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49:T40bf1,An overview of the Longevity Prize - Hypothesis Prize winners and submissions“What is the most promising but underappreciated or obscure area of longevity science we should pursue?Prize Description: "Over a century ago, many questionable experiments and surgical procedures were performed – heterochronic parabiosis (HCP) being one of them. This technique was revived by Irina Conboy at Stanford/Berkeley in the 200’s. Today, it’s one of the hottest (and most difficult to perform) models of aging research.Over a century of all the world’s biological knowledge is available to anyone taking the time to read the literature. There are cases where key discoveries are made in the past, but forgotten for long periods of time – only to be rediscovered. The hypothesis prize aims to resurface such discoveries and research areas, focusing our attention on the most promising directions.” Winners#1 Carlos Galicia (USA), galiciaa@usc.edu, University of Southern California / Buck Institute https://www.linkedin.com/in/cargalicia GPT-Summary: “The article proposes studying the rejuvenation process that occurs during embryogenesis as a promising opportunity against age-related decline. The study would involve identifying age-related changes in gametes, tracking those hallmarks of aging during embryogenesis, and harnessing the rejuvenation strategies used by the embryo. The article suggests using deep phenotyping, SMRT DNA sequencing, and multi-omic data analysis to gain insights into the rejuvenation processes. The article also discusses the potential clinical applications of the study's findings.” In 1930 Tracy Sonneborn published his observations on the reproduction of the flatworm Stenostonum Incaudatum. This flatworm reproduces asexually by fission and produces two daughter organisms. Sonneborn noticed that the daughter piece of the worm that requires more cell divisions to become a full-size worm, lives longer than the one that undergoes less cell multiplication (Sonneborn, 1930). This observation suggested an interplay between cell division and rejuvenation during development. Some species of planarian flatworms and hydra, thought to be immortal, undergo a similar process in which old cells are constantly being removed and replaced by new ones (Muller, 1996; Sahu et al., 2017). One of the reasons why cell division might be necessary for rejuvenation to occur is that the molecular components of a cell might not be able to be repaired indefinitely. In E. coli, after cell division occurs, the cell that inherits the old pole has a higher incidence of death (Stewart et al., 2005). Similarly, in budding yeast, the mother cell accumulates damage that is not passed down to the daughter cells and leads to the detriment of the mother cell (Egilmez & Jazwinski, 1989).In mammals, the major rejuvenation event that takes advantage of cell division occurs during embryogenesis, when gametes from adult individuals fuse to form a zygote that divides billions of times. Through this process, molecular damage and other age-related changes that occur to gametes are erased as new organisms that come from old parents are young. The rejuvenation processes utilized during embryogenesis present the most promising opportunity against age related decline, as they are the solution developed by evolution and the programs are already encoded in our DNA. To understand and harness the rejuvenation process that occurs during embryogenesis I propose three phases. The first phase would focus on identifying age-related changes in oocytes and sperm. Before we can study the mechanisms that drive rejuvenation during embryogenesis, we need to understand the molecular damage that accumulates with age in the cells that give rise to the embryo. This can be accomplished with deep phenotyping of oocytes and sperm collected across the lifespan of individuals. To study age related changes to the genome, one would focus on studying covalent modifications that occur to DNA as those have a higher chance of being passed down to the embryo. SMRT DNA sequencing and modular enzymatic labeling paired with nanopore sequencing would be able to detect 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, thymine dimers, uracil incorporation, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine at a single base resolution (Clark et al., 2011; Wang et al., 2017). These would make it possible to map these covalent modifications to specific sites in the genome and explore their dynamics during aging, similar to what has been accomplished with methylation clocks.For oocytes additional characterization is needed because they provide most of the cytoplasm for the embryo. Characterization of mitochondrial function and dynamics, mass spectrometry of soluble and insoluble protein fractions, RNA sequencing, unbiased metabolic and lipidomic profiling as well as quantification of products of oxidation and lipofuscin would provide useful insights. These data would allow us to understand the age-related changes that embryogenesis is equipped to reverse as well as provide valuable insight into age related changes in the quality of oocytes which is indispensable for the area of reproductive aging. Once age related changes have been identified in gametes, the second phase would focus on tracking those hallmarks of aging during embryogenesis to understand at what point they are rejuvenated. Transcriptomic, proteomic and metabolomic characterization of the embryos would also be obtained to gain deeper understanding of the mechanistic networks driving the rejuvenation processes. The third phase of the study would make use of these data to harness the rejuvenation strategies used by the embryo. The multi-omic data could provide candidate genes, peptides or molecules that correlated with the rejuvenation event. This would allow for planning of screens or other mechanistic studies specific to the rejuvenation event.FAQ:Isn’t reprogramming with Yamanaka factors already trying to harness the power of development?Yamanaka factors are believed to rejuvenate in a similar way to development. Nevertheless, induced pluripotent stem cells (IPSCs) generated with Yamanaka factors differ from pluripotent stem cells derived from embryos, suggesting different mechanisms. For instance, IPSCs are more susceptible to genomic instability than their embryonic counter parts (Zhang et al., 2018). It is possible that the embryo utilizes other rejuvenation strategies, and reprogramming resembles one of them but until a systematic study of embryogenesis is done, we don’t have assurances that Yamanaka factors are enough to produce a rejuvenation event equal to that of embryogenesis. By focusing our attention on the naturally occurring way of reversing aging, we could find new and better rejuvenation strategies.How is this a promising and obscure area of longevity science?The Gladyshev lab has shown the great potential of studying embryogenesis from an ageing perspective by showing methylation age reversal around gastrulation (Kerepesi et al., 2021). However, despite its great potential, very little effort is being directed towards understanding germline rejuvenation. A search on PubMed for germline rejuvenation produces 17 results while the amount of literature for other forms of aging research is vast.What would be the timeframe and budget required for this study?The timeframe and budget to complete this proposal would be highly flexible. For example, using Zebrafish or Xenopus, phase 1 and 2 could be completed in a year because collection of germ cells and embryos is relatively simple and older animals could be sourced from labs studying these organisms. However, obtaining germ cells and embryos at different stages from mice would be a more laborious process that could double the time to completion. The cost for data generation is equally flexible. With a small budget, exploratory experiments at very young and old ages could be performed to see what types of data are more promising and then increase temporal resolution on those. Significant progress could be made by an individual with $60,000-100,000 in funding in one year.How could these discoveries be taken to the clinic?The specifics of how to translate this newly gained understanding of embryonic rejuvenation would vary on a case-by-case basis. For example, if a well-defined transcriptional program is found to be correlated with rejuvenation of a specific hallmark of aging, one could attempt to modulate transcriptional networks through small molecules as has been done for drug repurposing with data from the L1000 project. If a hallmark can be measured at the single cell level and occurs at a stage of embryonic development where ESCs can be obtained, CRISPR screens could be used to identify targets able to modulate the program. Another important step would be to identify hallmarks that cannot be rejuvenated without cell division, this would create valuable insight into what form of rejuvenation would require more engineering. References:Clark, T. A., Spittle, K. E., Turner, S. W., & Korlach, J. (2011). Direct detection and sequencing of damaged DNA bases. Genome Integr, 2, 10. https://doi.org/10.1186/2041-9414-2-10 Egilmez, N. K., & Jazwinski, S. M. (1989). Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae. J Bacteriol, 171(1), 37-42. https://doi.org/10.1128/jb.171.1.37-42.1989 Muller, W. A. (1996). Pattern formation in the immortal Hydra. Trends Genet, 12(3), 91-96. https://doi.org/10.1016/0168-9525(96)81419-3 Sahu, S., Dattani, A., & Aboobaker, A. A. (2017). Secrets from immortal worms: What can we learn about biological ageing from the planarian model system? Semin Cell Dev Biol, 70, 108-121. https://doi.org/10.1016/j.semcdb.2017.08.028 Sonneborn, T. M. (1930). Genetic studies on stenostomum incaudatum (Nov. spec.). I. The nature and origin of differences among individuals formed during vegetative reproduction [https://doi.org/10.1002/jez.1400570104]. Journal of Experimental Zoology, 57(1), 57-108. https://doi.org/https://doi.org/10.1002/jez.1400570104 Stewart, E. J., Madden, R., Paul, G., & Taddei, F. (2005). Aging and death in an organism that reproduces by morphologically symmetric division. PLoS Biol, 3(2), e45. https://doi.org/10.1371/journal.pbio.0030045 Wang, F., Zahid, O. K., Swain, B. E., Parsonage, D., Hollis, T., Harvey, S., Perrino, F. W., Kohli, R. M., Taylor, E. W., & Hall, A. R. (2017). Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process. Nano Lett, 17(11), 7110-7116. https://doi.org/10.1021/acs.nanolett.7b03911 Zhang, M., Wang, L., An, K., Cai, J., Li, G., Yang, C., Liu, H., Du, F., Han, X., Zhang, Z., Zhao, Z., Pei, D., Long, Y., Xie, X., Zhou, Q., & Sun, Y. (2018). Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining. Cancer Commun (Lond), 38(1), 49. https://doi.org/10.1186/s40880-018-0313-0Kerepesi C, Zhang B, Lee SG, Trapp A, Gladyshev VN. Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging. Sci Adv. 2021 Jun 25;7(26):eabg6082. doi: 10.1126/sciadv.abg6082. PMID: 34172448; PMCID: PMC8232908. #2 Rakhan Aimbetov (Kazakhstan), r.aimbetov@gmail.com, Lead @ LabDAO and Hack-Age GPT-Summary: Maintaining accurate conversion of genetic information into protein structure is crucial for cellular function, but errors in transcription and translation can disrupt proteostasis and contribute to aging and age-related pathologies. Methylglyoxal (MGO) is a byproduct of glycolysis that can modify amino acids and impair protein function, and its production has been implicated in diabetic complications and organ decline. Additionally, the stiffening of the extracellular matrix (ECM) can lead to a loss of proteostasis due to increased glycolysis and subsequent MGO generation, potentially leading to ribosomal dysfunction and an increase in mistranslation rate. Shedding light on these mechanisms could pave the way for interventions to extend healthy life. Accurate conversion of genetic information into protein tertiary structure is one of the pillars of proper cellular function, and disruption of protein homeostasis (proteostasis) that leads to intracellular accumulation of misfolded proteins is a hallmark of aging and a contributor to age-related pathologies. The mechanisms of the aforementioned conversion are somewhat “noisy” – for transcription, the error rate is 10-5-10-4 (1/100,000 ribonucleotides are mismatched with the transcribed DNA template); for translation, the rate is 10-4-10-3 (1/10,000 mRNA codons are paired with a wrong amino acid). In addition, erroneous tRNA aminoacylation (when tRNA is coupled with an amino acid that does not match the anticodon) presents an extra source of mistakes in the flow of information from DNA to protein.Any deviation from the background error rate has its effect on the aging dynamics. It has been shown that certain long-lived organisms inherently benefit from a relatively more faithful translation (1–3), whereas experimental elevation of translation error rate in animal models shortens lifespan (4). It is therefore important to deconstruct the mechanisms that alter the fidelity of protein synthesis in order to devise strategies and interventions for extending healthy life.Methylglyoxal (MGO) is a byproduct of glycolysis with reactivity towards amines – arginine guanidino groups, lysine amino groups, and exposed protein N-termini. Non-enzymatic modification of amino acid residues by MGO leads to the formation of various chemical adducts and crosslinks (collectively known as advanced glycation endproducts (AGEs)) that may affect the susceptible proteins’ three-dimensional conformations (in other words, how they are spatially folded) and proper functioning (5). There are numerous reports that implicate MGO production in diabetic complications – cardiomyopathy, nephropathy, peripheral neuropathy, retinopathy, etc. – and age-related organ decline.Unfolded protein response (UPR) is a mechanism to circumvent the accumulation of misfolded proteins either in the lumen of the endoplasmic reticulum (ER stress) or in the cytosol (cytosolic UPR). It has been shown that cells grown in presence of high glucose display ER stress mediated by MGO (6). The authors conclude that the UPR is brought about by non-enzymatic post-translational modification of existing proteins by MGO. I propose an additional hypothetical mechanism: MGO-mediated glycation of ribosomes – i.e. ribosomal proteins, rRNAs – increases the error rate at the level of translation resulting in enhanced synthesis of polypeptides prone to misfolding.Extracellular matrix (ECM) is a heterogeneous but highly organized matter located in-between cells that provides structural support to tissues. Cells and their environment, represented by the ECM, communicate reciprocally to drive organogenesis during development and remodeling in response to insult. ECM composition and ensuing mechanical properties greatly affect cellular behavior. For example, mesenchymal stem cells commit to either bone or muscle lineages depending on substrate elasticity. Moreover, the accumulation of AGE-crosslinks (including but not limited to MGO-derived crosslinks) in matrix collagen has been shown to increase ECM stiffness and contribute to cardiovascular pathology. Cells continuously monitor the state of their environment, including its mechanical properties, via cell-surface receptors. Transduction of mechanical cues (mechanotransduction) is mediated by integrin receptors that probe the ECM elasticity and initiate a cascade of intracellular responses to fine-tune cellular behavior to the surrounding mechanical context. Increased ECM rigidity (stiffness) leads to translocation of the transcription coactivator YAP/TAZ from the cytoplasm into the nucleus where it binds to the transcription factor TEAD, driving expression of genes involved in tumorigenesis: those promoting growth, proliferation, survival, glucose uptake, glycolysis, epithelial-to-mesenchymal transition, apoptosis inhibition. Concurrently, I propose that enhanced glycolysis (and subsequent MGO generation) in response to the stiffened ECM leads to the loss of proteostasis at least partially due to ribosome glycation and an ensuant increase in mistranslation rate. The proposed mechanism of proteostasis disruption might have broader implications for the ECM organization. For instance, the substitution of key amino acid residues in tropoelastin has been shown to impede monomer coacervation and lead to aberrant elastic fiber formation (7).The hypothetical mechanism of ribosomal dysfunction has not yet been described in the literature. Shedding light on novel aspects of translation fidelity, especially in relation to metabolism, will draw a link between the tightly regulated proteostasis maintenance machinery and the stochastic nature of glycation, possibly paving the way to a new class of life-extending interventions.For the full project details, including preliminary data and further plans, please visit -- https://docs.google.com/document/d/1QLzipbn6umWGg2MfyyxEROxdzsWMWvXoiG_WBVFHzPQ/edit?usp=sharing #3 Shahaf Peleg (Germany), Group leader Leibniz Institute for Farm Animal Biology, shahafpeleg3@gmail.com, https://www.linkedin.com/in/shahaf-peleg-72790263/?originalSubdomain=de GPT-Summary: ”The article discusses how mitochondrial dysfunction is a central hallmark of aging and current interventions aim to reduce mitochondrial activity to extend lifespan and health span. However, this may impair the animal's ability to respond to stress and it is questionable whether such approaches could be translated into human longevity therapy. The article proposes a novel approach called external energy replacement, which involves using an engineered light-sensitive proton pump called mtON to generate ATP in the absence of oxidative phosphorylation. The authors hypothesize that using mtON technology in mammals will attenuate aging in the eye and skin by compensating for lack of ATP, reducing metabolic associated damage, and enabling people to eat less while maintaining physiological ATP levels. The authors believe this approach has great promise in attenuating human aging in a novel manner.” FBN "Mitochondrial dysfunction is a central hallmark of aging and many of the current interventions to extend lifespan and health span target the mitochondrial metabolism. For example, dietary interventions, genetic interventions (e.g., AMPK) and drugs (e.g., metformin and rapamycin) extends lifespan by, at least partially, targeting mitochondria. Generally, many of the interventions aim, quite surprisingly, towards a reduction of mitochondrial activity. A prominent example is the induction of mitochondrial unfolded protein response (mtUPR) during development, where reducing components of oxidative phosphorylation results in increased lifespan. However, it is noteworthy that such intervention reduces the physical activity of the animal in question and likely impairs the ability of the animal to respond to other type of stress. Therefore it is questionable whether such approaches could be translated into human longevity therapy.Here we hypothesize a novel and radical approach termed external energy replacement. What does it mean?It is established that plants and bacteria can capture the energy of light and translate it into energy in their cells, while animals must consume other organisms as food, in order to generate energy. Thus conventionally, evolution made a clear distinction between autotroph and heterotroph organisms. However, the idea of bringing proteins from evolutionarily distant organisms such as bacteria into eukaryotes can result in a leap in molecular biology. One well-known example is the bacterial CRISPR/Cas9 system, which was adapted to edit eukaryotic target genes. Another very recent example is introducing bacterial/fungal light-sensitive protein that generates proton gradients across in eukaryote mitochondria to generate chemical energy, which is a massive leap in molecular biology. In this hypothesis, we propose a major breakthrough in the field of metabolic aging. Specifically, we propose to test if mtON, an engineered light sensitive proton pump, will act as metabolic rescue for mammalian age-related mitochondrial dysfunction in the eye and the skin, where light is easily accessible. When activated by light, mtON pumps protons across the inner membrane of the mitochondria, thus contributing to the proton gradient that is essential to generate ATP. This genetic intervention practically enables a heterotroph to harness the energy of light to generate ATP in the absence of oxidative phosphorylation.We recently were the first and only scientists to show that the usage of mtON technology can increase lifespan in worms. Here, we hypothesize that using mtON technology in mammals will attenuate aging in the eye and skin. We hypothesize that using mtON will have several benefits. First, in an advanced form of aging, where mitochondria are dysfunctional and cannot generate enough ATP, that mtON treatment coupled with light can enable the aged eye and skin cells to generate youthful levels of ATP. Essentially, the light energy transformation will compensate for lack of ATP. Secondly, mtON technology may reduce the accumulation of metabolic associated damage. For example, we hypothesize that as ATP is now being partially generated by light, the cell would adapt and slow down its upstream metabolic (e.g., TCA cycle, glycolysis). Therefore, less biochemical reactions would take place, thus reducing the production of cellular waste, such as advanced glycation end-products, which contribute to the progression of aging. Also, as oxidative phosphorylation is less required, we hypothesize a reduction in the levels of reactive oxygen species (ROS), which may also benefit to generate less cellular damage during aging. Thirdly, it is possible that mtON would enable people to eat less while maintaining physiological ATP levels. As such, people may enjoy the benefits caloric restriction, which is believe to improved health span, while avoiding jeopardizing dysregulated ATP levels. This would be even more important once mtON technology would be upgraded to be used in inner organs beyond the skin and the eye.Taken together, our hypothesis of using light as an external energy replacement is quite revolutionary and show great promise of success in attenuating human aging in a novel manner." — Other interesting submissions John Hemming: “The Gompertz-Makeham law of mortality predicts mortality, with the Gompertz part relating to gradual health deterioration. Stem cell failure to differentiate may cause this, with a feedback loop involving Interleukin-10, which inhibits Nuclear Factor-κB and reduces inflammation from senescent cells. This loop may drive much health deterioration, fitting the Disposable Soma theory. Mitochondrial function is important in aging, and Heterochronic Parabiosis can explain its benefits.” Clarice Demarchi Aiello “The hypothesis proposes using tailored magnetic fields to control quantum signaling in cells for longevity's advantage. Weak magnetic fields, via the electron quantum property of 'spin,' have been shown to control many disease markers and cell activities. By designing novel instrumentation to directly measure and control spin states and their biological consequences, it may be possible to alter quantum signaling in cells to promote longevity. The long-term goal is to develop drugs and therapeutic devices that heal the human body via quantum signaling. The proposed instrumentation includes microscopes, electrophysiology setups, and scanning tunneling microscopes coupled to coils and radio-frequency microchips. These tools will allow for the visualization and manipulation of regeneration by weak magnetic fields, among other applications. Ultimately, the hypothesis seeks to control native electromagnetic-responsive pathways for personalized medicine and improved longevity.” John Jackson “Studies have found that certain animals that live a slower paced life have "protective traits" that help to extend lifespan. Can we study these protective traits more and implement them permanently within the human body?” Alexei Goraltchouk “Remedium Bio believes that Growth Factors can extend healthspan and lifespan, and are a promising solution to age-related diseases. Growth Factors have shown remarkable results in clinical trials, with repeat intra-articular injections of FGF18 reversing cartilage loss in Osteoarthritis patients. Remedium is developing a gene therapy based on FGF18 to restore a more youthful and healthy phenotype to arthritic joints.” Dian Ginsberg MD “The study aims to evaluate the safety and efficacy of administering young Fresh Frozen Plasma (yFFP) to aged subjects for therapeutic uses. The study will compare epigenetic, proteomic, laboratory, and functional results taken from before the initial treatment to results taken one month after the last treatment. The study will involve 30 patients, and each participant's starting condition will be identified using TruDiagnostic and Seer tests, laboratory markers, and functional markers. The primary outcome is the change in the epigenetic age or genome pattern, laboratory, and functional assessments of the yFFP treated patients. The study will take place at multiple sites in the State of Texas.” Kourosh Hojjati: “The writer believes that in the process of cell division, copying the ribosome is the most important factor, which becomes impaired with age. Finding an agent or stimulus that minimizes the problems of copying or prevents cells from reproducing with defects could lead to a longer life.” Daniel Bar “Epigenetic clocks measure DNA methylation at specific sites and offer an accurate measurement of biological and chronological age. Interventions that prolong healthspan and lifespan also slow the biological clocks. Identifying the direct mechanism behind the epigenetic clock reset is important to exploring the causality of epigenetic clocks. A list of candidate genes can be generated by integrating single-cell expression data, which can be tested individually or in combinations in cell culture for epigenetic reset.” Dmitry Dzhagarov (Dimitri Jagarovi) “The review proposes a new approach to treating age-related diseases using a combination of senolytics to remove aged cells and ROCK inhibitors and 5-LOX inhibitors to stimulate the regeneration of new cells. The review suggests that this combination of drugs can conditionally reprogram cells in situ, leading to proliferation of progenitor cells that can make up for losses. The review discusses the importance of senolytics and explains the difficulties in using mesenchymal stem cells for replacement therapy. The review explores the use of Conditionally Reprogrammed Cells (CRC) as a potential treatment for age-related diseases. The combination of ROCK and 5-LOX inhibitors may promote cell proliferation without promoting oncogenesis. The correct choice of Senolytic can significantly increase the effectiveness of such treatment. Treatment with such a combination of drugs should be short-term and repeated several times a year.” Suslova Alexandra: “The article explores the idea that the human mind has the potential to heal the body, citing examples such as the placebo effect and personal experiences. It suggests that the science of neurobiology should be studied further to understand this connection. The article also discusses studies on the influence of thoughts and emotions on the body, including experiments with DNA molecules. It highlights the importance of a positive mindset for a healthy and happy life, and the potential of changing one's brain to achieve this. The article concludes by emphasizing the need to study the structure of the brain and the influence of thoughts and emotions on the body for optimal health and well-being.” Varun Dwaraka: “The article discusses how epigenetic regulation through changes in gene expression can cause long-term effects on aging phenotypes, and how the stability of methylation marks on imprint control regions (ICRs) throughout life makes them ideal long-term records of early exposures. The article highlights the recent development of a custom Infinium methylation array to quantify DNA methylation patterns of the human imprintome and investigate any links between ICRs and age-related phenotypes, using DNA and multi-omic aging data previously collected from a collaboration with Partners Biobank and Brigham Women’s hospital. The article proposes three aims to investigate the relationship between ICRs, aging, and AD development, and to identify new aging biomarkers and possible therapeutic pathways.” Muneera Fayyad “This project aims to isolate mesenchymal stem cells from plucked hair follicles, which have high proliferation, differentiation ability, and efficient reprogramming potential. These stem cells will be differentiated into various lineages and employed in pre-clinical and clinical models to treat aging and degenerative diseases such as osteoarthritis, cartilage degeneration, and spinal cord injury. The use of an easily obtainable, autologous stem cell source like the hair follicle alleviates safety concerns associated with most stem cell-based therapies.” Albert Cheong: “The study aims to characterize the gut microbiome of centenarians and investigate the potential