans.

It is true the government funds research institutions and awards research grants. But the idea of preventing age-related diseases by addressing its underlying mechanisms is relatively new. There are not many experts among the decision makers in the grant system who can assess the breakthrough projects aimed at the hallmarks of aging and truly understand their potential. This is why these kinds of projects have less support from the government than the mainstream studies of a single disease like Alzheimer's or cancer.

In the case of government funding, the money goes from the taxpayer to the government treasury, where its future allocation is decided, and then to specific research institutions whose plan of research falls within the mainstream priorities. This makes it very hard for our community to influence the direction of research, which is a serious limitation indeed. Crowdfunding does not have this limitation, because it allows the public to connect with the researchers directly and support only the projects they believe are important. The amount of money collected during a crowdfunding campaign can be as much as a government grant (often even bigger), plus there is no need for the excessive paperwork typical for a government grant. This means that the researchers can focus on what they do best of all: their studies.

The number of ardent supporters of aging and longevity studies is relatively small due to the slow dissemination of information from scientists to the public. Most people still believe there is nothing we can do about biological aging, and so they see these studies as researchers simply feeding their scientific curiosity. Education regarding the plausibility and desirability to defeat aging takes time, patience and a lot of effort. It cannot be done by the scientists themselves (as their job is to work in the lab, not to make shows), and here is where advocacy groups and science popularizers should step in. However, people tend to forget that the best results can only be achieved if a group is well-organized, disciplined and uses evidence-based practices in all activities, from planning and management to crowdfunding, educating and lobbying. Steady progress requires a mindful and responsible approach from each person joining an advocacy group - which is sadly rarely seen.

Many members of our community prefer to profess their desire for indefinite lifespans directly, shocking the public. It is important properly explain the connection between aging and age-related diseases, and the causal relationship between aging prevention, health improvement, and longevity - longevity being a side-effect of better health. Being patient and addressing concerns people may have in relation to longer lives (like overpopulation, unequal access, boredom and others) is another important job which is rarely done properly, with enough supporting data to hand. Despite the fact that most of the sociological studies on public attitudes regarding life extension are available to read and have even been summarized by different members of the community, many people still refuse to explain the basics, or insist on using counterproductive radical messages, provoking additional skepticism and closing doors that would otherwise be open. Before starting a conversation with someone who is not familiar with the idea of healthy life extension, it would be useful to take a look at the existing data regarding how to make such conversation productive.

Alteration of the ribosome biogenesis and an overall protein synthesis rate decline have been observed to characterize aging process in many organisms, including humans. This decline could be an effect of the progressive deterioration in most cellular functions usually associated with aging, or it could be a concurrent factor in the process. If to date a general reduction of protein synthesis has been attributed to the decreased frequency of mRNA translation, current studies, reporting an involvement of epigenetic mechanisms in silencing a large fraction of the ribosomal RNA (rRNA) genes, with a consequent impairment of ribosomal DNA (rDNA) function, could lead to a new understanding of the phenomenon.

On the basis of this evidence, we investigated whether changes in the DNA methylation patterns of the rRNA gene promoter take place during the lifetime. DNA samples were extracted from whole blood collected from differently aged human individuals displaying different phenotypes according to cognitive, functional, and psychological parameters. We did not find a consistent statistically significant association between the methylation levels of the analyzed CpG sites with the age of the donor. On the other hand, although it is not associated with chronological aging, in middle/advanced-aged subjects the variability of CpG_5 methylation was found to be significantly correlated with both cognitive performances and survival in the 9-year follow-up period. This last result, which held multiple test correction, was further confirmed in the replication sample.

Our results seem to be particularly attractive, because they show a fine remodeling of the methylation profile associated with the biological aging rather than to the chronological age. The effects at molecular levels of the above association have to be clarified, but it is plausible to hypothesize that the decrease in the rRNA levels we observed late in life may be determined by the methylation of the CpG_5 site that in turn might be driven by multiple factors, including genetic variations, diet, environment, and the interindividual variation of the structure of rDNA cluster itself.