of using their microbiome profile as a method to increase human longevity. The study will involve sequencing the gut microbiome of 200 centenarians and comparing it to the gut microbiome of younger individuals. The study will also investigate the feasibility of developing interventions to modify the microbiome to promote healthy aging.” Alexandra Suslova: “The article explores the power of the mind to affect the body's healing process and the promising areas of study for longevity science. It discusses how thoughts and emotions can influence matter, including DNA molecules, and how the mind and body are in constant sync. The article presents scientific experiments that show the relationship between a person's mood and their health and longevity. It concludes that a person can heal themselves and destroy themselves, and that our diseases are mainly caused by stress hormones rather than genetics. The article emphasizes the importance of studying the structure of the brain and the influence of thoughts and feelings on the body for a happy and healthy life.” Ziwei Li: “The author is researching longevity by combining traditional Chinese medical theories, Taoism, and modern technological means of testing. They focus on the concept of inner alchemy, which is a Taoist practice of using the human body as a furnace and refining vital essence and Qi to reach immortality. The author believes that scientific tests can be used to quantify and record the changes that occur in the human body during the stages of inner alchemy. By combining Taoism, traditional Chinese medicine, and modern medical tests, the author hopes to find a natural way to enhance and transform human life, ultimately leading to a longer and healthier life.” Steven Fowkes: “Cellular hydration, which drops from 90% in the early stages of embryonic development to 50% in adults, is proposed to be a foundational aspect of development and aging. Potassium utilization, the ratio of cellular potassium to serum potassium, is suggested as a surrogate marker for cellular hydration, which can be easily and minimally invasively measured, making it a viable option for assessing this underappreciated aging-associated biomarker.” Denis Odinokov: “The weight loss industry is worth over $5.5 billion in the EU and muscle mass decreases while fat mass increases with age. A promising approach to weight management is changing the energy metabolism of adipose cells or supporting white-to-brown-fat conversion. Cell-free DNA fragments from adipose cells in the bloodstream can be used to track the ratio of white to brown fat cells as a novel metric for obesity research and healthy aging. A non-invasive laboratory method to measure this ratio is currently being developed.” Mikhail Batin: “Age-related changes in the extracellular matrix (ECM) are an important but underestimated area in life extension research. The accumulation of crosslinks in proteins, such as collagen and elastin, contributes to ECM rigidity, cell dysfunction, and chronic inflammation. The slow renewal of these proteins and the absence of reliable antiglycation mechanisms make glycation of long-lived proteins almost inevitable. Age-related changes in ECM proteins are associated with most other signs of aging and contribute to the formation of mechanisms involved in aging and age-related diseases. The structure of the ECM also contributes to neurodegenerative processes in aging. Thus, age-related changes in the ECM structure play a large but understudied role in aging.” Denis Odinokov: Stochastic damages to molecular structures lead to incomplete digestion of proteins and accumulation of indigested fragments, resulting in increased stiffness and malicious nano topography. Enzymatic activity against cross-linked collagen is limited, and targeting indigested fragments with ferromagnetic nano-tags and inducing local hyperthermia may be a better research direction. Every submission (above the threshold votes for participation)"Wolbachia is an alpha -proteobacterium, an intracellular symbiont of invertebrates - insects, isopods, arachnids and filaria. Its effects include cytoplasmic incompatibility, androcide, male feminization, and parthenogenesis. So far, Wolbachia has not been found in vertebrates, except for cases of infection with filariae, the obligate hosts of Wolbachia. Only one work indicated a positive analysis of the presence of Wolbachia DNA in biomaterial samples taken from a reptile, in the presence of a negative test for the presence of the obligate hosts of Wolbachia - filariae. The same work showed a high degree of Wolbachia infestation of ticks parasitizing reptiles. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397688/We show that Wolbachia successfully passed from ticks to reptiles and subsequently to mammals, with the preservation of most of its effects, to which one more was added - slowing down age-related changes, puberty and aging.https://www.facebook.com/valeriy.golub""Non-coding RNAs and agingMain efforts in aging studies are devoted to processes associated with protein-coding genes. But as we know, there are less than two percent of such genes in the human genome. The rest of the genome related to aging remains little-studied. It applies to a great extent to non-coding RNAs.Usually, all non-coding RNAs are divided into two large classes: microRNAs and long non-coding RNAs (lncRNAs).MicroRNAs are a class of small non-coding RNA molecules 18-25 nucleotides long, which are actively involved in the regulation of gene expression. The action of microRNAs is very diverse and appears to be closely related to many processes occurring in the body, including maintenance of the genome stability, immune responses, differentiation, proliferation, and apoptosis of cells.Numerous studies have shown a close link between changing microRNA levels and cardiovascular, cancer, and neurodegenerative diseases (i.e., major age-related diseases), as well as a direct role of microRNAs in the life span regulation.At the same time, according to current estimates, the expression of about 60% of human genes is directly related to the action of microRNAs, and the functions of most of them still remain undetermined.https://academic.oup.com/clinchem/article/55/11/1944/5629329https://pubmed.ncbi.nlm.nih.gov/27192016/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655276/https://pubmed.ncbi.nlm.nih.gov/28934394/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954352/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463915/In a number of studies, long non-coding RNAs (lncRNAs longer than 200 nucleotides) have also been shown to be involved in the processes associated with aging and longevity. Depending on their location and orientation in the genome, all lncRNAs are divided into seven different groups: sense, antisense, bidirectional, intergenic, intronic, enhancer, and promoter lncRNAs. They play an important role in many biological processes, such as transcription, post-transcriptional processing, and chromatin modification. Mechanisms used by lncRNAs to perform their function include interaction with other types of RNA or DNA, creation of a framework of subcellular domains or complexes, and regulation of protein activity or amount. In addition to being functional, the lncRNA transcript itself can also influence the structure of the nucleus, the epigenetic landscape, or the expression of nearby genes.Researchers describe the direct involvement of lncRNA in such aging-related processes as genome instability, cell aging, telomere shortening, chromatin remodeling, and development of main age-related pathologies.https://pubmed.ncbi.nlm.nih.gov/32565330/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880696/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641121/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140545/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509469/RNA editing (epitranscriptomics) is another understudied area closely related to aging. Nowadays, more than one hundred different RNA modifications are known.https://www.nature.com/articles/s43587-021-00058-yCircular RNAs (circRNAs) are another little-studied type of non-coding RNAs associated with aging and life span regulation. They are widely expressed in eukaryotes and have multiple functions. Recent studies show that levels of circRNAs change with age in various tissues in many species, from nematodes to mammals. It is no coincidence that a recent study proposed that changes in circRNA transcripts are another sign of aging.https://www.cell.com/trends/genetics/fulltext/S0168-9525(21)00126-8https://pubmed.ncbi.nlm.nih.gov/29753875/#:~:text=Recent%20profiling%20of%20circRNAs%20genome,role%20in%20the%20aging%20process%3FWith all that said, non-coding RNAs are still less studied by researches exploring protein-coding genes and aging.And as João Pedro de Magalhães, a well-known researcher of aging, notes in his work, the aging-related changes in the transcriptome, which encodes proteins, have been characterized in detail. But much less attention has been given to the non-coding part of the human genome, especially long non-coding RNAs. As a result, only a small number of known lncRNAs have been functionally characterized.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961210/""Pseudogenes. Mysterious Elements of the Genome, Regulation of Gene Expression, and Much MoreThe very name of these genomic structures - pseudogenes - implies that something is wrong with them and that they are very different from the regular protein-coding genes. This is indeed true. Pseudogenes used to be functional genes that subsequently lost the ability to code for a protein due to mutations that arose in their sequence.At this point a careful reader may immediately ask why such damaged genes were not selected by purge selection? Scientists have three answers to this question. In one case, a pseudogene can be a result of gene duplication (doubling), i. e. when one copy remained functional, and the second one became a non-functioning pseudogene due to damage. Since one copy of a regular functional gene proved to be sufficient for the life of an organism, the transformation of the second copy into a pseudogene did not go through the purge selection. It is estimated that 3,391 fully functional parental genes are associated with pseudogenes in the human genome. At the same time, almost two-thirds of the parental genes gave rise to only one pseudogene, and only a small part of the parental genes gave rise to dozens of pseudogenes each.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491395/In the second case, many such duplications are created by reverse transcription using retrotransposon enzymes. The result is intron-poor fossils devoid of promoters that are called ""processed pseudogenes."" In the human genome, processed pseudogenes are the most common type of pseudogenes due to a surge in retrotransposition activity in primate ancestors about 40 million years ago. The total number of such retrocopies in the human genome is estimated at more than 8,000 units.https://pubmed.ncbi.nlm.nih.gov/17424906/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC329124/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314676/According to the third scenario, a pseudogene could flap away from the selection if the loss of its function became not critical for the organism. A typical example of this setup is the pseudogene GULO, typical of higher primates. For millions of years, it synthesized vitamin C in the organisms of our distant ancestors. Mutation that disrupted GULO’s functioning coincided with our ancestors’ transition to a plant-based diet rich in vitamin C. Thus, the internal synthesis of the necessary vitamin was replaced by external intake. The GULO gene, having ceased to be critically important, successfully turned into a pseudogene. Remarkably, dogs and cats have processed GULO pseudogenes, being the result of retrotransposons’ activity. In humans, the GULO pseudogene is also reliably blocked epigenetically, to prevent any possibly functional part of this former gene from affecting the expression of other genes - e. g. the Clu gene, which is next to GULO and is associated with exceptional longevity in humans. By evolution, GULO is completely inactivated and no longer bothers anyone.https://link.springer.com/article/10.1007/s10528-013-9574-0Note that a changed diet has repeatedly contributed to the creation of pseudogenes. Thus, our very distant ancestors used to actively eat insects. For their digestion they had special enzymes that break down chitin and chitinases. After the extinction of the dinosaurs, our ancestors began to grow larger and move on to more high-calorie foods, refusing or greatly reducing eating of insects. As a result, some of the genes encoding chitinases successfully turned into pseudogenes.https://www.science.org/doi/10.1126/sciadv.aar6478However, not all pseudogenes became genetic fossils. Some of them can be quite active.First, they can regulate the expression of their parent gene, reducing the stability of its mRNA. For example, the MYLKP1 pseudogene, which is activated in cancer cells, generates non-coding RNA (ncRNA), which in turn inhibits mRNA expression of its functional parental gene, MYLK.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137860/Studies in Drosophila and mice have shown that small interfering RNAs (siRNAs) derived from processed pseudogenes can regulate gene expression through one of the epigenetic mechanisms - RNA interference.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206313/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981145/https://pubmed.ncbi.nlm.nih.gov/18404146/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708354/It has also been described that pseudogenes with a high degree of sequence homology to their parental genes can regulate their expression through the formation of antisense transcripts, such as the antisense long non-coding RNA (lncRNA) of the Oct4 pseudogene, which suppressed the expression of both the pseudogene itself and its parental gene.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2999937/On top of all this, pseudogenes can compete with their parent genes for microRNA (miRNA) binding, thereby modulating the repression of a functional gene via microRNA. For example, the PTEN pseudogene regulates the expression of its parent gene, a key tumor suppressor, through this mechanism of action. A part of the transcript derived from the pseudogene, PTENP1, acts as a miRNA bait that represses the parent gene. It is estimated that this mechanism can have a significant impact in this pathology.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206313/https://pubmed.ncbi.nlm.nih.gov/18455982/Another example from the same field is the DUXAP8 pseudogene. It generates a long non-coding RNA that promotes tissue degeneration by suppressing two suppressors, p21 and PTEN.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544748/Do pseudogenes play a role in aging? All available data indicates that they do, and their role is not unimportant. But, unfortunately, they have been little studied so far. For example, there is a study of lncRNA pseudogenes’ involvement in cellular aging.https://www.sciencedirect.com/science/article/pii/S1568163715300362Above, we have already described several examples of the participation of pseudogenes in the processes associated with mutagenesis. In addition, pseudogenes are involved in the formation of neurodegenerative pathologies, one of the main age-related diseases. Thus, the analysis showed that some pseudogenes increase their expression in Alzheimer's, Parkinson's and Huntington's diseases. At the same time, these pseudogenes shared about 80% of the common microRNA binding sites with their parental genes. According to scientists, these highly expressed but untranslated transcripts may act as regulators of the expression of their parental genes. And contribute to the development of neuropathology.The recently described T04B2.1 pseudogene plays a regulatory role and modulates the aggregation of α-synuclein and β-amyloid proteins, two key factors in neurodegeneration. Another pseudogene, ACTBP2, increases the permeability of the blood-brain barrier and exacerbates neuroinflammation and neurodegeneration in Alzheimer's disease.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484327/https://www.sciencedirect.com/science/article/abs/pii/S0006291X20322063https://www.nature.com/articles/s41420-021-00531-yIt is noteworthy that in long-lived naked mole rats, a very small number of pseudogenes, only 244 pseudogenes, were found in the genome after its full reading by Vadim Gladyshev's team. Same as transposons, 25%, compared to 37% in the mouse genome. As for pseudogenes, in naked mole rats predominate genes associated with the loss of functions that these animals apparently do not need underground, such as vision. Interestingly, there are pseudogenes associated with protein ubiquitination. Proteins are labeled with ubiquitin as being subject to degradation in the proteasome. For some reason, this pathway of proteostasis in mole rats is significantly reduced compared to mice.https://www.nature.com/articles/nature10533In the case of pseudogenes, one can clearly see how the diversity created in the course of evolution can have some ""side effects"".When gene duplication, one of the main mechanisms in the evolution of species, in addition to new functional proteins also creates their ""corrupted"" copies, aka pseudogenes, which can later actively participate in pathological processes. It was the case in duplication of the Notch2 gene involved in brain development of embryos. The first duplication of this gene occurred in a common ancestor of humans, gorillas and chimpanzees. However, the resulting copy of the Notch2 gene turned out to be non-functional. It has remained so in monkeys, but not in humans. About three million years ago, a new gene in the genome of our ancestors, the australopithecines, became functional. There were three more subsequent cases of its duplication. As a result, the Notch2NL gene family was formed. The products of these genes are critical for the formation of a large ""human"" brain with a more developed cortex.https://pubmed.ncbi.nlm.nih.gov/29856954/https://pubmed.ncbi.nlm.nih.gov/29856955/In general, it is safe to say that the definition of pseudogenes as ""genetic fossils"" is long outdated. Some of them may have certain functions important for the organism, functions related to aging, age-related pathologies and many other things""The Role of Organs in AgingModern aging research focuses largely on aging at the molecular and cellular level, however integrative approaches establishing the contributions of tissues and whole organs to aging and longevity are not as well studied. Current data suggests organs may age at different rates and it is well established in transplantations that different organs have different donor age effects [1-3]. It is therefore hypothesized that organs may exhibit varying degrees of influence on overall organismal aging rate and lifespan. The most straightforward method to test this hypothesis, and provide a potential future aging therapeutic path, would be the evaluation of heterochronic transplantations. Experiments involving heterochronic organ transplants have previously been performed with several organs including ovary, kidney, and thymus [4-6]. Additionally, heterochronic BMD and blood exchange have been well studied [7,8]. There is some suggestion that such transplants can impact lifespan, specifically ovaries, however extensive studies for many organs have not been performed [4]. Notably, while there is evidence heterochronic ovary transplantation increases lifespan, a study of heterochronic BMD transplantation failed to observe an effect on lifespan, providing further evidence for the hypothesis that some tissues and/or organs may exert greater influence on organismal aging and lifespan than others, while some may have no effect [4,7]. In addition to the organs and tissues previously mentioned, heart, liver, and pancreas transplants have also been performed in mice with many organ transplants having well established methods with high degrees of transplant success [9-11]. To test this hypothesis groups of aged mice (or rats) would be assigned to receive a heterochronic organ transplant from young animals for each individual organ to be evaluated, and potentially combinations as well, before being followed up for a battery of health measures, such as the frailty index, and lifespan compared to a sham operated group. Such a study would establish the capacity of each tested organ to influence the aging rate and lifespan of a whole organism, would provide translatable insights into the effects of donor age on transplantation, and potentially even future avenues for aging therapeutics. Additionally, it could establish the effects of an aged organismal environment on youthful organs, furthering our understanding of the role of systemic factors in aging. Citations:[1] Nie, Chao et al. “Distinct biological ages of organs and systems identified from a multi-omics study.” Cell reports vol. 38,10 (2022): 110459. doi:10.1016/j.celrep.2022.110459[2] Dayoub, Jose Carlos et al. “The effects of donor age on organ transplants: A review and implications for aging research.” Experimental gerontology vol. 110 (2018): 230-240. doi:10.1016/j.exger.2018.06.019[3] Lau, Ashley et al. “Mixing old and young: enhancing rejuvenation and accelerating aging.” The Journal of clinical investigation vol. 129,1 (2019): 4-11. doi:10.1172/JCI123946[4] Mason, Jeffrey B et al. “Transplantation of young ovaries to old mice increased life span in transplant recipients.” The journals of gerontology. Series A, Biological sciences and medical sciences vol. 64,12 (2009): 1207-11. doi:10.1093/gerona/glp134[5] Li D, Zhao D, Zhang W, et al. Identification of proteins potentially associated with renal aging in rats. Aging. 2018 Jun;10(6):1192-1205. DOI: 10.18632/aging.101460. PMID: 29907735; PMCID: PMC6046247.[6] Kim, Mi-Jeong et al. “Young, proliferative thymic epithelial cells engraft and function in aging thymuses.” Journal of immunology (Baltimore, Md. : 1950) vol. 194,10 (2015): 4784-95. doi:10.4049/jimmunol.1403158[7] Jazbec, Katerina et al. “The Influence of Heterochronic Non-Myeloablative Bone Marrow Transplantation on the Immune System, Frailty, General Health, and Longevity of Aged Murine Recipients.” Biomolecules vol. 12,4 595. 18 Apr. 2022, doi:10.3390/biom12040595[8] Conboy, Michael J et al. “Heterochronic parabiosis: historical perspective and methodological considerations for studies of aging and longevity.” Aging cell vol. 12,3 (2013): 525-30. doi:10.1111/acel.12065[9] Oberhuber, Rupert et al. “Murine cervical heart transplantation model using a modified cuff technique.” Journal of visualized experiments : JoVE ,92 e50753. 12 Oct. 2014, doi:10.3791/50753[10] Yokota, Shinichiro et al. “Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.” Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society vol. 22,4 (2016): 536-46. doi:10.1002/lt.24394[11] Cardini, Benno et al. “Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique.” Journal of visualized experiments : JoVE ,130 54998. 16 Dec. 2017, doi:10.3791/54998"There is a powerful need to include cellular hydration in the list of aging-associated biomarkers. Cellular hydration drops from 90% in the early stages of the developing embryo to 50% in adults. This makes it a foundational aspect of development, the ongoing, post-maturation continuance of which is hypothesized to be a mechanism of aging (The Neuroendocrine Theory of Aging and Degenerative Disease, Dilman and Dean, 1992). Due to the severity of the challenge of direct measurement of hydration, a surrogate marker is desired. Potassium utilization is claimed to be such a marker (Revici, 1961). Potassium utilization (the ratio of cellular potassium to serum potassium) is (1) cheap to measure with serum-testing equipment, (2) highly precise due to sequential measurment of two samples in the same device, thus eliminating independent calibration issues, and (3) minimally invasive (a single blood draw, split into two tubes, one of which is lysed by an exact ten-fold dilution with water). This makes it a perfect option for re-evaluating an old finding with new scientific and analytic methods for assessing an underappreciated but likely foundational aspect of aging."Devloping small climate controlled labs where effects of temperature/humidity/whether on aging & regular metabolism may be seen in case of human cells as well as rats. The inspiration came from the book by David P. D. Munns: ""Engineering the Environment: Phytotrons and the Quest for Climate Control in the Cold War"", and I propose these experiments to also include plant lifeLet us assume this experiment on surrounding climatic conditions on the ability to influence everyday metabolism takes places in two controlled chambers for a period of 17 months.Imagine two trons (A & B): having the exact same ecosystems and resources (just like a mini Earth ecosystem. However the average temperature and the atmospheric concentrations of different gases are made to alter differently. In tron A, the temperature is so programmed to increase at a steady rate from 20 to 30 degrees at the end of the experiment and the oxygen levels are made to also reduce from 21% to 15%. In tron B, the same changes might be made to occur except than the entire time would be of 34 months; so in an ideal scenario, the metabolism rates of rats at 8.5 months of experiment in tron A would be equal to the metabolism rate in tron B at 17 months.The experiment would help understand the process of climate change in case of plants as well which is the most vital in our zeal to survive eternally and would also give an accurate understanding of the metabolic changes with climate so that we may understand what are the best concentration of specific ingredients which might help reduce the process of natural aging.An alternative to this would be to remotely monitor hibernating organisms who are capable of reducing their metabolic rates and the specific techniques which might help humans achieve similar feat. This research proposal focuses more on the lifestyle and the environmental factors (external) rather than the internal unique constituents of each human which seems to be the primary research goals presently. External factor control would also be important but we would need to know whether cold climatic conditions really help humans (as is intuitively predicted by many) and the oxygen levels ideal of humans to optimize their lifespan""# Pauling’s Folly: Reactivation of Endogenous Vitamin C BiosynthesisJosh McNamara### IntroductionAnyone who has hung around a biochemistry lab for long enough has probably heard Linus Pauling invoked as shorthand for someone who veered toward quackery on a late career dementia quest. Running on the fumes of his eminence, Pauling is said to have bet it all chasing something to do with vitamin C. Here, I argue that Pauling’s vitamin C idea was well-founded in evolutionary logic, has accrued several decades worth of supporting evidence, and could increase physiological stress resistance and prolong lifespan. We can now, for the first time, really test the idea by genome-editing guinea pigs.### Vitamin C biosynthesisUp until roughly 40 million years ago, our proto-primate ancestors had the ability to synthesize vitamin C (also called ascorbic acid). This ability is preserved in most modern animals except for humans and their ape and monkey cousins, guinea pigs, and several bird and bat species. Humans and guinea pigs both independently lost the capacity for vitamin C biosynthesis due to inactivating mutations in the GULO gene, which produces the last enzyme in the vitamin C synthesis pathway. We and other species that have lost this ability must obtain vitamin C in our diets to avoid an eventually lethal case of scurvy.### Pauling’s orthomolecular hypothesisAround 1970 Pauling began to expound his theory that the recommended dietary vitamin C levels were high enough to prevent the acute effects of scurvy, yet were insufficient to promote optimal health [1]. He contended that the recommended amount was lower than what a traditional pre-modern diet would provide and ~100 times lower than what rats, horses, dogs, pigs, and our presumed proto-primate ancestors produce each day, adjusted for bodyweight. In contrast to contemporary recommendations of ~40 milligrams, Pauling recommended supplementing with several *grams* of vitamin C per day to keep up with this evolved deficiency in our physiology. The search for the optimal balance of nutrients became the founding principle of his Institute for Orthomolecular Medicine (ortho- = “straight” or “correct”).