How could the methylation changes of peculiar CpG sites be functionally involved in the functional decline characterizing the aging process? If the epigenetic modification of functional sites may hamper ribosomal biogenesis, this may drastically reduce the cellular protein synthesis, being ultimately responsible of those multisystem deficits occurring over the lifetime. Thus, an interdependence seems to exist between rDNA promoter methylation and the aging process, and in particular with the aging associated decay, and these sites may represent an potential evolutionary conserved biomarker of the rate of the aging process.

Nearly two decades ago, two key findings connected cytomegalovirus (CMV) with immune senescence. In 1999, researchers showed that CMV-positive and -negative humans exhibit dramatically different T cell subset ratios and that the effect seems to be increasing with aging. Around the same time, others described "memory inflation" driven by CMV in mice. Since then, numerous studies have been published investigating the associations between human, non-human primate or murine CMV with their respective hosts in the course of aging. The interest in the topic has been so sustained that it led to the establishment of CMV and Immunosenescence Workshops. This overview summarizes the state of the field before and the discussions at the 6th International CMV and Immunosenescence Workshop.

CMV, a member of beta-herpesvirus family, is the largest human virus. As is the case for other herpesviruses, following a brief acute infection period that elicits a typical CD8 T cell response, as well as CD4 and B cell responses, CMV establishes persistence that includes latency. Persistence/latency is established in reservoir cells that are distributed broadly across the organism. However, it is clear that the primary CMV infection is followed by a period of viral shedding and it remains unclear whether the virus, that is a master in immune evasion, ever really is truly latent in all its reservoirs or whether some cells produce it as a smoldering infection at low levels at most, if not all times. As a consequence, a large population of CMV-specific CD8, and to a lesser extent, CD4 T cells, is generated in response to cycles of viral reactivation (aptly called memory inflation). How exactly this memory inflation impacts the ability of an older immune system to function and provide defense against other infections is one of the key topics of interest.

The 6th International Workshop on CMV and Immunosenescence was organized with a primary goal to fill a gap identified at several recent meetings. Specifically, in addition to the topics reviewed above from the fifth workshop, it was felt that stronger attention must be focused on the biology of the virus itself and on its interactions with the host in the course of latency and reactivation. In fact, one of the greatest weaknesses of research into HCMV and immunosenescence comes from our inability to control and measure the state of viral activity. Another area of major interest is the impact of CMV on diseases on aging - whether and how the virus may be involved in modulating frequent age-related morbidities, in particular cardiovascular diseases (CVD), where there are strong epidemiological associations between CMV infection and morbidity and mortality from CVD.

In a provocative new perspective piece, researchers say that disease genes are spread uniformly across the genome, not clustered in specific molecular pathways, as has been thought. The gene activity of cells is so broadly networked that virtually any gene can influence disease, the researchers found. As a result, most of the heritability of diseases is due not to a handful of core genes, but to tiny contributions from vast numbers of peripheral genes that function outside disease pathways. Any given trait, it seems, is not controlled by a small set of genes. Instead, nearly every gene in the genome influences everything about us. The effects may be tiny, but they add up.

The researchers call their provocative new understanding of disease genes an "omnigenic model" to indicate that almost any gene can influence diseases and other complex traits. In any cell, there might be 50 to 100 core genes with direct effects on a given trait, as well as easily another 10,000 peripheral genes that are expressed in the same cell with indirect effects on that trait. Each of the peripheral genes has a small effect on the trait. But because those thousands of genes outnumber the core genes by orders of magnitude, most of the genetic variation related to diseases and other traits comes from the thousands of peripheral genes. So, ironically, the genes whose impact on disease is most indirect and small end up being responsible for most of the inheritance patterns of the disease.

Researchers have thought of genetically complex traits as conforming to a polygenic model, in which each gene has a direct effect on a trait, whether that trait is something like height or a disease. In earlier work on the genetics of height, researchers were surprised to find that essentially the entire genome influenced height. "It was really unintuitive to me. To be honest, I thought that it was probably wrong. I gradually started to realize that the data don't really fit the polygenic model. We started to think, 'If the whole genome is involved in a complex trait like height, then how does that work?'"

The polygenic model leads researchers to focus on the short list of core genes that function in molecular pathways known to impact diseases. So, therapeutic research typically means addressing those core genes. A common approach to gene discovery is to do larger and larger genome-wide association studies, but the team argues against this approach because the sample sizes are expensive and the thousands of peripheral genes uncovered are likely to have tiny, indirect effects. "After you get the first 100 hits, you've probably found most of the core genes you're going to get through genomewide association studies." Instead, the team recommends switching to deep sequencing the core genes to hunt down rare variants that might have bigger effects. "If this model is right, it's telling us something profound about how cells work that we don't really understand very well. And so maybe that puts us a little bit further away from using genome-wide association studies for therapeutics. But in terms of understanding how genetics encodes disease risk, it's really important."

Inhibitors of mTOR, including clinically available rapalogs such as rapamycin (Sirolimus) and Everolimus, are gerosuppressants, which suppress cellular senescence. Rapamycin slows aging and extends life span in a variety of species from worm to mammals. Rapalogs can prevent age-related diseases, including cancer, atherosclerosis, obesity, neurodegeneration and retinopathy and potentially rejuvenate stem cells, immunity and metabolism. Here, I suggest how rapamycin can be combined with metformin, inhibitors of angiotensin II signaling (Losartan, Lisinopril), statins, propranolol, aspirin and a PDE5 inhibitor. Rational combinations of these drugs can maximize their anti-aging effects and decrease side effects.

At first, the discovery of anti-aging properties of rapamycin was met with skepticism because it challenged the dogma that aging is a decline driven by molecular damage caused by free radicals. By now, rapamycin has been proven to be an anti-aging drug. In contrast, anti-oxidants failed in clinical trials and the dogma was shattered. In the last decade, anti-aging effects of rapamycin have been confirmed. Anti-aging doses and schedules can be extrapolated from animal studies. Well-tolerated doses with minimal side effects can be deducted based on clinical use of rapalogs. So optimal anti-aging doses/schedules can be suggested. Given that rapamycin consistently extends maximal lifespan in mice, rapamycin will likely allow mankind to beat the current record of human longevity, which is 122 years. Yet, rapamycin will not extend life span as much as we might wish to. Now is the time for anti-aging drug combinations. For example, metformin is currently undergoing re-purposing as an anti-aging agent. Several other existing drugs can be re-purposed. Now we can design an anti-aging formula, using drugs available for human use.

Rapamycin (or another rapalog) should be a cornerstone of anti-aging combinations, given its universal anti-aging effect and the ability to delay almost all diseases of aging. Rapamycin and metformin: Both drugs extend lifespan in animals and have non- overlapping effects. In addition, they may, in theory, cancel possible metabolic side-effects of each other. Rapamycin and statins: Rapamycin promotes lipolysis increasing blood levels of fatty acids. This, in turn, increases levels of lipoproteins produced by the liver. Rapamycin-induced hyperlipidemia is benevolent and reversible. Still, statins are already used to prevent rapamycin-induced hyperlipidemia.

The 7-drug combination can be tested in mice, especially in mice on high fat diet and in cancer-prone mice. If started late in life, the experiments will take just several months to evaluate the effect on lifespan and cancer incidence as well as weight, blood pressure, glucose, insulin, triglycerides and leptin. In humans, the treatment program can be initiated regardless of any pre-clinical studies, because all 7 drugs are approved for human use and some of them such as aspirin and statin are widely used for disease prevention anyway. The only what is needed is to watch for side effects. Especially, heart rate, blood pressure, and glucose levels should be monitored.