### A qualitative evolutionary rationaleThe implicit idea in this line of thinking is that genes evolve over millions of years like parameters in an optimization problem, tuning to each other and the environment through the influence of selective pressure. Random mutations de-tune these parameters and are much more likely to diminish overall fitness than increase it. Furthermore, deleterious mutations can fix (become universal) in a population in a “bottleneck” event, in which only a few individuals pass on their genes and only the maladaptive gene variant survives. Evolution exhibits substantial hysteresis / path-dependence, and re-tuning the rest of the genome to accommodate a newly-fixed maladaptive variant (“suppressor mutations”) might take millions of years or more. The fastest way to make up for a fitness loss would be to restore the ancestral wild-type gene or find a way to recapitulate its phenotype using diet, drugs, or other means.GULO inactivation might have been either a beneficial adaptation or a deleterious mutation that fixed in an archaic population consuming enough dietary vitamin C for it not to have mattered. If the latter, it follows that adjusting physiological vitamin C concentrations to the level that the rest of the genome had been tuned to for hundreds of millions of years may increase an organism’s overall fitness. Given that GULO inactivation likely occurred tens of millions of years ago, it is possible that suppressor mutations have since adapted humans and guinea pigs to any deficits that the mutation produced. Despite this, humans are thought to have only recently (on an evolutionary timescale) switched from a vitamin C rich diet to a vitamin C poor modern diet. High vitamin C consumption would weaken selective pressure for the evolution of GULO-null suppressors, and any fitness loss arising from this dietary change itself has had little time to select for suppressor mutations.### Vitamin C megadoses haven’t been medically usefulPauling co-authored a few vitamin C megadose studies that appeared to show promise in cancer, but nothing came of these protocols. One potential reason is that eating several grams of vitamin C per day has little impact on the active concentration in the blood and within cells because such super-doses are rapidly excreted in the urine. You simply can’t absorb vitamin C efficiently enough to get the active concentration that your dog enjoys. More recent studies have shown that intravenous dosing can achieve vitamin C levels much higher than the oral route. Some have even shown that intravenous vitamin C enhances checkpoint inhibitor immunotherapy efficacy in mouse cancer models and imparts a survival benefit [2].### Genetic interventionAside from eating or injecting vitamin C, we can now test the spirit of Pauling’s proposal more directly with new technologies such as CRISPR that enable us to edit mammalian genomes. Thanks to genome sequencing, we can also infer by comparison to other species what a functional GULO gene should look like for a given organism and mutate its genome to resurrect the long-dead gene.Some experimental results suggest that genetic delivery of vitamin C via repair of the GULO gene may provide benefits beyond what is possible with intravenous dosing. Budding yeast, a common model organism used in genetics research, can be mutated such that it can no longer synthesize several essential metabolites. These strains grow proficiently when the missing nutrients are supplied in their growth medium. However, restriction of the essential nutrient causes the strains to have shorter lifespans [3]. Experiments in mice have yielded similarly suggestive results. Researchers inactivated the GULO gene in mice and found that the animals quickly developed scurvy [4]. After supplementing their chow with vitamin C, the mice were able to grow and reproduce. Reducing the dietary vitamin C maintained viable mice but caused defects in the aortic wall. Would genetic rescue of GULO in a GULO-null animal such as humans or guinea pigs improve healthspan?### GULO rescue longevity experimentSeveral investigators have proposed genetic rescue of GULO-deficient animals, yet none have made a direct genomic repair of the gene. See [5] for elaboration. I propose to genome-edit the germ line of a guinea pig to restore native GULO function. Next, observe the animals and track their metabolic profiles and lifespans (~6 years on average) relative to control animals. One could give away 100 GULO+ guinea pigs and matched wild-type animals as pets on the condition of visits for regular exams and an autopsy. Perhaps there will be a difference in longevity. On a shorter timescale, one could measure a few correlates of lifespan such as time to sexual maturity, middle-age fecundity, resilience of cultured cellular explants, starvation resistance, strength and endurance, rates of tumorigenesis or vascular disease progression, and response to cancer immunotherapy. Similar experiments could be conducted in cultured human tissues with a repaired GULO gene. Even if these experiments do not produce a longevity boost, many investigators will be curious about the effects of reverting a DNA copying error that occurred in our lineage millions of years ago. Researchers have been pondering this experiment for decades, and we owe it to them to give it a try now that it is technically feasible.### Bonus: Phylogeny as version controlFrom this course we can generalize to an even more fanciful idea- using phylogeny as genetic version control. As discussed above, bottlenecks have been frequent in our evolutionary history, and we can infer that many fitness-reducing mutations have fixed in the human population. These mutations, like GULO-null, are universal and all functional alleles are on the other side of a species barrier. With genome sequencing, we can see over that barrier and infer “weakly perfect” ancestral genotypes from a composite of our phylogenetic relatives. See Kondrashov for a more thorough discussion of mutation, imperfect alleles, sign epistasis, and Bateson–Dobzhansky–Muller interspecies incompatibility [6].Many bacteria have found a solution to this problem through horizontal gene transfer. These organisms can achieve huge fitness gains by acquiring genes from distantly related species, as is commonly seen in the evolution of antibiotic resistance. Humans do not yet engage in gene transfer with other species, though genome editing enables it. Because random mutations are more likely to be deleterious than beneficial, it follows that reversion of fixed mutations is more likely to increase fitness than decrease it. As with GULO, we can use composites from other species to infer the sequence of potentially fitter human alleles and test whether they increase vitality and prolong lifespan in relevant model systems. ### References1. Pauling, Linus. Evolution and the need for ascorbic acid.https://doi.org/10.1073%2Fpnas.67.4.16432. Magrì et al. High-dose vitamin C enhances cancer immunotherapy.https://doi.org/10.1126/scitranslmed.aay8707 3. Gomes et al. Low auxotrophy-complementing amino acid concentrations reduce yeast chronological life span.https://doi.org/10.1016/j.mad.2007.04.003 4. Maeda et al. Aortic wall damage in mice unable to synthesize ascorbic acid.https://doi.org/10.1073/pnas.97.2.841 5. Cao et al. Ectopic Gene Expression to Restore the Ascorbate Biosynthesis Pathway in Vertebrates Lacking Functional Gulo.https://doi.org/10.23880/ggtij-160001086. Alexey Kondrashov. Crumbling Genome.https://doi.org/10.1002/9781118952146""Title: Ageing as run-on developmentHypothesis SummaryAgeing is widely thought to occur due to the accumulation of various forms of molecular damage [1]. What if, however, ageing changes are not primarily a result of a build-up of stochastic damage but are rather a product of regulated processes? Multiple facets of mammalian ageing follow predetermined patterns encoded in the genome as part of developmental processes. One provocative hypothesis is therefore that the developmental program, optimized for reproduction, inadvertently regulates ageing [2]. More precisely, my hypothesis is that gene regulatory programs during development trigger changes in cells and tissues that result in human ageing because, after reproduction, developmental programs have no evolutionary reason to change their predetermined trajectories and hence become detrimental.The hypothesis that ageing may be an extension of development dates back to the 19th century [3], and the notion that developmental factors can impact ageing has been debated for over a century [4], including as part of a program. In line with these largely unexplored concepts, I hypothesize that the majority, albeit not all, changes causing the functional decline that characterizes human ageing are triggered by a developmental program optimized for reproduction.The implications of seeing human ageing as at least partly deriving from developmental programs are multiple and far-reaching. This hypothesis challenges current paradigms in the ageing research field that view stochastic damage as the primary driver of ageing. Focusing on this very promising but largely unexplored hypothesis could lead to a potential paradigm shift in the field. According to my hypothesis, ageing is an information problem, and if we could instruct cells to reset the developmental programs this would result in cellular rejuvenation, as observed already in cellular reprogramming. Therefore, we need better models of gene regulation, of development and more broadly of how the genome and epigenome operate in space and time. As such this hypothesis also opens several promising directions and has important implications for developing interventions.In conclusion, I would like to resurface and focus on the hypothesis that the developmental program inadvertently regulates ageing.References:[1] López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013). The hallmarks of aging. Cell 153:1194-1217.[2] de Magalhaes JP (2012). Programmatic features of aging originating in development: aging mechanisms beyond molecular damage? Faseb J. 26, 4821-4826.[3] de Magalhães JP, Church GM (2005) “Genomes optimize reproduction: aging as a consequence of the developmental program.” Physiology 20:252-259.[4] Loeb J, Northrop JH (1917). What Determines the Duration of Life in Metazoa? Proc Natl Acad Sci U S A 3:382-386.""The Neuroendocrine Theory of Aging by V. Dilman.In 1960-70, Russian biologist Vladimir Dilman published a series of scientific papers in which he outlined his vision on the nature of aging in organisms.https://pubmed.ncbi.nlm.nih.gov/13816764/https://pubmed.ncbi.nlm.nih.gov/5686906/https://pubmed.ncbi.nlm.nih.gov/4425748/https://pubmed.ncbi.nlm.nih.gov/1218271/https://pubmed.ncbi.nlm.nih.gov/225186/In the Lancet in 1971, his article ""Age-associated elevation of hypothalamic threshold to feedback control, and its role in development, ageing, and disease"" was published. In it he detailed his hypothesis of the formation of aging and age-related pathologies, called by him the elevation theory of aging, and later renamed the neuroendocrine theory of aging. The basis of the hypothesis is the relationship between violation of homeostasis during the development of the body and aging, and an increase in the sensitivity threshold of the hypothalamus to feedback mechanisms.https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(71)91721-1/fulltextThe hypothalamus is the central organ in Dilman's theory of aging. Dilman suggested the key role of the hypothalamus in aging due to its important regulatory role at the junction of two body systems - endocrine and nervous. In this organ, control, regulation, communication, integration and homeostasis of metabolic and reproductive functions are carried out. The most important general principle by which this integration is achieved is the mechanism of negative feedback. As Dilman described in his hypothesis, maintaining a stable balance (homeostasis) is a key requirement for the life of an organism, but the development of the organism after its birth requires a violation of homeostasis, otherwise development is impossible. As a typical example, Dilman cites changes in the body during pregnancy. Cholesterol is critical for fetal development. You need a lot of cholesterol. To do this, a special genetic program is launched with the participation of several hormones. such as placental growth hormone. That suppresses the use of glucose by cells, resulting in the formation of the necessary conditions for increasing cholesterol levels. The placenta forms this evasion from homeostasis during pregnancy, because it is not included in the general negative feedback mechanism, when an increase in one hormone or substance will cause a compensatory response that dampens such an increase. After birth, the formation of an adult organism also requires a violation of homeostasis. Here, Dilman assigned a key role to the gradual increase in the hypothalamic sensitivity threshold to negative feedback mechanisms. A baby has only a small amount of sex hormones, so that they have an inhibitory effect on the hypothalamus. But in the process of development, the hypothalamus gradually increases its threshold of sensitivity to suppression by sex hormones, increasing its activity and stimulating the pituitary gland to further increase the activity of the gonads. But with aging, this increase in the threshold of sensitivity of the hypothalamus to negative feedback mechanisms leads to negative effects. Among which he primarily singled out a violation of energy homeostasis (insulin resistance and hyperinsulinemia, increased blood glucose, excess free fatty acids and cholesterol), which in turn leads to age-related pathologies, cardiovascular, neurodegenerative, etc. In his hypothesis, Dilman did not separate aging from age-related pathologies, considering them to be an interconnected process.What, in his opinion, could cause age-related changes in the hypothalamus? Among these reasons, he singled out:- Decrease in the level of hypothalamic neurotransmitters (in particular, catecholamines and serotonin); - Decreased number of hormone receptors in the hypothalamus;- Decreased secretion of pineal hormones (melatonin and polypeptide hormones of the pineal gland);- Accumulation of fat; decreased glucose utilization;- Accumulation of neuronal lesions caused by chronically elevated levels of cortisol due to prolonged stress;- Accumulation of cholesterol in the plasma membranes of neurons. The well-known gerontologist M. Blagosplonny in his theory of hyperfunction as a key factor in aging (based on the activity of the mTOR signaling pathway) emphasized its continuity with the theory of V. Dilman, which, in his opinion, is undeservedly forgotten today. And it is not even mentioned in works on the relationship between the hypothalamus and aging: “Vladimir Dilman suggested that aging is due to a progressive loss of sensitivity of the hypothalamus. This causes a progressive change in homeostasis, metabolic disorders leading to age-related diseases. There is an age-related loss of sensitivity of the hypothalamus to the negative feedback of certain hormones, such as estrogens and glucocorticoids. This explains the development of age-related diseases, including metabolic disorders and menopause. Unfortunately, the hypothalamic theory was far ahead of its time and too medical to be attractive to gerontologists. This theory, supported by excellent experiments, went unnoticed, only to be rediscovered recently.”https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765577/ In their brilliant work, the team of B. Cai et al., who described the systemic influence of the hypothalamus on aging, did not even mention the previous work of V. Dilman. B. Cai et al. revealed yet another yet unknown side associated with aging and the hypothalamus – microinflammation of the hypothalamus caused by an increase in the activity of the NF-kb signaling pathway.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756938/Today, no one doubts the very large influence of the violation of regulatory functions by the hypothalamus on aging. Аccording to V. Dilman, prophylactic and therapeutic measures should be directed towards the preservation of rhythmic function of the basic homoeostatic systems so that metabolic processes can be maintained at the level reached when development of the organism was complete. We need, therefore, the means to restore rhythmic activity of the homoeostatic systems by lowering the hypothalamic threshold to different types of homceostatic inhibition (i.e., by counteracting the process of the self-development of homreostatic systems).Means of this sort are not yet available, but from what we already know we may presume that the pineal gland can exert such influence (i.e., it can directly inhibit some of the functions of the hypothalamus and increase its sensitivity to intrinsic regulating factors), and we may anticipate that other pharmacological agents possessing these properties will eventually be found. In the meantime research should be directed towards the use of those prophylactic and therapeutic measures that may act on different stages of the elevating mechanism of ageing. In this respect studies should be made of: oestrogens; pituitary inhibitors, especially sigetin (meso-3,4-bis-(p-sulphophenyl)-hexane)’; androsterone ; D and L thyroxine; rational diet; aminoglutethimide, which can suppress both steroidogenesis and gonadotrophin secretion a5; phenytoin, capable of inhibiting adrenal function and gonadotrophic secretion and, possibly, of influencing the synthesis of R.N.A. ; and phenobarbitone and other antiepileptic and neurotropic preparations. More attention should be paid to phenformin, which eliminates compensatory hyperinsulinism and thereby reduces the cholesterol and triglyceride levels, and reduces excess body-weight in aged patients. With this in view, phenformin should be investigated with regard to its ability to suppress the development not only of atherosclerosis, but also of tumour formation. Anahormones, with their ability to induce antibodies, and to suppress the secretion of protein hormones or to compete with them at the level of target tissue, may be interesting tools for the study of age and pathological processes.The idea of using labelled immunoserum for selective retention of radioactive antihormonal antibodies in the hypophysis and hypothalamus should also be taken into consideration. More emphasis should be placed on the study of isohormones (e.g., those of stiocholanolone type) and of other ways which, by affecting the hypothalamic threshold to inhibition or by reducing compensatory reaction, might slow down the realisation of the genetic programme of ageing and related pathology.""Cellular senescence has recently became very famous hallmark of aging. Pro-inflammatory and matrix-degrading factors that are secreted by senescent cells are detrimental to health and participate in pathogenesis of various age-related pathologies. However, there still is no proven theory which explains why senescent cells accumulate with advancing age.In our work [1] I proposed a hypothesis that logically explains the accumulation of senescent cells as a result of non-enzymatic changes to the long-living proteins of the ECM: there's a growing body of evidence that cellular senescence (at least partly) is an anti-fibrotic cellular program. Cells stop to proliferate (thereby reducing the synthesis of collagen), secrete matrix-degrading enzymes that clear ECM debris and secrete pro-inflammatory cytokines that attract immune cells that clear degraded fragments of the ECM. My point is that the aged (due to cross-links accumulation) ECM has very similar properties as fibrotic ECM which in turn erroneously activates anti-fibrotic cellular program - cellular senescence. If this hypothesis is true then it has profound implications for the understanding biology of cellular senescence and could lead to a new class of drugs that prevent excessive accumulation of senescent cells without hampering the anti-fibrotics mechanisms.1. https://www.sciencedirect.com/science/article/pii/S1568163720302324""Proposal to test the Selective Destruction Theory of AgeingIn 1956 Harman proposed the free radical theory suggesting that molecular damage could accumulate to cause ageing (Harman, 1956). Then Kirkwood (1977) observed that evolution might allow damage to accumulate if energy spent on maintenance could be better utilised in other processes with a higher impact on fitness. These ‘common sense’ theories have dominated gerontology ever since; however, as our understanding of the mechanisms of ageing grows, it is becoming increasingly difficult to reconcile all aspects of ageing with accumulating damage. Long-lived worms and flies that accumulate more damage than their short-lived counterparts have caused some to ask if the free radical theory is dead (Pérez et al., 2009), while others have suggested that ageing via accumulating damage is contradicted by the rejuvenation potential of parabiosis and the ability of Yamanaka factors to return heavily damaged senescent and old cells to a youthful state (Katcher, 2015). We recently published a new ‘selective destruction theory’ (Wordsworth et al., 2022), which is not only consistent with these rejuvenation revelations but indicates the underlying mechanism. The concept is simple: cells in multicellular organisms undergo mutations or epimutations which cause them to grow at different rates. Importantly, these changes can result from point mutations in genes and promoters as part of replication without need for damage accumulation or oxidative stress. Mutants that grow and replicate faster than wildtype cells will have a selective advantage, eventually allowing them to overtake the tissue if they are not controlled. It has been suggested that this alone might be sufficient to make ageing and death inevitable in multicellular mitotic organisms (Nelson and Masel, 2017); however, these authors did not consider that organisms could employ a mechanism of counterselection against fast cells. As we modelled in our paper, when slower cells are capable of epigenetically slowing down fast cells or killing them, termed selective destruction, it prevents their takeover and even allows the spread of slower cells (Wordsworth et al., 2022). The result would delay multiple diseases such as cancer and fibrosis that become more probable with accelerated metabolism, but at the same time would result in a gradual metabolic slowdown. Importantly, if the fitness costs of metabolic slowdown are lower than those associated with the spread of fast mutants, as our models suggested, then selective destruction could undergo positive selection despite the induced functional decline. Multiple lines of evidence suggest this is occurring, from the decline in cell proliferation rate (Tomasetti et al., 2019) and energy expenditure (Pontzer et al., 2021) to the hallmarks of transcriptomic ageing which include reduced growth factor signalling, protein synthesis, and mitochondrial respiration (Frenk and Houseley, 2018).At the cellular level, the immediate consequence of metabolic slowdown will be a rise in ATP (due to reduced ATP hydrolysis). As ATP is tightly regulated to ensure constant availability, a decline in ATP usage necessitates a decline in ATP production to maintain ATP within the equilibrium range. Thus, a decline in cellular metabolism results in a decline in mitochondrial metabolism. However, our current modelling suggests that when mitochondria become sufficiently slowed, the shift in homeostasis becomes confused with the state of infection: infected cells slow ATP production to prevent energy utilisation by invading pathogens, inhibiting their replication and the spread of infection. We suggest that evolution has therefore linked reduced mitochondrial metabolism, which is frequently termed ‘mitochondrial dysfunction’, with inflammation by activating the NLRP3 inflammasome (Mishra et al., 2021). However, this mitochondrial dysfunction would not result from accumulated damage as is commonly believed, instead reflecting a homeostatic response. We therefore hypothesise that the pathway from selective destruction to inflammation is central to ageing and age-related disease. Selective destruction is therefore one of the few theories that provides both an evolutionary and mechanistic explanation for ageing, perhaps the only plausible theory independent of damage accumulation. If ageing is induced by cell communication as we suggest, it explains the rejuvenating effects of parabiosis, and is consistent with Yamanaka factor rejuvenation by removing the epigenetic controls that have slowed cell growth. However, our idea is still theoretical: the notion of cells within tissues slowing each other down is highly likely given the emerging importance of somatic cell competition (Kakiuchi and Ogawa, 2021), but the regulation of neighbouring cell growth is novel to this theory. Here we propose a series of simple cell culture experiments which could prove this communication is occurring, evidence its role in ageing, and elucidate the molecular pathways involved. Using labelled cells from old and young individuals with different growth rates, we will test if a low concentration of young cells grown in co-culture with old cells will be slowed compared to young cells in monoculture. If they fail to dominate despite an initially faster growth rate, then we can conclude that they have been slowed down or even killed by the older cells. To elucidate the mechanism, we have identified Notch signalling as a regulator of cell growth by juxtacrine signalling. Notch signalling from the surrounding tissue controls tumour cells (Demehri et al., 2009) and knockout results in tumours (Kimura et al., 2019), as we predicted would result from loss of selective destruction. We will therefore knockout Notch in our cell lines and observe what effect this has on young cells in co-culture with old cells, predicting that this might abrogate selective destruction and allow the faster young cells to dominate the co-culture. The prize money would be used to purchase the fluorescent and NICD knockout (to inhibit Notch) constructs and lentiviral agents as well as lab costs to carry out these experiments in our existing old and young dermal fibroblast and muscle stem cell lines, as shown in the following breakdown:Media and plastic consumables for cell culture 5,500Transfection reagent 850Disposable plastics and gloves 4,500General laboratory chemicals and media 2,000shRNA/cDNA constructs 1,000Bioimaging/cell sorting facilities charges 5,500In addition to the prize money helping to fund these initial experiments, we also hope that the recognition accompanied by this prestigious prize might build awareness of selective destruction theory and encourage collaborators with epigenomic experience to help us identify the effects of selective destruction in these co-culturing experiments on epigenetic clocks. If young cells are in fact being aged rather than simply slowed then we predict this will be reflected by CpG methylation at the sites identified by Horvath (2013) and others. If we can show evidence for selective destruction, the implications for ageing would be vast. Beyond providing a badly needed second theory, it would explain the limited efficacy of interventions such as calorie restriction and metformin after the regimens cease (Mair et al., 2003), as they are reducing basal inflammation while the underlying selective destruction is continuing unabated. Interventions aimed at altering cell communication and epigenetic regulation may prove significantly longer lasting, but must contend with the increased risk of overactivity disorders such as cancer and fibrosis. NB: This work would be conducted in collaboration with Viktor Korolchuk (panel member).""Quantum Biology for Longevity Research1-page hypothesis found in the link below:https://drive.google.com/file/d/1rfyAsx2cwGHQVjUwWbWftRZAFoOn2AP_/view?usp=sharing"At Remedium Bio, we believe that Growth Factors are the most promising and near-term solution to meaningful extension of healthspan and lifespan. While significantly under-represented in therapeutic research, Growth Factors have delivered remarkable results in placebo controlled clinical trials for a number of age-related diseases. Most recently, repeat intra-articular injections of the FGF18 protein have demonstrated the ability to reverse cartilage loss in Osteoarthritis patients relative to placebo control - a never before seen result in Rheumatology. As a classical disease of aging, Osteoarthritis affects 1 in 7 US adults and presents with progressive erosion of cartilage, eventually leading to debilitating pain and loss of function. Remedium is developing a novel FGF18 gene therapy with the aim of reversing cartilage loss to restore a more youthful and healthy phenotype to arthritic joints."With the promising potential of hundreds of autologous cell therapies currently in the pipeline, there has been increasing interest in finding stem cell populations that can be obtained in a less invasive manner and yet exert similar therapeutic properties. Studies over the past two decades or so demonstrated success in isolating multipotent and pluripotent stem cell populations from plucked hair follicles. The easy accessibility, high proliferation, differentiation ability, and efficient reprogramming potential associated with this stem cell source make hair follicle an ideal candidate for autologous cell therapy and regenerative medicine.In this project, we aim to isolate mesenchymal stem cells from the outer root sheath of the human plucked hair follicle. We will work towards differentiating them into osteogenic, adipogenic, chondrogenic, and neuronal lineages. Following validation and scale-up, we aim to employ them in various pre-clinical and clinical models to treat aging and degenerative diseases such as osteoarthritis, cartilage degeneration, and spinal cord injury. Use of a totally autologous stem cell source that is easily obtainable during adulthood, such as the hair follicle, alleviates the limitations and safety concerns associated with most stem cell-based therapies.""What if we could effectively harness cancer to live longer?Cancer is the immortal phenotype. If cancer can enable biological immortality, perhaps we should be more carefully looking at ways to harness cancer to promote longevity. This is a contrarian hypothesis, but the clinical framing around cancer as “the enemy” has likely prevented meaningful work exploring how cancer could potentially be leveraged from a longevity-enabling perspective. "" Antiaging research Theoretical Framework: Genomic stabilityThe novelty in this project is that it focuses on the quiescent1 stage of the cell cycle (fase G0), what we intend is to increase the period of time in which the cell is in the quiescence stage, with the premise that the quiescent period can be affected by epigenetic2, 5 factors. We do not intend in this case that the cell reproduces itself a greater number of times, or indefinitely, but that it increases the amount of time it works efficiently without dividing.In this project we agree with the theory of genetic damage3, considering aging as: a multifactorial process of generalized cellular degeneration, which has its origin in the damages that our genome experiences, having as a consequence one of three possibilities: the cells enter senescence, they become malignant or massive cell apoptosis; manifesting itself in a structural and functional insufficiency that finally leads to death. VISION:The health and longevity of the human species does not depend on a gene, a specific protein or a biomolecule “x”, but on the total genome integrity, both of coding genes and regulatory sequences. GENERAL OBJECTIVEStop Genetic Damage, achieving a radical increase in life expectancy in individuals under treatment and in their offspring, since it is genetic to transmit this benefit to succeeding generations increasing results. SPECIFIC OBJECTIVES.a) Achieve genomic stability4. b) Prevention of degenerative chronic illnesses with genetic component.c) To exceed with much the known longevity levels. HYPOTHESIS:By eliminating harmful element Carbon 14 of our genome we will be eliminating a major source of mutations and senescence3, which will give the genome greater stability and increase cell life.Our genome has adapted evolutionarily over the course of millions of years to the levels of natural radiation that exist in the environment, however, sometimes fast and drastic changes occur on our planet that cannot be assimilated as quickly by our genome, such is the case of the last glaciation, implied a drastic change in the terrestrial climate and consequently in the concentrations of C14 present in the atmosphere. In this project we intend to drastically reduce the levels of C14 present in the genome and in general throughout the organism.This project presupposes that the cell division cycle can be affected by stress factors of the genome (genetic damage) and that if we reduce C14 (stress factor) we can slow down the cell division process, obtaining longer-lived healthy cells and increase health, strength and longevity of the human species. There are multiple stressors of the genome, external, internal, genetic, epigenetic, but in this case we will focus specifically on the C14 element.Expected results:In vitro we expect to observe that the cell division process (human cells) is slowed down compared to control cultures; and that although slow, the cell division process in cultures with reduced C14 substantially exceeds the survival of the control culture.In vivo we expect a substantial increase in the strength, health and longevity of the species with which we will work, fruit fly and laboratory mice. We will observe results in successive generations, up to the tenth generation, and in the case of human cells, the age of the donor (newborn, young adult and elderly) will be taken into account due to the load of genetic damage.In this project we consider that if the stability of the DNA nuclear and mitochondrial is not achieved, all the tissues, organs and systems at the human body will end up failing. Our goal is to prevent the occurrence of mutations or any damage to our genetic code. 1.- https://www.nature.com/subjects/quiescence2.- https://www.genome.gov/genetics-glossary/Epigenetics3.-https://www.semanticscholar.org/paper/The-essential-mechanisms-of-aging%3A-Irreparable-of-Yin-Chen/7bf085463e44d3da5a6e2a95ed1f8976d2ab2f734.-https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/genetic-stability5.- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3481136/KindlyProfessor Ruben Lopez Cortes.Biology teacher.""link to text file in case copy/paste does not work:https://docs.google.com/document/d/1ehCXArbfy5CmuJ6KwWY_baxSh69nsANL4WQ93GX03dA/edit?usp=sharingCarbon Disulfide and AgingHypothesis: Endogenous carbon disulfide production changes during aging and these changes are associated with alterations in neurological and immune function.Rhyothemis princeps30 December 2022Carbon disulfide (CS2) is a volatile liquid at room temperature; its vapors are heavier than air [1]. Although CS2 is naturally produced by some anaerobic bacteria it is best known as an industrial solvent used in the production of rayon (also known as viscose) and as feedstock for the production of carbon tetrachloride and dithiocarbamate fungicides [1,2].Neurotoxic effects of CS2 inhalation were first observed from its initial use in rubber product manufacturing in the mid 19th century [3]. Intoxication from acute exposure, which Charcot termed “carbon disulphide hysteria”, can result in dizziness, nausea, headache, numbness, autonomic dysfunction, anorexia, loss of consciousness, and neuropsychiatric symptoms including anxiety, hallucinations, delusions, paranoia, hypersexuality, extreme emotional lability, and suicidal ideation [1,3,4]. Severe cases can result in death from respiratory arrest [4]. Chronic occupational exposure to CS2 has been linked to cardiovascular disease, kidney disease, loss of sexual function, menstrual cycle irregularities, early menopause, polyneuropathy, retinopathy, encephalopathy, parkinsonism and multiple system atrophy [1,3,4,5,6,7,8,9,10].Given the preceding litany of harms, it may be surprising to learn that CS2 is present in the breath of rodents [11]. In rats CS2 is appetitive and mediates social learning of food preferences [11,12]. CS2 can be detected in the breath of humans and elevated CS2 levels have been found in the exhaled breath of schizophrenia and cystic fibrosis patients; this may be the result of either higher rate of production or reduced rate of metabolism and excretion via the urine [13,14,15]. CS2 levels in urine have been found to fluctuate over the course of the menstrual cycle [16]. In humans, CS2 is present in the feces and its increase after supplementation with a synbiotic indicates production by gut microbiota [17,18]; bacteria in the lung may be another source of CS2 [15].Endogenous production of CS2 might not be so unexpected given that nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (HS2) are all toxic gasses that are also endogenously produced and, additionally, function as small molecule bioregulators (SMB) (also termed gasotransmitters) [19]. DeMartino et al. [19] have proposed that CS2 could be an SMB based on its similarities to NO, CO, and HS2 and that like these SMBs, the toxic effects of exposure to CS2 may be due to relatively high doses overwhelming regulatory or signaling processes in which it hypothetically normally participates. DeMartino et al. [19] suggest that in addition to the gut and/or lung microbiome “one should also consider the possibility of a dedicated, [human] enzymatic source.”The acute neurotoxic effects of CS2 and its dithiocarbamate (DTC) metabolites are mainly attributed to catecholamine disruption; following acute inhalation exposure rats show decreased brain levels of noradrenaline (NA) and increased dopamine (DA) [20]. Noradrenaline can be inactivated by protein cross-linking induced by DTCs, and CS2 and DTCs can inhibit dopamine beta hydroxylase (DBH), which catalyzes the conversion of dopamine to noradrenaline, leading to increase in the former and decrease in the latter [20,21]. Inhibition of DBH is the result of chelation of its cofactor, copper, by DTCs formed from the reaction of CS2 with amino acids and/or catecholamines [22]. Chronic CS2 exposure was reported to be associated with decreases in serum levels of both noradrenaline and dopamine, as well as copper and zinc [23]. DTCs also inhibit carbonic anhydrases and aldehyde dehydrogenases, which are also important to neurological processes [24,25]. The aldehyde dehydrogenase ALDH1A1 is strongly expressed in brain dopaminergic neurons where it functions to oxidize 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of DA, preventing the conversion of DOPAL to even more toxic compounds such as tetrahydropapaveroline [26]. ALDH1A1 inhibition could explain parkinsonian symptoms of chronic occupational CS2 exposure. An excess in endogenous CS2 levels above normal physiological levels caused by an increase in production by the microbiome or by a decrease in its rate of metabolism and excretion could exert harmful neurological effects through inhibition of ALDH1A1.De Martino et al. [19] propose that DTC and CS2 are in labile equilibria under physiological conditions and that CS2, which is lipophilic, can enter into membrane-bound catecholamine storage granules where it converts to DTC (a polar molecule unable to traverse membranes) and inhibits DBH - thereby exerting a regulatory influence on the production of NA from DA. It is difficult to predict if this would affect levels of these neurotransmitters in normative aging since their levels decline overall due to an age-dependent increase in activity of monoamine oxidases [27].It is worth noting, however, that while CS2 exposure is associated with parkinsonism and has been implicated in multiple system atrophy, large epidemiological studies have not found an association with increased risk of Parkinson disease [1,8,9,10,28]. People with ulcerative colitis, an inflammatory bowel disease, have increased risk of developing Parkinson disease [29]. In recent years there has been growing recognition of the role of the gut microbiome in both UC and PD, as well as other age-dependent neurodegenerative diseases [30,31]. As mentioned above, CS2 in feces can be increased by supplementation with a synbiotic [18]. Garner et al. [17] found CS2 present in 100% of fecal samples of healthy volunteers (n=30), but only in 61% in those of people with UC (n=18); CS2 was also reduced in Clostridium difficile patients, with only 41% (n=22) producing CS2. These results suggest that CS2 production may be an indicator of a healthy gut microbiome.DTCs inhibit the nuclear factor kappa beta (NF-κB) inflammatory signaling pathway [32]. DeMartino et al. [19] have proposed multiple mechanisms through which CS2 might also inhibit NF-κB. The NF-κB signaling pathway is thought to play an important role in the pathogenesis of UC and also PD and other neurodegenerative diseases [33,34]. NF-κB is a major signaling pathway involved in ‘inflammaging’ [35]. Chronic activation of NF-κB can induce cellular senescence and activation of longevity genes such as SIRT1, SIRT6, and FOXOs can suppress NF-κB signaling [35]. It is possible CS2 produced by the gut microbiome decreases intestinal inflammation through inhibition of NF-κB, reducing risk of UC, PD and perhaps even extending healthspan and lifespan. Interestingly, CS2 is present in cigarette smoke and smoking is associated with reduced risk of both UC and PD, though nicotine and carbon monoxide are usually named as the most likely active constituents [36,37].Occupational CS2 exposure is associated with endocrine and reproductive changes which include alterations in hormone levels, increase in low density lipoprotein, menstrual cycle irregularities, loss of libido, sexual dysfunction, changes to sperm morphology, and earlier onset of menopause [1,6,7,38]. Based on evidence from animal model studies, Printemps et al. [21] concluded that thyroid hormone disruption from CS2 exposure is most likely due to central nervous system toxicity rather than by a direct endocrine disruption mechanism.However, women chronically exposed to CS2 were found to have higher serum levels of dehydroepiandrosterone sulfate (DHEA-S) than controls [7]. Another study of rayon factory workers by Djuric et al. [39] found CS2 exposure may have caused an irreversible disturbance in sulfur metabolism [1,39]. Sulfation and desulfation are key mechanisms for regulating the availability and activity of steroid and thyroid hormones as well as neurotransmitters such as DA [40,41]. Sulfation of hormones renders them soluble for transport via the circulatory system whereas desulfation within cells converts them to their active form in most cases [40]. Sulfation and desulfation are affected by activities of sulfotransferases and sulfatases, respectively; availability of sulfur donor adenosine 3 ́-phosphate 5 ́-phosphosulfate (PAPS) is rate-limiting for sulfation [42]. Not surprisingly, some known endocrine disruptors such as acetaminophen and phthalates act through altering sulfation levels [42,43,44]. Since CS2 increases sulfation of DHEA, it may be considered an endocrine disruptor at exposures similar to those of rayon factory workers.If the deleterious effects of an SMB at toxic exposure levels implicate its bioregulatory activities at physiological concentrations, then modulation of sulfation by CS2 could be a particularly important aspect of its hypothetical regulatory roles in neurological function and aging. Reduction in DHEA-S is associated with Alzheimer disease (AD), whereas DHEA is not associated with AD [45]. Pérez-Jiménez et al. [46] demonstrated that inhibition of sulfatase increases lifespan and protects against age-related proteotoxicity in Caenorhabditis elegans, and also reduces β-amyloid aggregation and cognitive behavioral deficits in an Alzheimer disease mouse model. Interestingly, steroid sulfatase expression is upregulated by NF-κB [47], which leads to speculation that increase in DHEA-S from CS2 exposure is the result of decreased sulfatase expression through suppression of NF-κB. Note that NF-κB is Janus faced in neurodegeneration, having neuroprotective or harmful effects depending upon signal strength and type of NF–κB dimers activated [48].Sulfation also impacts the function of perineuronal nets (PNNs), which are specialized extracellular matrix structures that surround neurons [49]. PNNs are composed of chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, glycoproteins and glycosaminoglycans, such as hyaluronan [50]. PNNs function as molecular scaffolding that stabilizes and regulates the synapses they surround; they also protect neurons from oxidative stress and toxins such as β-amyloid (1-42) [50]. Aging is correlated with changes in sulfation patterns of PNNs which result in lowering of neuroplasticity and decreases in memory-forming ability [49,51]. Unfortunately, there appear to be no published studies on the effects of CS2 on perineuronal nets at the present time.In summary, CS2 is a product of the human microbiome which could plausibly impact aging and age-related neurodegeneration through the mechanisms discussed above. The hypothesis put forth here is that endogenous CS2 production changes during aging and these changes are associated with alterations in neurological and immune function. To test this hypothesis, a cohort study could be conducted with participants ranging in age from early 20s to late 80s that measures CS2 presence in feces and CS2 levels in exhaled breath. The study would also measure biomarkers of neurological aging and inflammation, as well as serum DHEA-S. Serum neurofilament light chain (Nfl) would serve as a biomarker of neurological aging; Nfl is a dynamic, robust biomarker of neuroaxonal damage that increases with age across the lifespan [52]. Biomarkers of inflammation would include high sensitivity C-reactive protein, interleukin 1β, interleukin 6, and tumor necrosis factor α.If one or more associations between CS2 levels and the listed biomarkers are found, this would not serve as evidence for a causal relationship. However, a positive finding would indicate whether further investigation into the potential impact of endogenous CS2 on aging is warranted and in that case, studies in animal models would likely be the next step. It should be noted that rodents and humans have an important difference with respect to CS2 signaling - humans lack the guanylyl cyclase D (GC-D) receptor [53]. In rodents, the GC-D receptor is required for CS2-mediated social learning of food preferences [11]. This could be an important point to consider in designing mouse or rat model experiments on CS2 and aging since the social environment affects the aging process [54].References1 - Abadin, H., & Liccione, J. J. (1996). Toxicological profile for carbon disulfide. U.S. Department of Health and Human Services. https://www.atsdr.cdc.gov/ToxProfiles/tp82.pdf2 - Australian Government, & Department of Climate Change, Energy, the Environment and Water. (2022, June 30). Carbon disulfide—DCCEEW. https://www.dcceew.gov.au/environment/protection/npi/substances/fact-sheets/carbon-disulfide3 - Blanc, P. D. (2016). Fake Silk: The Lethal History of Viscose Rayon (1st edition). Yale University Press.4 - National Research Council (US) Committee on Acute Exposure Guideline Levels; National Research Council (US) Committee on Toxicology. (2009). 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Total plasma exchanges of 1 liter saline/albumin and 2 liters of yFFP have been successfully performed, as have two 1-liter exchanges of yFFP.This study will compare epigenetic, proteomic, laboratory and functional results taken from just before the initial treatment to results taken one month after the last treatment. The comparative results from each study participant that has received 70% or more of a total of 2 liters of yFFP over a period not to exceed one month through intravenous or exchange methodologies, will be analyzed and reported.Results from all individuals will be correlated and compared, which is expected to allow the categorization of distinctive disease states to identify improvements derived from yFFP therapy that could apply to broad demographic and sex identified populations.The study is a 30-patient epigenetic, proteomic, laboratory and functional outcomes investigation designed to evaluate both the safety and efficacy of intravenous and exchange administered young Fresh Frozen Plasma at 25 ml/kg in no less than two doses over no more than thirty days.Each participant's starting condition will be identified by:- a TruDiagnostic test of the level of epigenetic methylation- a Seer test of blood proteomics -laboratory markers:- Leptin(an obesity signaling marker) -HbA1c(average blood sugar over 3 months)-Adiponectin(modulates glucose regulation and fatty acid catabolism-Troponin-(heart structural integrity) -NT-ProBNP(Heart function)-TNF alpha (assesses inflammatory responses in a large range of diseases)-Cystatin C (marker for kidney function)-IL6 a marker of aging, frailty, and disease -Functional Markers- standing balance testing-body composition (BMI)-waist circumference-Grip strengthJust prior to the commencement of their yFFP treatment(s), each participants starting condition will be identified by the above markers, with the same markers taken one month after the conclusion of their yFFP treatment(s).The primary outcome is the change, if any, in the epigenetic age or genome pattern as well as the laboratory and functional assessments of the yFFP treated patients.There will be multiple study sites participating throughout the State of Texas."Epigenetic clocks, based on measuring DNA methylation at specific sites, offer the most accurate measurement of biological and chronological age of any molecular-biology method known today. While causality for epigenetic clocks has not been established, interventions that prolong healthspan and lifespan also slow the biological clocks. Moreover, restoration of cell youth by partial reprogramming with a subset of Yamanaka factors resets the epigenetic age. However, these factors are unlikely to be the natural direct drivers of epigenetic clock reset. In mouse embryonic development, all factors are expressed until implantation (E4.5), and only a subset is expressed and only in specific cells past this point. However, epigenetic reset occurs predominantly in gastrulation (E5.5-E7.5) and in all cells. Thus, identifying the direct mechanism behind the epigenetic clock reset is of fundamental importance to exploring the causality of epigenetic clocks. By integrating single-cell expression data before, during and after the reset with extensive gene annotation, a list of candidate genes can be generated. These can be tested individually or in combinations in cell culture, for epigenetic reset without complete or partial reprogramming. "Age-related changes in the structures of the extracellular matrix.One of the most underestimated and important areas in life extension research is age-related change in the extracellular matrix. As early as 1942, Johan Bjorksten formulated his “The crosslinking theory of aging”. It is based on the assertion that aging is the result of the accumulation of intermolecular covalent bonds (crosslinks) in proteins, which increase very slowly. Examples of these proteins in the extracellular matrix are collagen and elastin.https://www.sciencedirect.com/science/article/abs/pii/0531556590900395Crosslinks are formed non-enzymatically (stochastically) and have a great influence on the physical properties of the extracellular matrix (ECM). In turn, the increased rigidity of the ECM will contribute to cell dysfunction through mechanosensory perception.https://www.nature.com/articles/s41586-019-1484-9In addition, advanced glycation end products can generate inflammatory responses through activation of the advanced glycation end products (RAGE) receptor, thus contributing to the formation of age-related chronic inflammation and age-related pathologies associated with inflammation.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661616/The extracellular matrix is mainly composed of collagen and elastin. Both proteins renew very slowly. The half-life of collagen in the skin, muscles and intestines is 74, 45 and 244 days, respectively. In human intervertebral discs, the half-life of collagen has been estimated to be at least 95 years.https://www.sciencedirect.com/science/article/pii/S002192582052841Xhttps://www.sciencedirect.com/science/article/abs/pii/030441659290095CElastin is another very highly stable molecule. Its functional non-renewable period in the human body is about 80 years and is comparable to the duration of human life. Therefore, any non-enzymatic modifications of elastin (such as glycation and carbamylation) will have serious consequences.https://www.jci.org/articles/view/115204The wide participation of sugars and their derivatives (such as methylglyoxal) in metabolic processes and the absence of absolutely reliable antiglycation mechanisms make glycation of long-lived proteins almost inevitable. Age-related decrease in elastin vessels in the ECM with simultaneous accumulation of collagen will contribute to the development of cardiovascular pathologies and accelerated aging. As expected, the decrease in elastin content imposes an upper limit on the functioning of the cardiovascular and pulmonary systems, which is estimated at about 100-120 years for humans.https://link.springer.com/article/10.1007/s10522-007-9122-6In general, according to the latest data, age-related changes in ECM proteins associated with non-enzymatic modifications are somehow associated with most other signs of aging. Often acting as a driver in the formation of mechanisms involved in aging and the development of age-related diseases. ECM aging forms ""vicious cycles"", positive feedback loops with other mechanisms of aging. such as inflammation and oxidative stress.https://www.sciencedirect.com/science/article/pii/S1568163720302324?via%3Dihub#bib1115In addition to non-enzymatic modifications of long-lived ECM proteins, its other structures also make a significant contribution to aging. Especially in the neurodegenerative processes characteristic of human aging. This applies to changes in the levels of hyaluronan, chondroitin sulfates, etc.https://www.nature.com/articles/s41380-021-01208-9?fbclid=IwAR0TET-rdro2p-Fm8Bdr9sP1tgYMLK665RpZWHlCYfNpItYsYTGIhZv92zsTherefore, it can be clearly stated that age-related changes in the ECM structure play a very large, but so far underestimated and poorly studied role in aging.""The substantial body of existing literature supports the leading role of the accumulation of stochastic damages of long-lived molecular structures in the aging process (https://pubmed.ncbi.nlm.nih.gov/32540391/).These damages (namely, Maillard reaction - non-enzymatic cross-linking of the nucleophilic amino group of the amino acids and carbonyl group of sugars, spontaneous protein-protein cross-linking (https://pubmed.ncbi.nlm.nih.gov/31794011/), racemization of aspartic acid residues) lead to incomplete digestion of proteins that compose the extracellular matrix, the nuclear pore, and other molecular structures with a slow or absent renewal.In the case of the extracellular matrix, probably, these indigested fragments accumulate over time and, due to a lack of specific enzymes, remain incorporated into the medium. Such accumulation fortifies the extracellular matrix and results in its increased stiffness and malicious nano topography (where needle-like fragments are poking out the collagen and elastin fibrils).The enzymatic activity of known collagenases against cross-linked collagen is limited (https://pubmed.ncbi.nlm.nih.gov/23957394/), and the efficacy of specific artificial enzymes or abzymes (https://diabetes.diabetesjournals.org/content/67/Supplement_1/1229-P) might be very low as they may not be able to penetrate the narrow space between cross-linked fiber due to their size.I may speculate that the development of ferromagnetic nano-tags exclusively targeting these indigested fragments might be a better research direction.Such nano-tags would stick to their targets (for example, to glucosepane-containing fragments), while the rest of them would shortly excrete from the body. Subsequently, an applied alternating magnetic field would induce local hyperthermia and destruction of fragments, the debris of which, along with used nano-tags, likely can be removed by macrophages.""Our proposal to treat age-related chronic degenerative diseases involves a cell-based gene therapy approach using genetically-matched differentiated embryonic stem cells (from somatic cell nuclear transfer; SCNT) for mitochondrial transfer therapy. See these publications from our group for details:https://pubmed.ncbi.nlm.nih.gov/35243258/https://pubmed.ncbi.nlm.nih.gov/23683578/""DNA methylation is currently the most promising molecular marker for monitoring aging and predicting life expectancy.However, the mechanisms underlying age-related DNA methylation changes remain mostly undiscovered, but scientists believe drug intervention could reverse epigenetic mechanisms that serve as regulators in diseases of aging. . Numerous studies over the past decade have strongly implicated epigenetic mechanisms in regulating the expression of genes involved in the regulation of several aging-related diseases, such as cancer and fibrotic disorders ((Ropero and Esteller 2007, Pang and Zhuang 2010), heart failure, neurodegenerative diseases such as Alzheimer's or Parkinson's, artheriosclerosis and other aging processes in different tissues (Hoeksema and de Winther 2016).(Lovrečić et al. 2013; Pagiatakis et al. 2019).Drugs that interfere with DNA methylation and histone deacetylation have been approved for clinical use by the Food and Drug Administration (FDA) (Jones et al. 2016).Epigenetics and therapeutic approachesExperiments in animal models of neurodegenerative disorders, diabetes or cataracts have confirmed the possible role of epigenetic drugs, as well as inhibitors of histone deacetylases and methyl donor compounds (Adwan and Zawia 2013).Drugs such as histone deacetylase (HDACi) inhibitors can cross the blood-brain barrier (BBB), thereby slowing the initiation and development of symptoms in animal models of neurodegenerative diseases (Coppedè 2014).Several structural classes of HDACi are under clinical trial for various diseases, including hydroxamic acids (vorinostat), cyclic peptides (depsipeptide or romidepsin), benzamides, and aliphatic acids (valproic acid (VA)) (Adwan et Zawia 2013).NutritionNutrition and food compounds, for example vitamin B, folate and methionine, are also known to affect epigenetic regulation and mechanisms.