The anti-aging formula is ready for human use. If one will wait until the life-extending effect will be shown in others, this individual will not be alive by the time of the result. Human clinical trials are needed to optimize the doses and schedules. However, unless we participate in clinical trials ourselves, we will not know how long participants will live because they are expected to outlive non-participants. If we want to live longer we should be participants in clinical trials. In the best scenario, this might allow us to live long enough to benefit from future discoveries of anti-aging remedies.

After nerve trauma, the standard clinical operating procedure is to oppose the two nerve ends and, when possible, suture them together. Ultimately, even with successful autografting, only 40% of patients regain useful function. Therefore, there is a clear, urgent, and unmet clinical need for an alternative approach that can match or exceed autograft performance. After peripheral nervous system (PNS) injuries, neurons respond rapidly by changing their activities and promoting a regenerative phenotype. At the distal nerve stump, Schwann cells (SCs) adopt a reparative phenotype. SCs, as well as infiltrating and resident macrophages, remove inhibitory debris, enabling new axons to sprout into the degenerated nerve. Although monocytes and their descendants (in particular, macrophages) have long been known to play an essential role in the degenerative process, only recently has their importance in positively influencing regeneration been recognized. Monocytes are abundant during nerve degeneration and regeneration and modulate the sequence of cellular events which can determine the outcome of the healing process.

After inflammatory insult, macrophages that accumulate at the site of injury appear to be derived largely from circulating monocytes. Entry of monocytes into the distal site of an injured nerve is enabled through up-regulation and release of a major monocyte chemokine, monocyte chemoattractant protein (CCL2) by SCs, which reaches its maximum 1 day after injury. Besides CCL2, the CX3CR1 ligand (fractalkine) can also recruit monocytes through the CX3CR1 receptor. In rats, two major subsets of monocytes have been identified. These two subtypes of monocytes can be recruited to injured tissues, where they can subsequently differentiate into classically activated (M1) or alternatively activated (M2) macrophages. These two phenotypes of macrophages represent a simplistic discrete depiction of a continuous spectrum between two activation states. Generally, M1 macrophages produce proinflammatory cytokines as well as high levels of oxidative metabolites, and M2 macrophages make the environment supportive for tissue repair by producing antiinflammatory cytokines that facilitate matrix remodeling and angiogenesis.

The plasticity of monocytes/macrophages makes them an attractive target for modulation in the context of nerve repair. A prior short-term study demonstrated that direct modulation of macrophages toward a prohealing phenotype, using interleukin 4 (IL-4), results in an increase in SC recruitment and axonal growth. The premise herein is that preferential recruitment of anti-inflammatory reparative monocytes to the site of injury will more effectively bias the immune microenvironment toward a prohealing response and in turn set off a regenerative biochemical cascade involving SCs and neuronal processes that leads to improved repair. Since CX3CR1 receptor is mainly expressed on antiinflammatory reparative monocytes, exogenous fractalkine, the ligand for CX3CR1, can be used to preferentially recruit these monocytes to the site of nerve injury and thus increase the subsequent ratio of prohealing to proinflammatory macrophages during the regeneration process.

Thus an immunomodulatory approach to stimulating nerve repair in a nerve-guidance scaffold was used to explore the regenerative effect of reparative monocyte recruitment. Early modulation of the immune environment at the injury site via fractalkine delivery resulted in a dramatic increase in regeneration as evident from histological and electrophysiological analyses. This study suggests that biasing the infiltrating inflammatory/immune cellular milieu after injury toward a proregenerative population creates a permissive environment for repair. This approach is a shift from the current modes of clinical and laboratory methods for nerve repair, which potentially opens an alternative paradigm to stimulate endogenous peripheral nerve repair.