- Polyphenols, for example, highlight the unique cooperation between the genome and the environment, especially at physiological concentrations (Remely et al. 2015).They only induce a significant effect if they are consumed in large quantities or if the levels of methyl donors are limited; however, possible toxicity resulting from the oxidation of polyphenols may occur and care should be taken when consuming these compounds (Fang et al. 2007).- Folate supplementation, for example, can inhibit the adverse effects of aging, such as uracil malincorporation, DNA methylation, protein methylation, mitochondrial deletion, and critical gene expression, and it It has been observed to protect against colon cancer (Jang et al. 2005; Kim 2005).The impact of folate deficiency on DNA methylation could be target site and gene specific and it is proposed that the progression of genomic and site specific DNA methylation due to deficiency in folate may vary (Mierzecki et al. 2015).5-Azacytidine or azacitidine (AzaC) is one of the most studied DNMT (DNMTi) inhibitors and is primarily used in the management of myelodysplastic syndrome (MDS), in which it acts as a methyl donor eliminator in vitro to weaken the effect of gene silencing on methylation.A 67-year-old man with high-risk MDS showed hematologic improvement three months after the first course of AzaC treatment (Takaoka et al. 2014).ConclusionEpigenetic changes play an important role in the progression of aging.Therefore we propose to develop and improve techniques to understand epigenetic regulation in normal aging. Studies should be carried out to restore chromatin dynamics and therefore proper gene expression, which could provide new therapeutic strategies if applied early and in combination with other therapies addressing all aspects of aging.""The Uberkinder Program: Spawning globally superior humans through repurposing of adult longevity and sports interventions to foetal and pre-adult phases of life.There are demonstrated and speculative reasons that suggest application of adult health and longevity interventions in the earliest phases of life can dramatically improve physiological function, including longevity and cognitive capacity. Many such interventions are highly accessible and GRAS, but efficacy and dosing have not been established for uberkinder application. The list of possibly effective interventions is long, and center around management of biological aging rate, genetic expression, and resilience enhancement. Uberkinder interventions will enhance overall physiology, and be extremely important as 'preventive' enhancements of longevity and disease resistance and recovery. Perhaps more importantly (and more speculatively) in our time of self-generated existential threats, it is likely that the interventions will enhance cognitive capabilities; spanning intellect, emotions and perception.Existing non-human mammal data points to dramatic results with simple interventions. Importantly, most envisioned initial interventions are very inexpensive; economically accessible to the vast majority of humanity. Research can begin almost immediately upon gaining IRB approval, with volunteer mothers.Although uberkinder interventions will not save my generation, they will be essential in the evolution of a safer, healthier, more creative, happier future, and likely go down in future history as amoung the most effective humanitarian use of resources, ever. ""The Longevity PrizeHypothesis PrizeHuman Very Small Embryonic-Like (hVSEL) Stem Cells and LongevityVSEL stem cells were first identified in murine bone marrow in 2006 and subsequently in human bone marrow1. Our own research has clearly shown the presence of CXCR4+, SSEA4+, Oct 3/4+, CD34-, CD133-, CD45-, Lin- hVSEL pluripotent stem cells in Platelet Rich Plasma (PRP) derived from human peripheral blood2. Other authors also describe hVSEL stem cells being present in human peripheral blood3 whilst some suggest that the hVSEL stem cells found in umbilical cord blood are an ‘aberrant and inactive’ population of cells4. It is evident that there are conflicting reports in the scientific and medical literature about the existence, viability and biological activity of hVSEL stem cells and more research is required to clarify this situation. Nevertheless, it is also clear that PRP contains hVSEL stem cells and that PRP is therefore a readily available source of pluripotent stem cells which is currently ignored by most people working in stem cell technology. The biological and therapeutic action of PRP does not currently include discussions on the importance of hVSEL stem cells in PRP in the overall efficacy of PRP treatments. The standardisation of the production of PRP is required to ensure homogeneity, safety and efficacy and carefully controlled clinic trials are needed using standardised preparations and methods to fully understand the full potential of PRP treatment5.The persistence of hVSEL stem cells throughout life, from young to old, has been reported6 suggesting a potential homeostatic mechanism which maintains the hVSEL stem cell pool throughout life. This is supported by our unpublished observations of there being a constant availability of hVSEL stem cells in the PRP of patients who have undergone multiple PRP collections. The bone marrow is the likely source of the hVSEL stem cells to maintain the peripheral blood hVSEL stem cell pool. This is supported by the observation of the mobilisation of hVSEL stem cells into the peripheral blood following acute myocardial infarction7. In further support of this concept, hVSEL stem cells have been shown to be present in human bone marrow itself and also in human leukapheresis products8. This supports the hypothesis of migration of hVSEL stem cells from the bone marrow to the peripheral blood during physiological homeostasis and during pathological stimuli. Similar studies in vitro have suggested that hVSEL stem cells are the ‘original embryonic stem cell’ highlighting the critical importance of hVSEL stem cells in normal embryonic development and subsequent physiological homeostasis9. All stem cell types are subject to both intrinsic and extrinsic stress, including the aging process, during normal physiology and in pathological states. Such stress can have detrimental effects, especially on rapidly dividing stem cells10.HypothesisOur hypothesis is as follows:• hVSEL stem cells appear to be biologically quiescent in normal physiology.• The QiLaser (formerly known as the SONG modulated laser) can activate hVSEL stem cells in PRP thus re-activating their biological role as pluripotent stem cells.• Aging is a multifactorial process but the stem cell pool reduction, and the stem cell niche degeneration, are assumed to play a critical role. This results in a significant depletion of the stem cell pool over time, and we propose that this is a key factor in the aging process.• If hVSEL stem cells are quiescent in normal physiology (which at the moment seems to be a reasonable conclusion from the evidence available), then QiLaser activated hVSEL stem cells (with pluripotent biological activity restored) could make an important contribution to replacing the depleted total stem cell pools and also to the repair of the age damaged total stem cell niches following aging. • If hVSEL stem cells are quiescent in normal physiology, then they represent pluripotent stem cells which have not aged since the time of embryonic development when they arose from the Primordial Germ Cell (PGC). This means that QiLaser activated hVSEL stem cells could repopulate all of the stem cell compartments and niches with stem cells and stem cell niche cells which have undergone no aging at all.• Telomere length in hVSEL stem cells in vivo may be conserved if hVSEL stem cells are truly biologically inactive in vivo. If this proves to be correct, then this will also enhance the longevity producing potential of pluripotent QiLaser activated hVSEL stem cells which will produce somatic cells with relatively long telomeres.Our hypothesis will be tested by carrying out a placebo controlled preliminary clinical study to assess the effect of QiLaser activated hVSEL stem cells on C-G DNA methylation and telomere length. The study will take place at Qigenix which is the Medical Clinic of Dr Ovokaitys. DNA methylation and telomere length studies will be carried out by a third-party collaboration.Professor Peter Hollands PhD (Cantab), Chief Scientific Officer, QiGeneration, Carlsbad, California, USA Dr Todd Ovokaitys MD, Chief Executive Officer, QiGeneration, Carlsbad, California, USAReferences1. Ratajczak MZ, Zuba-Surma EK, Machalinski B, Kucia M. Bone-marrow-derived stem cells--our key to longevity? J Appl Genet. 2007; 48: 307-19. doi: 10.1007/BF03195227. PMID: 17998587.2. Hollands P, Aboyeji DR, Ovokaitys T. The action of modulated laser light on Human Very Small Embryonic-Like (hVSEL) stem cells in Platelet Rich Plasma (PRP). CellR4 2020; 8: e2990 DOI: 10.32113/cellr4_202012_29903. Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Ratajczak J, Kucia M. Very small embryonic-like stem cells: characterization, developmental origin, and biological significance. Exp Hematol. 2008; 36: 742-751. doi:10.1016/j.exphem.2008.03.0104. Danova-Alt R, Heider A, Egger D, Cross M, Alt R. Very small embryonic-like stem cells purified from umbilical cord blood lack stem cell characteristics. PLoS One. 2012; 7: e34899. doi:10.1371/journal.pone.00348995. Everts P, Onishi K, Jayaram P, Lana JF, Mautner K. Platelet-Rich Plasma: New Performance Understandings and Therapeutic Considerations in 2020. Int J Mol Sci. 2020; 21: 7794. doi:10.3390/ijms212077946. Sovalat H, Scrofani M, Eidenschenk A, Hénon P. Human Very Small Embryonic-Like Stem Cells Are Present in Normal Peripheral Blood of Young, Middle-Aged, and Aged Subjects. Stem Cells Int. 2016; 2016: 7651645. doi: 10.1155/2016/7651645. PMID: 26633977; PMCID: PMC4655065.7. Zuba-Surma EK, Kucia M, Dawn B, Guo Y, Ratajczak MZ, Bolli R. Bone marrow-derived pluripotent very small embryonic-like stem cells (VSELs) are mobilized after acute myocardial infarction. J Mol Cell Cardiol. 2008; 44: 865-873. doi: 10.1016/j.yjmcc.2008.02.279. PMID: 18430437; PMCID: PMC26923868. Sovalat H, Scrofani M, Eidenschenk A, Pasquet S, Rimelen V, Hénon P. Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells. Exp Hematol. 2011; 39: 495-505. doi: 10.1016/j.exphem.2011.01.003. PMID: 21238532.9. Virant-Klun I, Skerl P, Novakovic S, Vrtacnik-Bokal E, Smrkolj S. Similar Population of CD133+ and DDX4+ VSEL-Like Stem Cells Sorted from Human Embryonic Stem Cell, Ovarian, and Ovarian Cancer Ascites Cell Cultures: The Real Embryonic Stem Cells? Cells. 2019; 8: 706. doi: 10.3390/cells8070706. PMID: 31336813; PMCID: PMC6678667.10. Mierzejewska K, Heo J, Kang JW, Kang H, Ratajczak J, Ratajczak MZ, Kucia M, Shin DM. Genome-wide analysis of murine bone marrow derived very small embryonic-like stem cells reveals that mitogenic growth factor signaling pathways play a crucial role in the quiescence and ageing of these cells. Int J Mol Med. 2013; 32: 281-90. doi: 10.3892/ijmm.2013.1389. PMID: 23708325; PMCID: PMC3776718."Testing and using Unpatentable Natural Remedies will certainly be overlooked. I've written and tweeted this repeatedly. I would like to build a gym facility to record the efforts of people learning to breath better. I'm using cardio machines and full gym to increase muscularity and VO2max. I have a GS 1712 = Doctorate in Education, I beat cardio vascular disease in 7.5 years I got a 'clear/normal' heart reading. Nearly impossible but I'm gifted, autistic and with my Phd in Edu I know what I did to fix my heart, and the 'cardio' element of living longer. At 63 I'm doing 12 minutes of Bruce Protocol with bad lumbar, I'm injured - it's incredible. I used Mg, Turmeric, ALA, Potassium, Amino Acids, Collagen Peptides, B vitamins, now using Ashwaganda and Fenugreek for testosterone boost. I want to build a center in Southern Arizona [outside Tucson 30 min, 85602 zip] to partner with my local gym, we need to add a pool, sauna and not much else to begin. I want to charge $1200 year/$100 month to work with people on treadmill, stepper, versa climber, bikes and exercise 6 days/week to reach MAX VO2. We track progress and take vitals weekly. I'll write a report weekly. When I was a professor at the phone company they always said, 'don't ever bet against Anderson' - I've always had big ideas. I'm a world class troubleshooter - very open minded - the problem can be anything - the solution anything is possible. I also want to ADD CBD for telomer and Magnesium for telomer - testing. I need sauna because we rub on Mg in the sauna, pool and the rest is available at the gym next door. They will do my membership and I'll let them have swimming/sauna that they lack. Anyway CBD and Magnesium won't be tested by the BIGS. Thank you -Neo- Oren Anderson"Apolipoprotein A-1 Milano is a missense naturally occurring variant (rs28931573) of the apolipoprotein A1, discovered in a small population of Valerio Dagnoli of Limone Sul Garda, a small village in northern Italy, which causes a reduction in the risk of cardiovascular disease.Despite promising pre-clinical results, a clinical trial of a synthetic ApoA-1 Milano protein in patients with atherosclerosis failed to produce significant results in 2016. Since then, there have been no new clinical trials conducted.However, the prophylactic use of ApoA-1 Milano-based drugs hasn't been studied yet, probably due to the intravenous route of their administration.In light of that, the reported positive effects of ApoA-1 Milano full-length proteins produced by generically modified rice and delivered via oral administration (https://pubmed.ncbi.nlm.nih.gov/29907443/) raise the question of whether it is possible to construct vectors for inducible and constitutive ApoA-1 Milano expression in Lactobacillus.""RAPID REDUCTION OF EPIGENETIC AGEHypothesis:Given that viable stem cell numbers decline dramatically throughout life even while cellular replacement needs increase, the endogenous proliferation of stem cells should provide the most direct and comprehensive approach to restoring cellular maintenance and reducing epigenetic age.It is known that stem cells are quiescent due to high expression of uncoupling protein 2 (UCP2), which allow protons to dissipate through the inner mitochondrial membrane without producing ATP. It is also known that mitochondrial fusion will bias stem cell division to proliferation. Thus by simultaneously supplying a fusion supplement and a blocker for UCP2, quiescent stem cells should awaken and begin proliferating, subsequently replacing senescent cells as needed.A proposed method can be divided into three parts:A. Filling stem cell niches: Blocking proton leakage through UCP2 pores with C60* activates stem cells while mito fusion directs them to proliferation. B. Replacement of senescent cells: Mitochondrial fission/apoptosis using senolytics removes senescent cells, which are replaced by cells derived from proliferated stem cells of part A, at the direction of natural paracrine signaling. C. Maximizing epigenetic age reversal: Supplying a demethylase promoter during parts A and/or B further reduces aberrant methylation and epigenetic age.Proof of Concept:1. A 68 year old individual who had previously used a fullerene C60/oil supplement over several years showed a baseline epigenetic age of 0.5 years above his chronological age, suggesting that C60 used alone had no long-term epigenetic benefit.2. After treatments employing C60 according to Hypothesis parts A and B, his epigenetic age dropped 12 years over a period of about 2 years. And after treatment with parts A-C, further declines in epigenetic age were noted, reaching a maximum decline of 28 years. His appearance and general sense of physical well-being improved in accordance with the measured epigenetic age reduction.*The fullerene C60 dissolved in oil is proposed herein as a UCP2 blocker. The use of C60 to extend rodent lifespans was the subject of three papers. The first (in 2012, PMID 22498298) showed that feeding rats C60 in oil increased rat lifespans by 90% over controls, and attributed the increase to the antioxidant properties of C60. The next two (in 2021, PMID 33123847 and PMID 33849306) attempted to replicate this result in mice, but found no increase in lifespan. The discrepancy can be resolved by postulating a different MOA for C60 — UCP2 blocking rather than ROS quenching — and a different feeding regime for the three experiments. Although none of the papers precisely described how their test animals were fed, it is likely the research group of the first paper fasted their rats overnight (as they did in a previous C60 toxicology study), while research groups of the next two papers likely did not, as there was no suggestion to do so in the first paper. Rodents have a metabolic rate about six times that of humans, so a state of fasting-induced mitochondrial fusion would have been easily achieved in the first study, but no fusion would have occurred in next two. As noted in the proof of concept trial, C60 sans fusion produced no epigenetic reversal in the human subject, and likewise no increase in rodent lifespan.""RESTORATION OF MITOCHONDRIAL FUNCTIONHypothesis:Methylation of mitochondrial DNA loops (mtDNA) can be expected to produce an energetic deficiency that is difficult to treat, as normal cellular QC does not remove methylation, while methyltransferase insures its persistence. Herein it is proposed that cellular QC can be tricked into removing methylated and sub-functional mtDNA by briefly forcing ATP output to zero.Background: Mitochondrial quality control involves PINK1/Parkin labeling of fissioned mitochondria with zero surface potential (ΔΨm). Labeled mitochondria are then removed by recycling in lysosomes (mitophagy). Mitochondria with methylated mtDNA may have lower than normal ATP output due to deficient enzyme synthesis, but are not removed as surface potential doesn’t go to zero. Such sub-functional mtDNA may have a survival advantage over unmethylated mtDNA due to fewer ROS and a lower mutation rate, and thus may come to dominate the cell.Proposed treatment method: Fissioned mitochondria having a single loop of sub-functional mtDNA will also have a smaller reserve of enzymes. If enzyme synthesis stops during biogenesis, they may run out faster than normally functioning mitochondria. ATP production will briefly cease and surface potential will go to zero. PINK1/Parkin will then label them for mitophagy. Contracting and expanding the cellular mtDNA population by cycling fission/biogenesis with fusion/biogenesis should remove sub-functional mtDNA in an iterative fashion, in which the mtDNA removed per cycle is limited by lysosomal capacity. Adding a demethylase promoter should accelerate the process.Oral supplements for promoting fission, fusion, biogenesis and demethylase are readily available.Proof of Concept:The cyclic method proposed above was trialed by a subject who suffered persistent mitochondrial damage from statin use years before. Fission/biogenesis/demethylase was alternated with fusion/biogenesis/demethylase on a daily basis (using a “reps to failure” exercise to monitor progress). Reps to failure during fission were initially 43 percent lower than during fusion*, but as cycles were completed, fission reps rose and ultimately matched fusion reps after three weeks, suggesting that sub-functional mtDNA had been removed.*Fusion reps were expected to be initially higher than fission reps due to enzyme sharing.""Protein glycation is a non-enzymatic post translational modification in which sugars or reactive carbonyls react with amino acids to form Advanced Glycation End products (AGEs). AGEs constitute a structurally diverse group of molecules linked to malignant ageing and age-related diseases. The human body can eliminate only early glycation intermediates, but not AGEs. As a result, AGEs accumulate in our body as we age and exert their pathophysiological effects via interaction with cell-surface receptors and by altering the structure and function of proteins. AGEs are ligands for the receptor for AGEs (RAGE), a multi-ligand receptor associated with oxidative stress, cellular damage and chronic inflammation. Despite implications of AGEs in age-related pathologies, mechanistic studies are limited by the lack of precision molecular tools to reverse AGE modifications. In this project, we aim to expand the ageing research toolbox by engineering enzymes with the ‘new-to-nature’ capacity to catalyse AGE reversal. Enzymes are perfectly suited for this application, as they can enhance challenging transformations with exacting selectivity and deliver these reactions in cells. Developing AGE-repairing biocatalysts remains a significant challenge due to the structural diversity and site-specificity of AGE formation. Although there are currently no naturally occurring enzymes known to repair AGEs as their native function, protein engineering can expand their catalytic repertoire and tailor them to biotechnological and clinical applications. We will create precision molecular tools for ageing research by addressing the following aims: (1) Biocatalytic reversal of AGEs. We will develop novel AGE-repairing enzymes by repurposing promiscuous biocatalysts and extending their substrate scope by rational design.(2) Engineering of AGE-reversing biocatalysts. Using a hybrid experimental and computational approach, based on machine learning-guided directed evolution and droplet microfluidics, we will dramatically improve the activity, stability and biocompatibility of the AGE-repairing enzymes to meet high biotechnological standards. Additionally, structural and functional characterisation of enzyme variants will be performed in order to understand the basis of improvements along the evolutionary trajectory.(3) Cellular impact of biocatalytic AGE reversal. Last, we will extend the functionality of the enzymes to AGE reversal in cells, and will determine the impact of AGE reversal on RAGE signalling and inflammation.The availability of precision molecular tools with the ability to repair AGEs is envisioned to revolutionise the fields of ageing and glycation damage, by allowing researchers to uncover causal relationships that drive ageing and diseases via accumulation of AGEs. Finally, the superior biocatalysts developed in the study could pave the way towards enzyme therapeutics for the treatment of AGEs and related pathologies.""Hepatocyte growth factor for aging and age-related diseasesPersistent low level injury is the underlying cause of modern society’s most prevalent and costly diseases (1). Pulmonary disease, liver disease, kidney disease, diabetes, intestinal disorders, neurological disease, atherosclerosis, autoimmune disease, even aging itself, all have the common denominator of persistent low level injury. While there are many routes to each individual malady (obesity, chemicals, autoimmunity) the response to ongoing low level organ damage is very consistent. Non-epithelial cells are activated and proliferate to repair and limit the damage. Cells of the immune system, macrophages, and myofibroblasts are all recruited to the site. In the face of ongoing damage, the repair process never completes and the functional cells of the organ are slowly replaced by extracellular matrix resulting in fibrosis. There is an urgent need for a mechanism to limit inflammation, stimulate regeneration and reverse existing fibrosis.The response to tissue damage is complex but two cytokines play a central role in initiating repair and then terminating the response. TGFβ is released from the surface of cells in response to injury. This initiates the repair process, acting through a series of TGFβ family receptors, mobilizing events to reestablish the epithelial barriers(2). HGF and its receptor, c-met, are subsequently induced, stimulating division and differentiation of new epithelial cells and eventually shutting down the TGFβ response(3). In the face of ongoing damage, TGFβ signaling becomes dominant, suppressing transcription and activation of HGF and leading to replacement of functional tissue with fibrous extracellular matrix leading to fibrosis and aging. A major transcriptional target for TGFβ is plasminogen activator inhibitor (PAI1) (4). PAI1 inhibits both urokinase type plasminogen activator (uPA) and tissue plasminogen activator (TPA). Both uPA and TPA are able to convert the HGF precursor, proHGF, into the two chain active HGF (aHGF) (5, 6). PAI1 inhibits activation of proHGF. Defective activation of proHGF due to high PAI1 has been demonstrated in bleomycin induced pulmonary fibrosis, kidney and liver fibrosis, and muscle regeneration (7–11).Generalizing the idea that poor HGF synthesis and activation is detrimental to the organism, HGF may have been overlooked in relation to aging itself. HGF is implicated in three interesting age-related examples. The most startling is the finding of an Old Order Amish cohort in Berne, Indiana, who carry a defect in SERPINE1, the gene coding for PAI1 (12). Homozygotes have a mild hemophilia-like disorder due to overactive uPA and TPA. The heterozygotes have about half the normal level of PAI1, a reduced incidence of cardiovascular disease, a lower circulating insulin level and live, on average, about ten years longer than their normal matched controls. HGF is known to reduce cardiovascular disease and plays a role in insulin and glucose homeostasis (3, 13). It is possible, but unproven, that the reduction in PAI1 produces a somewhat higher level of HGF activation, resulting in the improved phenotype.A second instance for a possible role for HGF in aging is found in Werner syndrome (14). This is a late onset progeria caused by a mutation in the WRN gene, coding for a DNA helicase. Tu, et al, used CRISPR to correct the gene defect in induced pluripotent stem cells derived from Werner syndrome patients (14). When these cells were differentiated into mesenchymal stem cells, they were found to produce dramatically higher levels of HGF, resulting in improved angiogenesis and wound healing. The authors suggest that at least some of the aging phenotype in Werner syndrome is due to poor synthesis of HGF and thus poor stromal/epithelial communication. Werner syndrome patients most commonly succumb to cardiovascular disease (15). HGF is known to reduce cardiovascular disease (3). The third example comes from a recent paper demonstrating high levels of PAI1 synthesis in fibroblasts derived from patients with Hutchinson-Gilford progeria (16). The authors showed that these fibroblasts had a myofibroblast phenotype with high synthesis of extracellular matrix proteins, defective mitochondria and poor cell cycle progression. Inhibition of PAI1 with a small molecule inhibitor restored a normal phenotype, reducing synthesis of fibronectin and collagen, restoring the mitochondrial structure and stimulating DNA synthesis. The authors suggest that PAI1 inhibition could relieve some of the symptoms of Hutchinson Gilford progeria.Hypothesis: Aging and age related diseases are caused by poor synthesis and activation of HGF subsequent to elevated TGFβ signaling. HGF, or an HGF mimetic, could cure age related diseases, increase longevity and relieve the symptoms of both Werner syndrome and Hutchinson Gilford progeria. Experiment – A simple experiment to test this idea could be done using the same Hutchinson Gilford progeria derived fibroblasts described by Catarinella, et al (16). These cells and their controls are available from the Progeria Research Foundation and the Coriell Institute. Fibroblasts and myofibroblasts are a main source of HGF. As determined by SDS gel electrophoresis and immunoblotting of serum free supernate from the cultures, normal fibroblasts will have primarily active HGF while the progeria derived fibroblasts will have primarily proHGF. Inhibition of PAI1 with TM5441, as done by Catarinella, et al. will demonstrate increased concentrations of active HGF. Addition of an antibody to HGF or to c-met will block the effect of TM5441 on the cells. Finally, addition of recombinant human HGF to the cells will have the same effect as TM5441. References1. Furman, D., Campisi, J., Verdin, E., Carrera-Bastos, P., Targ, S., Franceschi, C., Ferrucci, L., Gilroy, D. W., Fasano, A., Miller, G. W., Miller, A. H., Mantovani, A., Weyand, C. M., Barzilai, N., Goronzy, J. J., Rando, T. A., Effros, R. B., Lucia, A., Kleinstreuer, N., and Slavich, G. M. (2019) Chronic inflammation in the etiology of disease across the life span. Nat Med. 25, 1822–18322. Katsuno, Y., and Derynck, R. (2021) Epithelial plasticity, epithelial-mesenchymal transition, and the TGF-β family. Dev Cell. 56, 726–7463. Gallo, S., Sala, V., Gatti, S., and Crepaldi, T. (2015) Cellular and molecular mechanisms of HGF/Met in the cardiovascular system. Clinical Science. 129, 1173–11934. Samarakoon, R., Overstreet, J. M., and Higgins, P. J. (2013) TGF-β signaling in tissue fibrosis: Redox controls, target genes and therapeutic opportunities. Cell Signal. 