Researchers have found the first direct evidence that autoimmunity - in which the immune system attacks the body's own tissues - plays a role in Parkinson's disease, the neurodegenerative movement disorder. The findings raise the possibility that the death of neurons in Parkinson's could be prevented by therapies that dampen the immune response. "The idea that a malfunctioning immune system contributes to Parkinson's dates back almost 100 years. But until now, no one has been able to connect the dots. Our findings show that two fragments of alpha-synuclein, a protein that accumulates in the brain cells of people with Parkinson's, can activate the T cells involved in autoimmune attacks. It remains to be seen whether the immune response to alpha-synuclein is an initial cause of Parkinson's or if it contributes to neuronal death and worsening symptoms after the onset of the disease. These findings, however, could provide a much-needed diagnostic test for Parkinson's disease and could help us to identify individuals at risk or in the early stages of the disease."

Scientists once thought that neurons were protected from autoimmune attacks. However, in a 2014 study, researchers demonstrated that dopamine neurons (those affected by Parkinson's disease) are vulnerable because they have proteins on the cell surface that help the immune system recognize foreign substances. As a result, they concluded, T cells had the potential to mistake neurons damaged by Parkinson's disease for foreign invaders. The new study found that T cells can be tricked into thinking dopamine neurons are foreign by the buildup of damaged alpha-synuclein proteins, a key feature of Parkinson's disease. "In most cases of Parkinson's, dopamine neurons become filled with structures called Lewy bodies, which are primarily composed of a misfolded form of alpha-synuclein."

In the study, the researchers exposed blood samples from 67 Parkinson's disease patients and 36 age-matched healthy controls to fragments of alpha-synuclein and other proteins found in neurons. They analyzed the samples to determine which, if any, of the protein fragments triggered an immune response. Little immune cell activity was seen in blood samples from the controls. In contrast, T cells in patients' blood samples, which had been apparently primed to recognize alpha-synuclein from past exposure, showed a strong response to the protein fragments. In particular, the immune response was associated with a common form of a gene found in the immune system, which may explain why many people with Parkinson's disease carry this gene variant.

Researchers hypothesizes that autoimmunity in Parkinson's disease arises when neurons are no longer able to get rid of abnormal alpha-synuclein. "Young, healthy cells break down and recycle old or damaged proteins, but that recycling process declines with age and with certain diseases, including Parkinson's. If abnormal alpha-synuclein begins to accumulate, and the immune system hasn't seen it before, the protein could be mistaken as a pathogen that needs to be attacked."

One of the most frequent questions we at LEAF hear is this: when will innovative therapies to delay, stop and eventually reverse age-related damage become available? This is not an easy question to answer, because the pace of progress depends on many factors - predominantly, funding. Fundamental studies on aging are not well-funded and the accumulation of knowledge necessary to proceed from lab work to clinical trials and then clinical practice does not happen fast enough. Government funding is more often allocated to mainstream areas, such as research on single diseases. Business, for its part, does not show much interest in fundamental science, because usually there is no final product that can be sold. The only other source of funding is the general public. But most people are not yet sufficiently informed about the plausibility of bringing the aging processes under medical control and do not share the values of our community. Sometimes (to be frank, pretty often) activists trying to engage the public in an enlightening conversation can encounter resistance or even rejection.

Learning requires active, conscious participation. This means that students will seek and absorb information if they are interested, or they will ignore it if they do not see any personal benefit in it. So what do most people want? It's life extension right? Wrong! Studies show that when people are asked "how long would you like to live?" with no other conditions specified, people added around 5 to 10 years to the average life expectancy for their country and that was it. If we do not first explain the aging processes, the connection with age-related diseases and the repair based solutions that lead to healthy longevity, people think longevity means a longer life of prolonged ill health and frailty. Who wants to spend another ten or twenty years in a wheelchair or in a care home? And this is exactly the image most people have when these words are used. This is what the expression "life extension" means to the majority of people - and this is why we should avoid beginning the conversation with it.