25, 264–2685. Mars, W. M., Jo, M., and Gonias, S. L. (2005) Activation of hepatocyte growth factor by urokinase-type plasminogen activator is ionic strength-dependent. Biochem J. 390, 311–3156. Naldini, L., Tamagnone, L., Vigna, E., Sachs, M., Hartmann, G., Birchmeier, W., Daikuhara, Y., Tsubouchi, H., Blasi, F., and Comoglio, P. M. (1992) Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. Embo J. 11, 4825–48337. Eitzman, D. T., McCoy, R. D., Zheng, X., Fay, W. P., Shen, T., Ginsburg, D., and Simon, R. H. (1996) Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest. 97, 232–2378. Hattori, N., Mizuno, S., Yoshida, Y., Chin, K., Mishima, M., Sisson, T. H., Simon, R. H., Nakamura, T., and Miyake, M. (2004) The Plasminogen Activation System Reduces Fibrosis in the Lung by a Hepatocyte Growth Factor-Dependent Mechanism. Am J Pathology. 164, 1091–10989. Dworkin, L. D., Gong, R., Tolbert, E., Centracchio, J., Yano, N., Zanabli, A. R., Esparza, A., and Rifai, A. (2004) Hepatocyte growth factor ameliorates progression of interstitial fibrosis in rats with established renal injury. Kidney Int. 65, 409–41910. Wang, H., Zhang, Y., and Heuckeroth, R. O. (2007) PAI‐1 deficiency reduces liver fibrosis after bile duct ligation in mice through activation of tPA. Febs Lett. 581, 3098–310411. Sisson, T. H., Nguyen, M.-H., Yu, B., Novak, M. L., Simon, R. H., and Koh, T. J. (2009) Urokinase-type plasminogen activator increases hepatocyte growth factor activity required for skeletal muscle regeneration. Blood. 114, 5052–506112. Khan, S. S., Shah, S. J., Klyachko, E., Baldridge, A. S., Eren, M., Place, A. T., Aviv, A., Puterman, E., Lloyd-Jones, D. M., Heiman, M., Miyata, T., Gupta, S., Shapiro, A. D., and Vaughan, D. E. (2017) A null mutation in SERPINE1 protects against biological aging in humans. Sci Adv. 3, eaao161713. Fafalios, A., Ma, J., Tan, X., Stoops, J., Luo, J., DeFrances, M. C., and Zarnegar, R. (2011) A hepatocyte growth factor receptor (Met)−insulin receptor hybrid governs hepatic glucose metabolism. Nat Med. 17, 1577–158414. Tu, J., Wan, C., Zhang, F., Cao, L., Law, P. W. N., Tian, Y., Lu, G., Rennert, O. M., Chan, W. Y., and Cheung, H. H. (2020) Genetic correction of Werner syndrome gene reveals impaired pro‐angiogenic function and HGF insufficiency in mesenchymal stem cells. Aging Cell. 19, 13–2015. Kato, H., and Maezawa, Y. (2022) Atherosclerosis and Cardiovascular Diseases in Progeroid Syndromes. J Atheroscler Thromb. 29, 439–44716. Catarinella, G., Nicoletti, C., Bracaglia, A., Procopio, P., Salvatori, I., Taggi, M., Valle, C., Ferri, A., Canipari, R., Puri, P. L., and Latella, L. (2022) SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson–Gilford progeria syndrome. Cell Death Dis. 13, 737""Isocaloric twice-a-day-feeding improves brain agingScientific backgroundAs we age, our brain changes. Cognitive capabilities and motor control are detrimentally affected. At a microscopic level, our brain accumulates the age-pigment lipofuscin, leading to neuronal loss, and reactive gliosis with an accumulation of astrocytes and microglia1. Western diet, characterized by a high intake of saturated fats and refined carbohydrates, induces overweight and metabolic changes, which exacerbate these aging features. Diet, neuronal loss, and gliosis have been linked to most of the known neurodegenerative diseases. Caloric restriction (CR) extends health and lifespan in multiple organisms2. Despite its remarkable benefits, humans adhere poorly to CR. Other strategies try to emulate CR, for instance, intermittent fasting or the isocaloric twice-a-day (ITAD) feeding, which benefits are mediated by autophagy stimulation. From those, ITAD-fed model mice consume the same food amount as ad libitum controls but at two short windows early and late in the diurnal cycle. This has proven metabolic and cognitive benefits. This is a strategy easier to implement in humans than CR or intermittent fasting. However, ITAD-fed benefits in brain aging have not been explored. We hypothesize that ITAD feeding ameliorates features of brain aging, particularly, those produced by western diets. We aim to test the improvement of aging markers, particularly neurodegenerative ones, using the ITAD strategy in high-fat diet (HFD) fed mice, that emulate the western diet.Preliminary workWe have conducted a pilot study in a reduced group of mice to test that ITAD-feeding benefits metabolism, and behavioral and motor skills. 6-month-old C57BL6 mice were fed for 12 months with HFD, half with the ITAD-feeding strategy and the other half ad libitum. ITAD-feeding strategy feed mice with the same calories as ad libitum but in just two meals (two time periods) separated by a long starvation period, 10-12 hours. ITAD-feeding reduces mice weight and triglycerides, insulin, and leptin in serum samples. Thus, our model improves whole animal metabolism, as it has been described before3. Regarding behavior, we have found a motor improvement, analyzed by rotarod and grip strength test, less anxiety and more wiliness to explore, by open field, and a reduction of hindlimb clasping. All these tests showed a clear improvement of key aging behavioral features thanks to the ITAD diet.Aims and work programAim 1: Test ITAD-feeding benefits with macroscopic analysis, including behavioral and motor skills.6-month-old C57BL6 mice are fed for 12 months with HFD, half with the ITAD-feeding strategy and the other half ad libitum. Mice in a regular chow diet are kept in control, with ITAD and ad libitum diet. We are monitoring feed intake and weight. After the 12 months, we will conduct behavior analysis, e.g., open field, rotarod; and we analyze metabolic markers in serum, e.g., insulin, leptin to check that the ITAD-feeding is working. We are performing a life expectancy test, so we will keep mice fed with all the strategies previously described until they die. Thus, we will clarify if the ITAD-feeding strategy prolongs life spam.Aim 2: Decipher ITAD-feeding molecular mechanism to improve brain health.Mice in ITAD-feeding, ad libitum, and controls will be divided into two groups: histological and for biochemical analysis. We will collect different tissues (brain, liver, muscle, adipose tissue) to evaluate the impact of ITAD-feeding benefits on them. Histological analysis: to evaluate neurodegeneration we will use neuronal (MAP2, Tuj1) and apoptotic markers (TUNEL, Caspase3). To understand the causes of cell death, we will analyze lipofuscin accumulation, gliosis (GFAP and Iba1), microglial activation markers (CD86, CD206), and other disease markers (amyloid-β peptides).Biochemical analysis: CR and other similar feeding strategies rely on autophagy activation to exert their benefits. Autophagy is the cellular clearing and recycling program that degrades cytoplasmic content in lysosomes. Autophagy decline is well known during aging, which contributes to waste accumulation. To analyze whether the ITAD-feeding system stimulates autophagy in the brain we will perform flux analysis in different brain areas (cortex, hippocampus, and hypothalamus).Impaired autophagy contributes to neuroinflammation through the accumulation of damaged structures (Aβ, P-Tau, and mitochondria) and the activation of microglia. Then, we will analyze inflammation (NF-Kβ, TNF-α, interleukins, by western and ELISA) and microglia activation markers (CD86, CD206). Also, we will address how brain improvement benefits the whole of mice's well-being. Thus, specific pathologic, inflammatory, and autophagic markers will be evaluated in the other tissues (liver, adipose tissue…). Our design provides the proof-of-concept needed to establish ITAD-feeding as a plausible therapy to improve brain aging, particularly with western diets. As such, it would be of great relevance when ITAD-feeding is implanted in a future clinical trial.Impact, including interdisciplinarity The overall burden of disease is assessed using the disability-adjusted life year (DALY), which expresses the years of healthy life lost due to disability (YLDs) or lived in less than full health5. Age-related diseases are a very important factor causing DALYs, particularly neurological diseases, including dementias, which are affecting over 7 million people in Europe, costing approximately €130 billion per annum6. Caloric restriction has been proven effective to reduce the hallmarks of aging in mice and humans, including improving memory loss7. However, human adherence to these diets is very complicated. In our project, we will evaluate the benefits of a change of habits to brain aging. We maintain the same calories but are divided into just 2 meals. The intervention we are proposing to reduce aging brain symptoms won’t increase the cost to the health system, as it will not require any additional treatment. Therefore, this project has the potential to significantly reduce the impact of neurodegenerative diseases and improve the quality of life, particularly in elderly people, also reducing the neurodegenerative disease burden on the health system.Timeline of activitiesThe mice are already under the ITAD-feeding strategy, so this project will be finished in 2023 year, as proposed in the Gantt chart. We will finish the first aim during the 3 first months of 2023 and the second aim will be performed during the following 8 months. The manuscript is expected to be sent at the end of November 2023.""Mitochondrial dysfunction is a central hallmark of aging and many of the current interventions to extend lifespan and health span target the mitochondrial metabolism. For example, dietary interventions, genetic interventions (e.g., AMPK) and drugs (e.g., metformin and rapamycin) extends lifespan by, at least partially, targeting mitochondria. Generally, many of the interventions aim, quite surprisingly, towards a reduction of mitochondrial activity. A prominent example is the induction of mitochondrial unfolded protein response (mtUPR) during development, where reducing components of oxidative phosphorylation results in increased lifespan. However, it is noteworthy that such intervention reduces the physical activity of the animal in question and likely impairs the ability of the animal to respond to other type of stress. Therefore it is questionable whether such approaches could be translated into human longevity therapy.Here we hypothesize a novel and radical approach termed external energy replacement. What does it mean?It is established that plants and bacteria can capture the energy of light and translate it into energy in their cells, while animals must consume other organisms as food, in order to generate energy. Thus conventionally, evolution made a clear distinction between autotroph and heterotroph organisms. However, the idea of bringing proteins from evolutionarily distant organisms such as bacteria into eukaryotes can result in a leap in molecular biology. One well-known example is the bacterial CRISPR/Cas9 system, which was adapted to edit eukaryotic target genes. Another very recent example is introducing bacterial/fungal light-sensitive protein that generates proton gradients across in eukaryote mitochondria to generate chemical energy, which is a massive leap in molecular biology.In this hypothesis, we propose a major breakthrough in the field of metabolic aging. Specifically, we propose to test if mtON, an engineered light sensitive proton pump, will act as metabolic rescue for mammalian age-related mitochondrial dysfunction in the eye and the skin, where light is easily accessible. When activated by light, mtON pumps protons across the inner membrane of the mitochondria, thus contributing to the proton gradient that is essential to generate ATP. This genetic intervention practically enables a heterotroph to harness the energy of light to generate ATP in the absence of oxidative phosphorylation.We recently were the first and only scientists to show that the usage of mtON technology can increase lifespan in worms. Here, we hypothesize that using mtON technology in mammals will attenuate aging in the eye and skin. We hypothesize that using mtON will have several benefits. First, in an advanced form of aging, where mitochondria are dysfunctional and cannot generate enough ATP, that mtON treatment coupled with light can enable the aged eye and skin cells to generate youthful levels of ATP. Essentially, the light energy transformation will compensate for lack of ATP. Secondly, mtON technology may reduce the accumulation of metabolic associated damage. For example, we hypothesize that as ATP is now being partially generated by light, the cell would adapt and slow down its upstream metabolic (e.g., TCA cycle, glycolysis). Therefore, less biochemical reactions would take place, thus reducing the production of cellular waste, such as advanced glycation end-products, which contribute to the progression of aging. Also, as oxidative phosphorylation is less required, we hypothesize a reduction in the levels of reactive oxygen species (ROS), which may also benefit to generate less cellular damage during aging. Thirdly, it is possible that mtON would enable people to eat less while maintaining physiological ATP levels. As such, people may enjoy the benefits caloric restriction, which is believe to improved health span, while avoiding jeopardizing dysregulated ATP levels. This would be even more important once mtON technology would be upgraded to be used in inner organs beyond the skin and the eye.Taken together, our hypothesis of using light as an external energy replacement is quite revolutionary and show great promise of success in attenuating human aging in a novel manner.""Background/Motivation of StudyA relevant term in this context is cellular energy homeostasis and by extension, energy metabolism. While different theories of aging consider different variables and factors – this is one that is recurrently observed in most cases(K. Jin & Rose, 1988). Complex biological constructs are open thermodynamic systems that are subjected to constant exchange of energy components with the environment(Flatt & Partridge, 2018; Kowald et al., 2020; López-Otín et al., 2013; Maklakov & Immler, 2016). Cellular homeostasis and survival in general are threatened with age due to the loss of capacity of organisms to maintain a balance in generation, usage and storage of biochemical energy. The energy component is seminal to this understanding because it links the aging process to a seemingly distant function – sex and reproduction. While aging is generally considered for somatic tissue maintenance, reproduction is another fundamental process that shares the same variables and objectives as aging – survival, proliferation and maintenance(da Costa et al., 2016; Gems, 2014; K. Jin & Rose, 1988; Kowald et al., 2020). Organisms need to allot a proportion of their system energies to reproduction, that impacts other metabolic processes – particularly fitness and aging. Therefore, in a dimorphic system where reproduction is handled heterogeneously, the aging process should be expected to be perturbed divergently as well. This notion is supported by a large body of existing literature where a significant bias is observed in mammals where female have longer maximum lifespans as well as average lifespans than males(Benayoun et al., 2020; Bonduriansky et al., n.d.; Garratt, 2019; Gems, 2014; Sampathkumar et al., 2020; Tower, 2006). However, this is not true in all cases with clusters of contradictory data spanning across different species and experimental conditions. Moreover, the underlying molecular processes behind aging are just as confounding as the sexual dimorphism behind them. Apart from the last few years of research, very little effort has been previously invested in studying the sexual disparities in senescence – further extending the gap of knowledge required to be addressed in the molecular and cellular levels. Over decades of empirical research, the biological aging process in cellular and molecular terms have been illustrated through various inter-connected components known as the hallmarks of aging(López-Otín et al., 2013). While the hallmarks of aging themselves are often considered as driving forces in aging, there are certain molecular pathways and metabolic networks that act as intermediate regulators of these endpoints(Childs et al., 2015; Fahy et al., 2019; Pagiatakis et al., 2019; Ribarič, 2012; Serrano, 2017; Soto-Gamez et al., 2019). Among these, a clear energy metabolism associated pattern is observed – constructing a complex network of genes and gene products across carbohydrate and fat metabolism, oxidation, inflammation, stress response, immune-senescence, nutrient sensing, starvation and autophagy, apoptosis, cellular proliferation and epigenetic regulation. Among these gene products, some major regulators of energy homeostasis and aging have been broadly investigated as therapeutic targets. 5' AMP-activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR), insulin and insulin-like growth factor-1 (IGF-1), Sirtuin family proteins (SIRT1-SIRT7), forkhead box transcription factors (FOXOs) and different kinases construct an evolved survival-proliferation regulating network utilizing other aging associated processes(Arpón et al., 2019; Bonkowski & Sinclair, 2016; Imai & Guarente, 2014; J. Kim & Guan, 2019; Lewinska et al., 2020; Michan & Sinclair, 2007; C. Zhang et al., 2020). While literature provides substantial evidence on sexual bias in the expression and impact of these regulators, the nature and direction of the bias is often contradictory(Benayoun et al., 2020; de Arellano et al., 2019; Fagan et al., 2020; Garratt, 2019; McCartney et al., 2019; Regan et al., 2016; Sampathkumar et al., 2020; Tower, 2006; Tower et al., 2020).The current project proposal takes these different aspects and notions into account in order to re-evaluate the current concept of a sexually homogenous molecular biological aging process. In order to fully understand the aging process, we must trace back the evolutionary mechanisms in aging and sexual dimorphism as well as the cellular, molecular, metabolic and epigenetic elements contributing to this divergence. Genetic analysis supports the notion of sexually divergent expression patterns of aging associated alleles and transgenes. These examples hint at a female driven selection mechanism that influences males to inherit genes and by extension, traits that do not necessarily favor them in longevity and fitness (""Mother's Curse"" in mitochondrial functionality, X chromosome optimization for females, i.e. different forms of sexually antagonistic pleiotropy). Some genes and by extension, traits, asymmetrically evolved in this sex-specific manner in turn, evidently aid a selected sex in life extension. Other avenues of sexual dimorphism in longevity genes extend to nutrient sensing and fat metabolism in aging, adaptive homeostasis and hormesis, endocrine regulation of senescence and gonadectomy, and epigenetic regulation/markers(Salekeen et al., 2021). We have attempted to derive an evidence-based hypothesis on the sexually dimorphic relation of a key regulatory protein families relevant to human longevity.HypothesisIn order to develop a model that attempts to explain an integral aspect of the sexually dimorphic nature of the biological aging process, we may define some key considerations to be made as follows: a) Should there be a sexual divergence of a particular outcome of inheritance, it must be either divergent from the beginning or the trait must be introduced to a population as a common trait until it is sexually and naturally selected to be sex-limited due to simultaneously advantageous and detrimental effects in a sex-specific manner.b) Should there be an evolutionary event of sexual divergence in the core processes of biological aging, the role of at least one key survival-proliferation switch element must also be sexually divergent.c) Should there be a divergence event in the aging associated machinery, the likelihood of it occurring in mitochondrial function and energy metabolism should be significantly higher.d) One key junction of sexual dimorphism and biological aging mechanisms is the endocrine regulation of fitness and longevity. Therefore, should there be an underlying mechanism to this divergence, it should be linked to sexual endocrine dimorphisms and provide significant advantages to one sex while causing deleterious effects to the other.e) Should there be a dimorphism and decoupling event, it should accommodate the evidence of post-pubescent/post-reproductive onset of benefits/detrimental effects in different sexes.f) Should there be an element driving sexual dimorphism, it should be epigenetically responsive to stimulants, and be reproducibly tracked.g) Finally, should there be an evolutionary divergence event in the aging metabolic circuit; it should be conserved through elements present in a wide range of genetically distant organisms.Based on the evidence from previous investigations and these key considerations, we propose a model where key survival-proliferation protein families which are highly conserved and work as response element to epigenetic stimuli to be sexually dimorphic in their roles. While the presence of these genes/proteins are sex-nonspecifically beneficial to longevity and fitness, there is a clear disparity on how much the sexes depend on these proteins to maintain homeostasis and function. Therefore, the hypothetical model states that Survival-Proliferation switch proteins provide survival benefits in a sex-nonspecific manner but the dependence on these proteins in driving metabolic networks after reaching reproductive maturity is evolutionarily decoupled from male/female longevity.Proposed Testing Model and ImplicationsProviding a testing model for a hypothesis like this is complicated – less because of complex features and more because of the sheer lifespans of test subjects or models. Using Yeast, Drosophila and C. elegans are particularly of interest due to their short lifespans. Regulatory protein homolog knockout models or inhibitors can be used and different stress conditions can easily be simulated because of their short lifespans(Taormina et al., 2019). On the other hand, their evolutionary distance from mammals raises issues on the applicability of the findings in more complex biological systems such as humans or rodents. Contrastingly, testing in human subjects require multiple generations of data to fully interpret and establish the evolutionary decoupling and its translational applications therewith. Nevertheless, to simplify the hypothesis testing, we may consider a simple model using murine variants.One proposed testing model would be to simplify the hypothesis in a two-phase testing process. The first phase would be to disprove that selected regulatory switch proteins (eg: SIRTs, FOXOs, IGF, PARPs etc.) are sex-nonspecific in providing longevity benefits in model organisms under different stress conditions, respective of reproductive stage. A simple approach to this would be to take different aged groups of male and female model organisms with knockout variants and subject them to stress conditions and observe whether the benefits are sex-limited. In this context, not only lifespans of subjects should be considered – but also other biohorological markers of longevity including inflammation and senescence associated biomarkers. If the results are found to benefit both sexes but only cause detrimental effects on one after reproductive maturity, we can infer that the model is fundamentally correct.The subsequent stage would be to establish which aging associated molecular pathways do the screened proteins/genes perturb in a sex-specific manner. Notably, decoupling from certain regulatory genes also imply the presence of a possible bypass mechanism that assists in sustaining a dimorphic system. Identification of such bypass mechanisms require complex systems-level approaches to identify hidden links or molecular associations among different protein pathways. While designing such models can be complicated, similar studies exploring the evolutionary, sexual and genetic prevalence of such associations and constructing metabolic networks for AMPK, FOXOs and SIRTs as well as aging-associated disease networks have been undertaken before(Mostafavi et al., 2018; Sharma et al., 2012; Soltow et al., 2010; Webb et al., 2016). Secondly, another consideration to be made is that not all tissue systems have the same epigenetic profile with regards to survival and proliferation(Basisty et al., 2020; Chen et al., 2016; Galkin et al., 2020; García-Giménez et al., 2017). Hence, the expression and regulation of this decoupling mechanism may not be pan-tissue. In order to address that possibility, samples from different tissue systems and organs need to be verified before the hypothesis can be proved or disproved. Based on available technology, high throughput analysis of single cell transcriptomics and metabolomics can be applicable in determining such tissue-specific biases. This would be refined according to a trade-off between the parallel development in sequencing, multi-omics, and imaging technologies Nevertheless, these are major factors to be considered when designing studies to test different aspects of this hypothesis. Detailed and integrated multi-disciplinary approaches exceeding cell biological techniques need to be developed in future undertakings to fully explore how and to what extent these sexual divergence may exist in model organisms.In conclusion, a clear evidence of one statement can be inferred from the available literature, and that is the lack of necessary data in the context of sexual dimorphism in fundamentally understanding the biological aging process. While this area has only been interrogated in recent years, the available evidence already hints at significant prevalence of dimorphic mechanisms involved in human aging. Moreover, the roles of primary aging associated regulatory factors responsible for oxidation, inflammation, immune modulation, tissue homeostasis and proliferation deem it imperative to establish the sexually dimorphic roles and dependencies associated to develop effective, safe and sustainable anti-aging therapeutics and to revisit a majorly underexplored area in the field.ReferencesArpón, A., Milagro, F. I., Santos, J. L., García-Granero, M., Riezu-Boj, J. I., & Martínez, J. A. (2019). Interaction among sex, aging, and epigenetic processes concerning visceral fat, insulin resistance, and dyslipidaemia. Frontiers in Endocrinology, 10(JULY), 1–9. https://doi.org/10.3389/fendo.2019.00496Basisty, N., Kale, A., Jeon, O. H., Kuehnemann, C., Payne, T., Rao, C., Holtz, A., Shah, S., Sharma, V., Ferrucci, L., Campisi, J., & Schilling, B. (2020). A proteomic atlas of senescence-associated secretomes for aging biomarker development. 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A molecular network of the aging human brain provides insights into the pathology and cognitive decline of Alzheimer’s disease. Nature Neuroscience 2018 21:6, 21(6), 811–819. https://doi.org/10.1038/s41593-018-0154-9Pagiatakis, C., Musolino, E., Gornati, R., Bernardini, G., & Papait, R. (2019). Epigenetics of aging and disease: a brief overview. In Aging Clinical and Experimental Research (pp. 1–9). Springer. https://doi.org/10.1007/s40520-019-01430-0Regan, J. C., Khericha, M., Dobson, A. J., Bolukbasi, E., Rattanavirotkul, N., & Partridge, L. (2016). Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction. ELife, 5(FEBRUARY2016). https://doi.org/10.7554/eLife.10956Ribarič, S. (2012). Diet and Aging. Oxidative Medicine and Cellular Longevity, 2012, 741468. https://doi.org/10.1155/2012/741468Salekeen, R., Diaconeasa, A. G., Md. Billah, M. M., & Islam, K. M. D. 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Integrative and Comparative Biology, 50(5), 844–854. https://doi.org/10.1093/ICB/ICQ094Soto-Gamez, A., Quax, W. J., & Demaria, M. (2019). Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions. In Journal of Molecular Biology (Vol. 431, Issue 15, pp. 2629–2643). Academic Press. https://doi.org/10.1016/j.jmb.2019.05.036Taormina, G., Ferrante, F., Vieni, S., Grassi, N., Russo, A., & Mirisola, M. G. (2019). Longevity: Lesson from model organisms. In Genes (Vol. 10, Issue 7). MDPI AG. https://doi.org/10.3390/genes10070518Tower, J. (2006). Sex-specific regulation of aging and apoptosis. Mechanisms of Ageing and Development, 127(9), 705–718. https://doi.org/10.1016/j.mad.2006.05.001Tower, J., Pomatto, L. C. D., & Davies, K. J. A. (2020). Sex differences in the response to oxidative and proteolytic stress. Redox Biology, 31, 101488. https://doi.org/10.1016/j.redox.2020.101488Webb, A. E., Kundaje, A., & Brunet, A. (2016). Characterization of the direct targets of FOXO transcription factors throughout evolution. Aging Cell, 15(4), 673. https://doi.org/10.1111/ACEL.12479Zhang, C., Xie, Y., Chen, H., Lv, L., Yao, J., Zhang, M., Xia, K., Feng, X., Li, Y., Liang, X., Sun, X., Deng, C., & Liu, G. (2020). FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice. Aging, 12(2), 1272–1284. https://doi.org/10.18632/aging.102682"" Key to solving the problem of aging is engaging more minds towards the problem. This means increasing efforts at educating potential researchers and thought leaders on the nature of the science, the current longevity technology strategies and the many open questions still remaining. This is true for undergraduate education, but it is even more essential to target a younger audience, high school and possibly earlier, so that an initial seed of curiosity for this field is planted and can compete with all the other interesting areas of study in biology and other fields. In our last five years of working with high school age students and engaging this age group in original life science and physical science research, we have found that this is true for a number of other fields untouched by secondary school education, including natural product total synthesis [organic chemistry], green materials science, quantum mechanics, all of which the typical high school student is unacquainted with, and may view as not only unfamiliar but also esoteric. While the Next Generation Science Standards (NGSS) have attempted to distill the fundamental first principles of science that undergird how any discipline operates, standard public school biology curriculum does not include any substantial unit on aging or, in general, central theories of disease or decay. It is astonishing that there are units on the creation of life, the different types of living beings, how it is sustained, the makeup and structure of organisms as they grow and function, and some, but very little, aspects of damage and disease but nothing substantial about why organisms fundamentally degrade and die. Generally speaking, most modern life science units tend to incorporate into teaching natural systems or first principles a “reversal foil” to “natural systems”, i.e. anthropogenic greenhouse gas pollution is a reversal foil to natural nutrient cycles in the environment, and likewise human autoimmune or endocrine disorders are presented as a reversal-foil subtheme in the cell signaling module. Notably, while there is an entire unit on ‘growth and development’, there is no such ‘reversal foil’ that exists for this unit. This vacuum in completing the circle between the closely interrelated disciplines of developmental biology and aging research is therefore an incomplete picture. This may be tied to hesitancy in standard curricula in presenting the world as filled with unsolved problems, which more often than not prefer to pedagogically establish a world of pure “facts” that aren’t further questioned or explored – despite the reality that is the very purpose of all research. This presentation of the world as solved is a greater fallacy that spans the teaching of many disciplines, but outside of solving this fundamental issue of education, we ask what can be done specifically about bringing the known “facts” of aging biology into high schools. There is still much to understand about the process of aging, yet there have been major strides towards revealing the core aspects of how the aging process is measured, motivated and manifested which should still be discussed. Interventions of course remain somewhat controversial because of the general public’s stance on trying to alter the aging process, but ultimately not much time need be explicitly spent on discussing solutions. This is because simply characterizing the nature and manifestation of aging as just another tangible, biological process, not just a philosophical dogma, will naturally lead kids to question about ways to manipulate, alter or reverse aging. This may especially hit home because, sadly, they may be at a point in their lives where they are starting to see the ravages of age in their grandparents and possibly even their parents. In our experience educating kids for the last five years, we find that the instruction needn’t be heavy handed, just simply present what is known and what are the open problems and let the kids start questioning things themselves. Furthermore, the unit needn’t be particularly long, it can stretch from a couple weeks to maybe a month. All that is required is a teaser and then the natural demand from this exciting field will create a need for more extensive courses in colleges. As our organization works by giving high schoolers exposure to the latest fields of biological and biochemical investigation, we can appreciate how exciting the field of aging is among the many biotechnology initiatives around it, right here in the bay – some of whom we work with. When the concepts are explained the kids naturally gravitate towards it as a pressing and open problem and so we would like to propose a challenge to create a standardize and easily adoptable aging unit that can be incorporated into the high school curriculum in a manner that fits into our organization’s vision in training students in original hands-on research while also supplementing the core pillars of growth and developmental biology as presented in the NGSS framework. In terms of the approach of introduction, though a chapter in the curriculum textbook might at first glance seem to be the most straightforward and canonical approach, our experience with the local schools shows this may be difficult given the amount of recent and historic politics over what goes into kid’s textbooks. Instead, we’ve found that a more digestible and tangible strategy would be to create a contained experiment module, that is cheap, consistent and effective at conveying the core modus operandi of aging research as a unifier of the chronic failure modes in the body. In addition, the module should fit well into the standard curriculum and one area we have seen that has been particularly emphasized in the NGSS curriculum as of late is cell signaling. This might present a good opportunity to get a foot in the door so to speak by branding one aspect of aging as a failure of systemic cell signaling. An example contained and reproducible experiment module we could propose would be one on learning about and deriving senescent cells. These can be simply achieved after growing cells for a long time or having an inducible cell line which the students can maintain and chart the population doubling to find when they no longer cycle. Additionally they could also then apply the very standardized pH dependent beta-galactosidase stain to confirm the cells are senescent or qPCR can be used to measure telomere length, if resources permit. Furthermore, a rudimentary cytokine measurement can be done to understand the senescence associated secretory phenotype or SASP which is the critical “aging signaling” component. Though these are often done in full multiplexed panels to show the global change in signaling, a couple cytokines can be chosen and evaluated with simple Elisa based tests that are cheap and usable like a standard COVID test. This would demonstrate how a fundamental process like cell division – widespread in a number of cell types - can lead to change in cell state and cell signaling with global consequences. This can then be used as a springboard for talking about other types of signaling in aging like the results of the parabiosis experiments or ways to intervene for senescent cells like senolytics. And then a further brief dive into just the literature on some of the other areas like the hallmarks, caloric restriction and epigenetic reprogramming can complete the unit. This achieves the concrete and reproducible example point, fits into the cell signaling curriculum and teases the larger field and purpose of the science, with the ultimate goal being an early introduction to the questions posed by aging research, its integration into other core disciplines in the life sciences, and its potential therapeutic and scientific impact on the lives and livelihoods of people in society. We therefore propose inquiry-driven, project-based research as an entrypoint of young people into the arena of aging biology, first conceptualized as completing the circle to an unfinished story of developmental biology, and further actualized through hands-on research experiences for high school age kids.""Age-associated Neurodegeneration Induced by Mitochondrial Dysfunction, ROS Accumulation, Oxidative Stress, and Telomere ErosionOne of the most crucial and yet most obscure areas of longevity science is the study of age-associated neurodegeneration which contributes to debilitating conditions (such as Alzheimer's disease (AD), Parkinson's Disease (PD), Parkinson's Disease Dementia (PDD), Cerebrovascular Disease (CVD), Lewy Body Disease (LBD), Mild Cognitive Impairment (MCI), Progressive Supranuclear Palsy (PSP), and Amyotrophic Lateral Sclerosis (ALS)) that prevent one from aging with dignity. Achieving longevity without achieving an optimum quality of life is contrary to the aim. Considering that most age-associated pathologies have been linked to Mitochondrial dysfunction, ROS accumulation, oxidative stress, and telomere erosion, it will be helpful to further analyze the factors that can induce the abnormal activity of these mechanisms in order to better understand the potential counteracting mechanisms that can help with inhibition of such dysfunctions. Thus far, the unspecified/unidentified underlying factors contributing to the onset of these conditions have been making it far more challenging to investigate them in great detail. Moreover, the fact that the booming market of anti-aging products is mainly concentrated on the aesthetic aspects of aging, also contributes to overlooking the more detrimental conditions. However, what is the point of aging gracefully in appearance, if these neurodegenerative conditions are preventing mankind from aging with dignity?Various limitations associated with the study of these neurodegenerative conditions prevent scientists from thoroughly investigating them in order to identify the precise underlying factors and to develop feasible and effective treatments. Therefore, it is important that the prospective research on these conditions begin by addressing the limitations that have prevented scientists from making advancements in these studies thus far.""Most anti-aging strategies in geoscience currently focus on druggable targets and hallmarks of aging to address tissue damage. Since there is quite a diverse repertoire of macromolecular forms of damage, including some that are irreversible like DNA mutations, drugs and therapies aimed at targeting endogenous cellular repair and maintenance machinery are probably not enough to slow aging to the point of significantly extending maximal lifespan. An alternative approach to correcting existing forms of damage is to try and replace existing aged tissue with new tissue. One of the challenges to this approach is replacing brain tissue, since it is responsible for mediating self-identity and therefore not possible replace all at once without the loss of existing sense of self. However, it is possible that by gradually replacing discrete regions of the brain at a time, namely within the neocortex (which makes up the bulk of the brain and is the center of higher order processing functions), we can still manage to replace the aged brain tissue without significantly altering the activity related individual self-identity at any given point in time.The Hébert lab is working to achieve this aim of neocortical replacement and has already demonstrated, along with other groups, that integrating new neocortical cells into existing brains is possible in mice. One of the major goals of the lab is to now take this further and construct neocortex from pluripotent stem cells at the tissue level with the correct architecture, all the right cell types, and supporting ECM environment. This should make for neocortical transplants more functional with greater replacement potential beyond simply dissociated neural precursor cells.One of my projects is centered around characterizing the iPSC-derived neocortical cells we are generating through our directed differentiation protocols using scRNAseq. Part of this involves finding out which timepoint(s) in fetal development (as shown by comparison to fetal cortical scRNAseq datasets from different ages) they most resemble at different points in the differentiation protocol. This information is relevant to understanding both what fetal age to look at as a guide to reverse engineer the neocortical tissue transplants and to see how long it takes to differentiate our cells to particular fetal ages we want to test the transplant efficacy at. Beyond this, I am going to characterize the general transcriptional fidelity of our iPSC-derived cells to fetal cortical tissue cells as well as compare the pattern of trajectory using RNA velocity-based approaches.My other project seeks to use CRISPR-Cas9 Gene editing to create hiPSC cell lines that express, specifically in neurons, inhibitory DREADD receptors that allow for selective silencing of the targeted cells through the administration of DREADD receptor ligand. This will be important to test the integrative capacity of the neurons within our reconstructed neocortical transplants. This is because if the transplant neurons encode useful behaviors then silencing them should result in the transient loss of those behaviors in our animal models. ""Each target was reduced individually using siRNA-mediated knockdown in the presence or absence of DOT1Li. Depletion of Fosl1 resulted in cell death with three different siRNAs (data not shown) and was not analyzed further. For each of the other five targets, siRNA-mediated depletion was robust and resulted in a similar fold change compared with that obtained by DOT1Li (Figure S5A). When siRNA for each target was combined with DOT1Li, mRNA levels were further reduced (Figure S5A). Hic1, Hoxd12, and Twist2 depletion did not enhance reprogramming in the presence or absence of DOT1Li (Figures S5B and S5C). Meox2 depletion alone or combined with DOT1Li slightly decreased NANOG+ colony formation (Figure S5B) but did not affect bona fide colonies that remained after doxycycline withdrawal (Figure S5C). Depletion of Nfix alone increased the occurrence of NANOG+ colonies 2-fold compared with a 3-fold increase with DOT1Li (Figure 5E, left). However, Nfix depletion did not lead to a robust formation of bona fide colonies, as DOT1Li treatment increased transgene-independent colonies 11-fold, whereas only a 4.8-fold increase was observed with siNfix (Figure 5E, right). When combined with DOT1Li, Nfix depletion enhanced transgene-independent colony formation (Figure 5E, right). Thus, Nfix depletion acts in conjunction with DOT1Li to promote reprogramming.To interrogate whether these genes are barriers of the process, we overexpressed MEOX2 and NFIX individually in MEFs prior to the induction of reprogramming (Figure S5D). Exogenous MEOX2 expression inhibited reprogramming on its own but did not affect DOT1Li-mediated reprogramming (Figure S5E, left). Surprisingly, MEOX2 increased stable colony formation in the absence of DOT1Li (Figure S5E, right). These contrary effects of Meox2 depletion and overexpression on reprogramming may be related to its heterogeneous expression in the reprogramming population (Figure 5C) and the increase in proliferation in MEFs overexpressing MEOX2 (data not shown). Ectopic expression of NFIX during reprogramming reduced DOT1Li-mediated NANOG acquisition below control levels and prevented stable colony formation (Figure 5F), suggesting it is a potent reprogramming barrier independent of DOT1L activity. Thus, the downregulation of Nfix acts in an additive manner to increase pluripotency by contributing to the DOT1Li phenotype.By comparing the results of the depletion experiments (Figures 5E, S5B, and S5C) with the pattern of expression from single-cell analysis (Figure 5C), we find that DOT1L acts in a collaborative manner with Nfix that is still expressed at later stages of reprogramming. Hic1, Fosl1, and Twist2 are downregulated in most cells by day 3 of reprogramming, and Meox2 is upregulated in only a portion of reprogramming cells (Figures 5C and 5D). Taken together with the temporal effectiveness of DOT1Li at the mid stages of reprogramming (Figure 1D), DOT1Li sets the stage for enhancing reprogramming efficiency along with the downregulation of persistently expressed genes such as Nfix.Go to:DiscussionDOT1L is crucial for mammalian development, yet its role in cell-fate determination is still unknown. Here, we find that DOT1L is a barrier to pluripotency acquisition of MEFs throughout reprogramming but acts most strongly during mid-reprogramming (Figure 1). Stages of reprogramming when starting from MEFs include an early inactivation of the somatic program, an important component of which is the mesenchymal genes (Li et al., 2010; Samavarchi-Tehrani et al., 2010). Inhibition of DOT1L was reported to enhance human fibroblast reprogramming by facilitating MET (Onder et al., 2012). The reverse process, epithelial to mesenchymal transition (EMT), is prevented in other systems, such as renal (Liu et al., 2019) and breast cancer cell lines (Cho et al., 2015), by DOT1Li. We demonstrate that DOT1L control of cell identity extends far beyond epithelial transitions. Although inhibition of DOT1L increases Cdh1 expression in our mouse reprogramming studies, it also enhances reprogramming of keratinocytes that do not have to undergo MET and MEF reprogramming post-MET (Figure 4). This corroborates our recent discovery that the epithelial and mesenchymal programs are independently regulated in the presence of DOT1Li during reprogramming using scRNA-seq (Tran et al., 2019).In the course of assessing DOT1L transcriptional regulation, we identified contributions of the reprogramming barrier Nfix that maintain cellular identity with DOT1L (Figure 5). NFIX is a transcription factor important for neural (Pekarik and Belmonte, 2008) and muscle development (Pistocchi et al., 2013). In addition, NFIX is required to maintain murine hair follicle stem cell enhancer function (Adam et al., 2020), and its depletion increases the number of pluripotent colonies (Yang et al., 2011). From our previous analysis of reprogramming with scRNA-seq, we ordered single cells in a trajectory (Tran et al., 2019). We found a major branchpoint where cells stalled and did not complete the transition to iPSCs (Tran et al., 2019). Nfix was found to be a branchpoint gene such that downregulation was required to continue in the reprogramming trajectory, further suggesting that it may have a specific role in mid-reprogramming. Of the identified DOT1L targets, Nfix continues to be expressed in cells later in reprogramming and gains H3K79me2 after OSKM induction (Figures 5B and 5C), suggesting DOT1Li may facilitate its downregulation by preventing the gain in H3K79me2 enrichment at later reprogramming time points. This result is in contrast to previous findings that DOT1Li enhances pluripotency acquisition by downregulation of the established transcriptional program (Onder et al., 2012). It is important to note that depletion of Nfix does not reach the reprogramming efficiency of DOT1Li, and the effect is additive with DOT1Li (Figure 5E). Thus, downregulation of reprogramming-associated factors may contribute but is not solely causal of the DOT1L reprogramming phenotype.We find that modulation of DOT1L-DE genes does not substitute for DOT1L catalytic inhibition (Figures 4 and and5),5), suggesting that H3K79me may have a role in reprogramming beyond transcriptional regulation of single genes. During development, only four genes were DE in Dot1l KO mouse c-kit+ cells sorted from embryonic day 10.5 (E10.5) yolk sacs, where profound phenotypic alterations in vascular morphology and erythrocyte maturation were observed (Feng et al., 2010). These small transcriptional changes in key factors like Nanog during reprogramming or Gata2 during erythrocyte maturation suggest that loss of H3K79me may alter global epigenetic profiles rather than local expression profiles. For example, loss of H3K79me2 allowed for spread of H3K27me3 on downregulated genes in leukemia cells (Deshpande et al., 2014). H3K79me2/3 are enriched at certain intronic enhancers in leukemia cell lines and regulate future deposition of H3K27ac (Godfrey et al., 2019), a modification associated with enhancer activity. In neural differentiation, DOT1Li decreased accessibility of a subset of enhancers (Ferrari et al., 2020). In support of further epigenetic alterations, both upregulated and downregulated genes can be decorated by H3K79me2 before DOT1L depletion in development of the cerebral cortex (Franz et al., 2019). Thus, H3K79me may affect histone modification, depending on chromatin context.We observed that many more genes are upregulated rather than downregulated in their steady-state expression by DOT1Li during reprogramming (Figures 1 and and4).4). Many of the upregulated genes are expressed in other lineages and are not modified by H3K79me2 in MEFs or ESCs (Figures 3 and S3D). Alternatively, these genes may be regulated indirectly by the binding of DOT1Li-DE transcription factors, such as HOXD12 or HIC1. The aberrant expression of lineage-specific genes may promote reprogramming, a notion that can be further investigated by single-cell transcriptional analysis. Regardless, the boost to reprogramming cells by DOT1Li seems to outweigh the burden of spurious lineage gene expression. Taken together, our results demonstrate that DOT1L activity functions beyond steady-state alterations to the somatic transcriptome; rather it collaborates with reprogramming-associated factors to safeguard cell identity.Go to:Experimental proceduresCell isolation and cultureMale and female MEFs were isolated on E13.5 from embryos that were homozygous for the Oct4-2A-Klf4-2A-IRES-Sox2-2A-c-Myc (OKSM) transgene at the Col1a1 locus and either heterozygous or homozygous for the reverse tetracycline transactivator (rtTA) allele at the Rosa26 locus, as previously described (Tran et al., 2019). MEFs were grown in DMEM, 10% fetal bovine serum (FBS), 1× non-essential amino acids, 1× GlutaMAX, 1× penicillin/streptomycin, and 2-mercaptoethanol (4 μL/500 mL). Feeder MEFs were maintained and isolated as above from DR4 mice genetically resistant to geneticin (G418), puromycin, hygromycin, and 6-thioguanine. Feeder cells were irradiated with 9,000 rad after three passages. ESCs V6.5 were grown on feeder MEFs in KO DMEM, 15% FBS, 1× non-essential amino acids, 1× GlutaMAX, 1× penicillin/streptomycin, 2-mercaptoethanol (4 μL/525 mL), and leukemia inhibitory factor. Keratinocytes were isolated from reprogrammable mice 4 days postnatal as previously described (Li et al., 2017) and cultured in EpiLife medium with 60 μM calcium (Thermo Fisher Scientific MEPI500CA) with the EpiLife defined growth supplement (Thermo Fisher Scientific S0125). 293T cells were acquired from ATCC and grown in DMEM and 10% FBS. Mice were maintained according to the UW-Madison institutional animal care and use committee (IACUC)-approved protocol.A detailed description of all materials and methods is provided in the supplemental information.Data and code availabilityThe accession number for all RNA-seq datasets reported in this paper is National Center for Biotechnology Information Gene Expression Omnibus (GEO): GSE160580.Go to:Author contributionsC.K.W. performed experiments and bioinformatic analysis, C.K.W. and R.S. wrote the manuscript and acquired funding, and R.S. conceived and directed the project.Go to:Conflict of interestsThe authors declare no competing interests.Go to:AcknowledgmentsThis work was supported by a UW-Madison Stem Cell and Regenerative Medicine Center postdoctoral award to C.K.W., a UW-Madison Fall competition, and a Shaw Scientist award to the R.S. lab. We thank Dr. Roice Wille for R script design, Stefan Pietrzak and Brenton Halvorson for scRNA-seq analysis, Dr. Jason Tchieu for the NFIX vector, and Dr. Konstantinos Chronis and the R.S. lab members for critical reading of the manuscript.""As the interest in longevity research increased in recent years, so did the numbers of publications on the subject that are available online. While results and conclusions from earlier publications are often referenced and discussed in later articles, the datasets underlying these results are usually analyzed only once, for the purposes of the original publication.Thus, one of the most underappreciated areas of longevity research is not any single particular publication, but the opportunity of recycling available “single-use” datasets of previous publications. Generating aggregated datasets that are used to repurpose, reanalyze, and reinterpret data in a broader context can provide deeper and more robust insights than would be possible by analyzing a single dataset.Current data availability policies of scientific journals require uploading and sharing of all relevant datasets in online repositories such as Gene Expression Omnibus (GEO). This includes datasets such as DNA methylation array data and RNA-seq gene expression data, which are typically generated using widely available methods and instruments (e.g. Illumina platforms) and analyzed using standardized, i.e. comparable, analysis workflows. This leads to the accumulation of extremely large datasets. However, although the datasets are available online for everyone, they are not easily accessible and usable.Many datasets, however, are standardized enough to be compared in principle, but not standardized enough to be easily analyzed in a meaningful way by the no-code research community.Machine learning algorithms are especially suited to find patterns in datasets that 1) are large, 2) have many parameters, and for which 3) an interplay of a multitude of parameters shapes the outcome. As aging is characterized by the functional deterioration of highly complex intra- and intercellular networks, it is in particular suitable for machine learning applications. To enable meaningful aggregated data analysis driven by state-of-the-art machine learning applications, certain quality criteria must be defined and met for individual datasets that are to be included. In particular, a standardized format and annotation is required because quality control, preprocessing, and formatting of data is usually the most time- and resource-intensive step in bioinformatics analyses. Facilitating the analysis of aggregated data by establishing an accessible online tool for the no-code research community should provide the basis for a closer connection between bioinformatics and bench scientists. This online tool would make these aggregated datasets accessible so that they can either be analyzed online within the provided tool or easily fed into customized machine learning pipelines from the research community. As aggregate data analysis provides more statistical and explanatory power to evaluate large integrated datasets, this will facilitate the identification of novel cellular and molecular candidate factors to generate new hypotheses about how these candidates influence and shape aging. To confirm promising drug targets and signaling pathways, these identified candidates should then be further characterized in vitro and in vivo.An application example of this approach would be to aggregate datasets generated from numerous datasets that include data from healthy volunteers who are typically part of many studies as a control group, but for whom additional information such as age or another clinical age-related biomarker or outcome is available.In addition, aggregated data could be used to generate regularly updated reference datasets that can be made available to the research community to improve contextualization of new findings by promoting comparability of results (e.g., through cross-validation of models).In principle, the aggregate data analysis approach can be applied to data from all -omics fields, including RNAseq, proteomics, epigenetic clock research. Importantly, this approach is not limited to datasets originally generated for aging research, but can also incorporate datasets from other fields that can also be applied to aging because they contain relevant data such as age or a specific treatment method.Importantly, input and suggestions from the broader longevity research community are critical to shaping this approach of aggregate data analysis to reach its full potential.In summary, the rediscovery and reuse of previously published data currently requires considerable bioinformatics expertise and is not being pursued in a coordinated and standardized approach in the field of longevity. Therefore, data must be made available to laboratory scientists in a meaningful way.Aggregate data analysis of previously published datasets combined with subsequent confirmation of the testable hypotheses derived from them in vitro, in vivo, and ultimately in clinical trials will help identify and validate biomarkers and mechanisms of aging and longevity.""Accurate conversion of genetic information into protein tertiary structure is one of the pillars of proper cellular function, and disruption of protein homeostasis (proteostasis) that leads to intracellular accumulation of misfolded proteins is a hallmark of aging and a contributor to age-related pathologies.The mechanisms of the aforementioned conversion are somewhat “noisy” – for transcription, the error rate is 10-5-10-4 (1/100,000 ribonucleotides are mismatched with the transcribed DNA template); for translation, the rate is 10-4-10-3 (1/10,000 mRNA codons are paired with a wrong amino acid). In addition, erroneous tRNA aminoacylation (when tRNA is coupled with an amino acid that does not match the anticodon) presents