At the same time, sociological studies show that if the possibility of perfect health throughout life is introduced into the equation from the very beginning, people show much more interest and support for the idea of ​​prolonging life. People literally switch from one camp to the other, those who just did not want to live for more than 80 years now decide they want to live to more than a hundred, and those who just wanted to get to 120 are suddenly ready to live to 150 or beyond. People are ready to support the development of new medical technologies, even scary ones like gene therapies or gene editing, under the condition they are going to be used for treatment. However their use for the prolongation of life belongs to the category of "human enhancement" and as such the idea is most often rejected. So it is highly recommended for advocates to add that these technologies will help treat or prevent serious chronic diseases, while extended lifespans will just be a possible nice side-effect.

Epigenetic clocks provide powerful tools to evaluate nutritional, hormonal, and genetic effects on aging. What can we learn from differences between species in how these clocks tick? One of the most fascinating findings in human aging is that it is associated with highly reproducible DNA methylation (DNAm) changes. DNAm levels at age-associated CG dinucleotides (CpG sites) can be integrated into epigenetic age predictors, which provide robust biomarkers to estimate chronological age. With the advent of more and more publically available DNAm profiles, such aging signatures were further developed to facilitate higher precision in age predictions, particularly for blood samples. Probably the most commonly used epigenetic aging signature is based on DNAm levels at 353 CpG sites and facilitates relatively precise age predictions for many human tissues: the median "error" (MAE), defined by the median absolute difference between DNAm age and chronological age, is usually less than 4 years.

Now - about 6 years after the first epigenetic clock paper - similar age predictors have been established for mice. Again, they were initially described for defined murine tissues, specifically liver and blood, taking into account the fact that there are notoriously large differences in the epigenetic makeup of cells from different tissues. However, it is also possible to derive a multi-tissue murine DNAm age predictor, in analogy to the most commonly used human clock. The signature is based on 329 CpGs and has been validated for cortex, muscle, lung, liver, and heart tissue. Overall, the multi-tissue age predictor reached a MAE of less than 4 weeks, although how it performs in other tissues has yet to be shown.

Studies indicate that the epigenetic clocks of mice tick faster than those of humans. This can be anticipated because the maximum life-span of mice (about 2 years) is much shorter than it is in humans (about 85 years). If the molecular changes of aging are linked to life expectancy and generation time, then this might support the notion that aging reflects a controlled evolutionary process. However, there is still an open debate on whether aging is due to an accumulation of cellular defects, or is driven by a developmental mechanism. Either way, comparison of epigenetic clocks in mice and men will provide new insights into the regulation of age-associated DNAm.

Direct comparison of age-associated CpGs in mice and men indicated that there is a moderate but significant association between the two species. It is not always trivial to identify orthologous CpG sites, and further interspecies comparison will be required to better understand similarities and differences of age-associated genomic regions. However, the overlap of age-associated CpGs in age predictors for human and mice seems to be rather low, and hence epigenetic clocks need to be trained specifically for different species. In terms of function, age-associated CpGs in humans and mice seem to be enriched in genes that are involved in morphogenesis and development. However, in both species age-associated DNAm changes are not generally reflected at the gene expression level - and thus the biological relevance remains largely unclear.

Mouse DNAm clocks provide powerful tools to study longevity interventions in one of the most relevant model organisms for aging research. These signatures were initially trained to correlate with the "real" chronological age of mice - but aging rates may differ between individuals. In fact, there is evidence that epigenetic clocks rather reflect the biological age, which is related to the perceived aging process of an organism. In analogy, it was previously demonstrated that human DNAm age is related to life expectancy: accelerated epigenetic age is associated with higher all-cause mortality. This finding has been validated in various additional cohorts and with different epigenetic age predictors. Furthermore, human epigenetic aging rates have been shown to be significantly associated with sex, race/ethnicity, and some disease risk factors. In mice, there was no clear difference in predicted DNAm age of male and females. However, ovariectomy, which reduces the average life span in female rats, results also in significant age acceleration. Caloric restriction or dietary rapamycin treatment, both of which result in increased life expectancy of mice, reduced epigenetic age. In humans, specific diet seems to have a less pronounced impact on epigenetic age, but there is significant association of DNAm age and body mass index (BMI). Apparently, different parameters can affect biological aging in mice and men.