an extra source of mistakes in the flow of information from DNA to protein.Any deviation from the background error rate has its effect on the aging dynamics. It has been shown that certain long-lived organisms inherently benefit from a relatively more faithful translation (1–3), whereas experimental elevation of translation error rate in animal models shortens lifespan (4). It is therefore important to deconstruct the mechanisms that alter the fidelity of protein synthesis in order to devise strategies and interventions for extending healthy life.Methylglyoxal (MGO) is a byproduct of glycolysis with reactivity towards amines – arginine guanidino groups, lysine amino groups, and exposed protein N-termini. Non-enzymatic modification of amino acid residues by MGO leads to the formation of various chemical adducts and crosslinks (collectively known as advanced glycation endproducts (AGEs)) that may affect the susceptible proteins’ three-dimensional conformations (in other words, how they are spatially folded) and proper functioning (5). There are numerous reports that implicate MGO production in diabetic complications – cardiomyopathy, nephropathy, peripheral neuropathy, retinopathy, etc. – and age-related organ decline.Unfolded protein response (UPR) is a mechanism to circumvent the accumulation of misfolded proteins either in the lumen of the endoplasmic reticulum (ER stress) or in the cytosol (cytosolic UPR). It has been shown that cells grown in presence of high glucose display ER stress mediated by MGO (6). The authors conclude that the UPR is brought about by non-enzymatic post-translational modification of existing proteins by MGO. I propose an additional hypothetical mechanism: MGO-mediated glycation of ribosomes – i.e. ribosomal proteins, rRNAs – increases the error rate at the level of translation resulting in enhanced synthesis of polypeptides prone to misfolding.Extracellular matrix (ECM) is a heterogeneous but highly organized matter located in-between cells that provides structural support to tissues. Cells and their environment, represented by the ECM, communicate reciprocally to drive organogenesis during development and remodeling in response to insult. ECM composition and ensuing mechanical properties greatly affect cellular behavior. For example, mesenchymal stem cells commit to either bone or muscle lineages depending on substrate elasticity. Moreover, the accumulation of AGE-crosslinks (including but not limited to MGO-derived crosslinks) in matrix collagen has been shown to increase ECM stiffness and contribute to cardiovascular pathology.Cells continuously monitor the state of their environment, including its mechanical properties, via cell-surface receptors. Transduction of mechanical cues (mechanotransduction) is mediated by integrin receptors that probe the ECM elasticity and initiate a cascade of intracellular responses to fine-tune cellular behavior to the surrounding mechanical context. Increased ECM rigidity (stiffness) leads to translocation of the transcription coactivator YAP/TAZ from the cytoplasm into the nucleus where it binds to the transcription factor TEAD, driving expression of genes involved in tumorigenesis: those promoting growth, proliferation, survival, glucose uptake, glycolysis, epithelial-to-mesenchymal transition, apoptosis inhibition. Concurrently, I propose that enhanced glycolysis (and subsequent MGO generation) in response to the stiffened ECM leads to the loss of proteostasis at least partially due to ribosome glycation and an ensuant increase in mistranslation rate. The proposed mechanism of proteostasis disruption might have broader implications for the ECM organization. For instance, the substitution of key amino acid residues in tropoelastin has been shown to impede monomer coacervation and lead to aberrant elastic fiber formation (7).The hypothetical mechanism of ribosomal dysfunction has not yet been described in the literature. Shedding light on novel aspects of translation fidelity, especially in relation to metabolism, will draw a link between the tightly regulated proteostasis maintenance machinery and the stochastic nature of glycation, possibly paving the way to a new class of life-extending interventions.For the full project details, including preliminary data and further plans, please visit -- https://docs.google.com/document/d/1QLzipbn6umWGg2MfyyxEROxdzsWMWvXoiG_WBVFHzPQ/edit?usp=sharing"" Humanity's most important biological discoveries, of course, have had to do with the origins of life itself. Furthermore, if we are aiming to reverse aging and prevent death, it would make the most sense to solve this problem, with it anti-thesis, the origin of life. Via the discoveries of the egg cell, the sperm cell, and the ensuing zygote which forms during fertilization, and the further ensuing process of embryogenesis, capable of faithfully creating healthy cells of every type and form, from what were once ""biologically aged"" sex cells in adult human beings, humanity has already discovered the fundamental process for reversing aging and preserving immortality, which naturally has allowed for the healthy and immortal continuation of cellular lineages and the human species. Therefore, my hypothesis for the cure to aging and death is simply that we must recapture the potential of the processes at the origin of life, as it occurs between generations via fertilization. More specifically, my hypothesis is that the fundamental minimum combination of rejuvenation processes that the scientific community has discovered to occur during fertilization, is a combination of 1) Epigenetic reprogramming; 2) DNA repair; and 3) Autophagy/Mitophagy; And, that furthermore, therefore, an in-vitro rejuvenating processing of adult stem cells, screened for high initial genetic quality, should at least enact the simultaneous combination of all 3 fundamental processes of reproductive rejuvenation (""3FPRR""), which may thus successfully and fully rejuvenate a small selection of an adult's stem cells, which can then become the starting material for manufacturing large quantities of autologous, non-immunogenic, non-cancerous, cellular replacement therapies. It should be further noted that the 3FPRR are synergistic to the extent that DNA Repair and Epigenetic Reprogramming enhance each other, as do Autophagy/Mitophagy and Epigenetic Reprogramming, and therefore the potential of any single 3FPRR processes by itself is even further diminished compared to my proposal for the minimum 3FPRR.In order for this vision to practically work to truly reverse aging and avoid biological death, these rejuvenated & reprogrammed stem cells (""RRSCs"") would need to be delivered in such a way and as part of a system capable of in-vivo cellular replacement. I propose that this initially could be done via a 2 step process of 1) initially tagging a random and very small subset (ie. .05%) of all cells, evenly distributed throughout the body, with dual aptamer homing ligands, for attracting both engineered macrophages, and RRSCs; 2) administering the RRSCs to home to the distributed tagged cells, whereby they could engraft, and if necessary, spontaneously differentiate, and become functional resident tissue, enacting cell replacement with single cell fidelity; and 3) administering engineered CAR macrophages to target and phagocytose the tagged old cells; thus, completing the cycle of fully removing aging and having a functional cell by cell level replacement process. Furthermore, this vision requires the RRSCs to be able to be manufactured in an unlimited supply, which can be achieved by a combination of telomerase enforced immortalization, and periodic RRSC re-induction. A portion of RRSCs can be kept after early passaging, via cryopreservation, to mimic oocyte quiescence, and provide a more ideal source of future RRSC re-induction. Ideal culture conditions for the virtually infinite manufacturing of iPSCs has already been databased, and will work for this vision, and therefore insurmountable infinite manufacturing limitations are in no way a serious threat. This vision furthermore requires the RRSCs to be primed for in-vivo applications, with non-cancerous regeneration of all lineages. This should ideally be done via the Division of Regenerative Labor (""DRL""), which is a novel concept I have coined, meaning the use of both a LT-HSC lineage derived from RRSCs, and the use of a multipotent MSC (""MMSC"") lineage derived from RRSCs. The LT-HSC lineage is easy to understand, as we have already proven over the last 30 years that cellular replacement, and therefore the immortalization and reversal of aging, for the hematopoietic lineage at least, is a viable model, as widely proven by successful bone marrow transplantations. The MMSC lineage approach is less understood but viable nonetheless, and should obviously be the approach taken given the proven success of LT-HSC cell replacement therapy which can be mimicked, to cover the remaining gap toward the end goal of Total Body Cell Replacement (""TBCR""). These MMSCs would have to have the ability to migrate, home, engraft, survive, spontaneously differentiate into all non HSC lineage cells throughout the body, and not form cancers. Thankfully, we can mimic another natural solution, which is an under-appreciated discovery, namely the discovery of SSEA3+ MSC subset cells, which have already proven in dozens of studies to have all of these exact properties. These SSEA3+ MSC cells, which have been dubbed by their founding research team and in the literature as ""MUSE"" cells, have been shown to be able to migrate effectively to areas of cell death, damage and inflammation, without getting trapped in the lungs like traditionally studied MSCs, and they can spontaneously differentiate via clues from the local niche, into functional tissue of every non-HSC lineage based organ and tissue type so far studied (brain, spinal cord, skin, lung, liver, kidney, pancreas, heart, endothelium, etc.), and they can persist for years, again, unlike traditionally studied MSCs. Therefore, we can differentiate RRSCs into MMSCs, which at the very least copy these traits of endogenously existing, regenerative SSEA3+ MSCs, but ideally which are over-engineered and evolved upon this basis, via specific culturing and priming as discussed, to optimize cell replacement goals, and therein be able to cover, alongside with the LT-HSCs, every type of cell needed to accomplish TBCR, and the only true, ethical, understandable, and currently viable form of fundamentally and totally reversing aging, and curing biological death. The endogenous SSEA3+ MSCs also provide us initial pathways to mimic to ensue that these undifferentiated stem cells do not cause tumors, unlike the direct use of iPSCs, while still maintaining a virtually pluripotent potential. The cancer preventing pathways discovered thus far may have to do mainly with the fact that these SSEA3+ MSCs have low levels of telomerase, low levels of OCT4 and Nanog, a low ratio Lin28 to Let7, and a high level of the tumor suppressor CDKN2A. Therefore in addition to priming the RRSC derived MMSCs for cell replacement, they should be primed to be non-cancerous, although it should be assumed, and not completely avoided, that a small cancer risk will inevitably be a part of regenerative therapies. By further understanding these stem cells, we will nevertheless continue to get a better grasp on targetting cancer stem cells, and curing cancer, and cancer is not a fundamental limit to reversing aging, especially if we already are working on a viable cell replacement therapy that could work to replace cancer cells as well as normally aged cells, again very similarly to copying the successful model for curing cancerous HSCs of the bone marrow, which are simply eliminated and replaced with healthy HSCs. In order to enact the reprogramming of cells, I propose humanity should likely universalize the use of a universally accessible, viable, simple, cheap, and highly effective adult stem cell, which can be isolated from a voided human urine sample. Dozens of research groups have already shown the high efficiency of Urine Derived Stem Cells (""UDSCs"") to be reprogrammed into iPSCs. UDSCs express high levels of embryonic surface markers SSEA4 & PDX1, pluripotent marker OCT4, and even significant telomerase levels, unlike other adult stem cells. They express MSC markers such as CD44 and CD90, but are epithelial in identity, and are therefore logically, but are also already experimentally proven to, have higher reprogramming efficiencies, as it is known that the Mesenchymal to Epithelial (""MET"") transition is a bottleneck and barrier to iPSC induction from the low efficiency and more commonly used fibroblasts or MSCs. Furthermore the MET transition during traditional iPSC induction is a major source of genetic instability and raises cancerous risks of such a stem cell lineage. Therefore, collecting a simple urine sample from any human being on earth, and isolating these cells with an already high degree of stemness, as the starting source for RRSCs is logical and viable. This isolation could be further enhanced by using various means of further filtration to select a smaller UDSCs sub-population which have the highest degree of genetic quality, and the least damage. As we know from the immortal germ line and embryogenesis, even a single cell could be enough if handled correctly. In order to enact the 3FPRR it is important to show sufficient knowledge and capacity to faithfully recreate the end result of a cell lineage capable of healthy immortality. I have done the research to create the initial concepts, and provide experimental starting grounds, to achieve this assurance. 1) Epigenetic reprogramming; While lauded as possibly the greatest discovery in cell biology, the nobel prize winning traditional process of iPSC induction using the 4 transcription factor cocktail ""OSKM"" is not ideal. I propose a more universal, simpler, cheaper, faster, more efficient(higher success rate), and safer method, which would pair well with UDSCs, towards achieving RRSCs for TBCR. In recent years, small molecule and chemical replacements to these genetic transcription factors have been disclosed. I have compiled them into the most exhaustive list to date, and have significant detailed backup research on their more precise reprogramming roles and potentially ideal media concentrations. With further high throughput combinatorial testing optimization for optimal synergy, concentrations and temporal requirements, I believe the compounds on this list to already suffice for the job needed. The list is currently; 0) Low Oxygen, 1) NaB > 1.5) VPA, 2) CHIR99021 > 2.5) 6-bromoindirubin-3-oxime, 3) 616452(TGF-B Inhibitor), 4) Tranylcypromine, 5) Forskolin, 6) DZNep, 7) TTNPB(RAR agonist), 8) Repsox > 8.5) Dasatinib > 8.5') PP1 >8.5'') iPYrazine, 9) cyclic pifithrin-α (a P53 inhibitor), 10) A-83–01, 11) thiazovivin, 12) Trametinib, 13) PS48 14) DHEA 15) Lactoferrin, 16) Spermine, 17) H2S, 18) Purmorphamine > 18.5) SAG, 19) Oxysterol, 20) 2-methyl-5-hydroxytryptamine, 21) D4476, 22) Melatonin , 23) Vitamin C + 24) Alpha KetoGlutarate, 25) Beta-Hydroxybutyrate, 26) SB43 +> 26.5) PD03 (SYNERGY), 27) JQ1 > 27.5) iBET-151, 28) Y-27632 > 28.5) Thiazovivin, 29) FGF2 / 30) Collagenase, 31) PD173074, 32) Kenpaullone, 33) SU5402(3i>2i), 34) OAC1, 35) SC1 ""Pluripotin"", 36) Bayk8644, 37) Parnate, 38) Suberoylanilide hydroxamic acid, 39) AMI-5, 40) 2, 4-Dinitrophenol, 41) Fructose 2, 6-bisphosphate, 42) N-oxalylglycine, 43) BIX-01294 44) AM580 > 44.5) CD437(RAR agonists), 45) 100mm NaCl(hyperosmolarity), 46) Rapamycin(autophagy), 47) SMER28(autophagy) 2) DNA Repair; Although the great discovery of DNA, the code and source of generating biological life, has been lauded as one of, if not the most important discovery in biology, and what furthermore literally defines us, not enough work has been done to focus on repairing DNA to reverse aging. It is obvious to any cell biologist, and should be easily explained to any human being, that DNA damage is the most fundamental damage to a cell, and therefore is a fundamental driver of aging. Although cells have DNA repair mechanisms, not all damage is repaired. In oocytes, which play the most important role in anti-aging processes though-out the human species, there is very clearly the most outsized upregulation of multiple mechanisms to ensure genetic quality, check for DNA damage, and enact DNA repair, which we would be wise to learn from, mimic and build upon when trying to solve the problem of aging. I have compiled many of the most important DNA repairing enzymes, molecules and pathways into an exhaustive list, and have significant detailed backup research on their more precise repair roles, interactions and modes of action. With further high throughput combinatorial testing optimization for optimal synergy, concentrations and temporal requiements, I believe this list to already suffice to prove a completely higher level of quality and possibly even the potential for infinite healthy cell replacement. The list is currently; 1) MGMT: (Direct Reversal of DNA Alkylation Damage), 2) AlkB: (Direct Reversal of DNA Alkylation Damage), 2.5) ALKC & ALKD, 3) CHK1 & CHK2, 4) NUDT1, 5) GADD45, 6) SSB1(SINGLE STRAND DNA DAMAGE REPAIR + Facilitates TERT Recruitment to Telomeres and Maintains Telomere G-Overhangs), 7) ATRX, 8) RAD51 (Synergy with iPSC induction), 9) XRCC1 (single strand breaks), 10) SMUG1 (Base excision repair enzyme that removes uracil and oxidized pyrimidines), 11) OGG1, 12) NTHL1, 13) DJ1, 14) Glutathione, 15) SIRT6-PARP-NAMPT, 16) PGP, 17) BRD4, 18) PRDM14(DNA demethylation + TET mediated BER) , 19) TDP1 (single strand breaks, and formaldehyde induced DNA cross links), 20) BRCA1, 21) 53BP1, 22) XRCC4 (NHEJ in double strand breaks).Perhaps one of the most important discoveries related to the 3FPRR is that Histone Deactylase Inhibitors enhance DNA damage repair during epigenetic reprogramming, and may be attributed to histone acetylation at specific sites that enhance DNA repair. It is further known that the chromatin remodeling during epigenetic reprogramming also promotes DNA damage repair, and the general knowledge that cells with increased stemness such as in iPSCs and Zygotes have more open and relaxed chromatin, which is more accessible, not only to epigenetic modification, but surely at the same time, to DNA repair recruitment. This provides us with an ""opening"" in every sense of the word, a gift of an ideal opportunity to both enact epigenetic reprogramming and enhance DNA repair at the same time, which are both required for true rejuvenation. This process would make sense biologically as well, as this is the time in life when the window for true rejuvenation occurs. Enhancing DNA repair during this step is further synergistic because it helps to avoid cancer risks as well, which is necessary for the downstream use of RRSCs, and epigenetic reprogramming is known to cause genetic instability and DNA damage, which therefore logically requires us to enhance DNA repair with the addition of exogenous repair enzymes, molecules and repair pathway activators. Moreover, studies across long-lived species across the biological kingdoms show enhanced DNA repair to both underly low cancer rates, and reduced aging, as a conserved mechanism, and therefore at the very least, enhancing humanity's capacity to enact successful DNA repair, both in-vitro and possibly in-vivo, through the manufacturing and practical applicative expertise with DNA repair mediators, such as those listed above, will undoubtedly be a 100% surefire way to help us towards longer maximal lifespans. 3) Authophagy/Mitophagy; There has been much fanfare regarding fasting and caloric restriction in anti-aging research, and much of this research has delegated a large portion of the benefits to autophagy. Furthermore, there are other large groups of prominent and outspoken anti-aging thought leaders and researchers who are interested in mitochondrial health and related effects of oxidative stress. It is vital to note that mitochondria have their own DNA as well, and therefore mitochondrial DNA quality enhancement must be a part of the DNA repair process in the 3FPRR. Mitochondria do share much of the same DNA repair pathways and machinery as the nucleus, which could be applied to them as well in the 3FPRR for adult stem cells, but there is another, and perhaps more fundamental mechanisms for enhancing mitochondrial DNA quality, which is through the process of mitophagy. Mitophagy is the process by which damaged mitochondria are removed and/or recycled from a cell, so as to dilute poor genetic quality copies, and allow the higher quality mitochondria to reproduce. (Mitophagy Flux goes hand in hand with mitochondrial Fusion and Fission Flux, which also allow for a dilutive effect and selective enhancement of Mitochondrial DNA quality.) Research has shown that Oocytes in specific, as well as Zygotes and Embryonic stem cells, have significantly enhanced pathways and mechanisms for Mitophagy, to ensure healthy and immortal mitochondrial lineages are also passed down generation to generation. ""Pink1 "" is an example of a mitophagy enhancer that also synergistically promotes epigenetic reprogramming, as are UCP2 and ""GAS6"". FZO1 effects are mediated by the mitophagy receptor Atg32, and another important mitophagy receptor is OPTN, which also enhances an embryonic identity. PHB2 is an inner mitochondrial membrane mitophagy receptor that also regulates embryonic identity. Nix is another selective autophagy freceptor for mitochondrial clearance. BNIP3 is another important pathway that mediates mitophagy. Moreover, studies have shown that fundamentally mitochondrial clearance is essential during the process of iPSC reprogramming, and more specifically ATG3 dependent autophagy has been shown to mediate mitochondrial quality control during the acquisition of induced Pluripotency. As a single applicable intervention example, Melatonin is a molecule that has been shown to enhance epigenetic reprogramming, DNA repair, authophagy and mitophagy, and therefore represents the ideal of 3FPRR anti-aging. Unsurprisingly melatonin is perhaps one of the oldest and most fundamental molecules in biology and is severely decreased and dysregulated in the elderly, while its in-vivo therapeutic use has been shown to be beneficial in thousands of studies across nearly every disease model, including extension of lifespan. During the 3FPRR enforcing and enhancing mitophagy could be done synergistically by at the least enhancing general cellular autophagy, which will provide other synergistic benefits and would be advisable. Autophagy is a logical and actual target for immortal achievements in RRSC not only because of its role in mitophagy and mitochondrial quality, but because it is able to clear an old and damaged proteome, which also paves the way for a new proteome to be developed as part of the epigenetic reprogramming and assumption of a new identity. Autophagy is a fundamental mechanism for protection against the otherwise would be damaging levels hypoxia as well, which is generally favored for conditions of epigenetic reprogramming and pluripotent cell culturing, and enhanced autophagy has been shown to maintain stemness and repress the development of senescence, or even reverse it, in otherwise would be senescent stem cells. Enhanced general autophagic flux, via the FOXO1 pathway related Ulk1, Beclin1, Atg5, Atg3, and LC3 has been shown to maintain Embryonic identity as well. Supporting this, knockdown of LC3 in ESC also shows to reduce pluripotency. Phosphatidylethanolamine could be a useful in-vitro treatment molecule, as it is structurally necessary for autophagy machinery, and has been shown to enhance autophagy and longevity, and is required at the early stage of epigenetic reprogramming anyway, providing another synergistic opportunity. Phosphatidylcholine(PC) is another essential molecule for embryonic identity and pluripotency, and plays a function in autophagosome formation. The primary lysosome biogenesis and autophagy transcription factor HLH-30/TFEB has also been shown to increase lifespan, and could be a helpful target for 3FPRR autophagy induction. Rab32 is another target which increases autophagy, and, in addition, synergistically the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, and improving the induction of iPSCs through the enhancement of lipid biosynthesis. TFEB is another fundamental target pathway linking enhanced autophagy to lysosomal biogenesis. Beclin 1 is also an autophagy inducer which is synergistcally essential for embryonic identity as well, and interestingly has shown to be able to reverse neurodegenerative accumulation or amyloid proteins in alzheimer's models, and generally support adult neurogenesis. Activation of the Sigma-1R receptor to induce autophagy and enhance proteostasis is another appealing pathway, and even reduces stress between the endoplasmic reticulum (ER) and mitochondria. SIRT6, which is involved in DNA repair also suppresses senescence and induces autophagy, as another attractive target. Ultimately my hypothesis and supporting research is approachable and simple, but is the most sensible and reliable method for humanity to attain true reversal of aging. Total body cellular replacement with reprogrammed and rejuvenated stem cells via the 3 fundamental processes of reproductive rejuvenation, is what we should be investing in. We could always evolve upon the basis of this organic approach, but in my hypothesis this is the minimal requirement, and potentially fully sufficient approach to start with. In a sense, this hypothesis also would advise that support for other approaches to reverse aging are incomplete, and therefore it is important to be clear on one's goal of simply extending human lifespan and, or, healthspan, with “anti-aging” interventions, or fundamentally and finally attempting with experimental, practical, and engineering based advancements on a platform biotechnology to attain a fundamental and complete solution to aging itself. Comparing all other options to completely cure aging, there seems to be nothing that compares, as the greatest biological problem requires a total replacement of the problem with a solution, down to every last cell, throughout the entire body. ""We would like to submit our proposal. Please find a shared link below for reviewing a PDF format.Thank you.https://www.dropbox.com/s/08na5w5bpdqe95a/Proposal%20for%20the%20Longevity%20Hypothesis%20Prize_BioAtla%20Inc.pdf?dl=0""The Uberkinder Program (UP) aims to inclusively foster innately stronger, smarter, happier, healthier and more enduring adults, in an extremely cost-effective fashion, based upon two tenets: 1) Construction of an optimized adult requires maximization of neural and physical capacity and fitness, which are rooted in the bio-physical environment during early developmental2) Proactive health and longevity measures reliably offer reduced need for, and superior results from reactive repair measuresEvolution never delivers an optimum possible set of features; rather, it reflects countless compromises between past environments, limitations of genetics, immunity, and bodily structure, developmental circumstances, and pure chance. Additionally, the genetic diversity that mitigates unpredictable existential threats also yields progeny that are poorly adapted to current circumstances. On the bright side, there are social and bio-physical factors that we increasingly can optimize. ‘Pre-adult’ bio-physical factors are the preponderant focus of UP, and include parental reproductive fitness at conception, plus bio-physical developmental environments during gestation and puberty. These factors significantly influence physical, neural and emotional capacities at maturity, and thus are strong determinates of adult limitations that can only be partially mediated by social factors (emotionally nurturing home life, good education, etc.). Minus social influences, powerful evidence suggests that the most economical and humane approach to lasting health, comfort, social harmony and productivity is non-invasive optimization of pre-adult development, growth and repair factors that are the soil in which community behaviors are planted. Discovery, characterization, optimization, approval and universal implementation of these pre-adult factors is the sole goal of UP.UP begins with vitalized and broad integration of known and easily accessible bio-physical boosters of pre-adulthood (e.g., avoiding alcohol during pregnancy; good sleep habits), but that only scratches the surface of what can be done. Indeed, even the cited, well-known boosters are imprecise, and have not been validated in detail. It might be that a single cc of 80-proof liquor every pregnant day is a net positive; that a 20-minute mid-morning and mid-afternoon nap, plus six hours sleep at night yields better outcomes than an eight-hour stretch at night. How can you know? Where is the data? What about a forty-minute nap after lunch that includes that one cc? Studies behind popular pediatric advice almost never offer fine detail, but the stakes are high, with seemingly minor outcome improvements (e.g., a 1-point average IQ increase) potentially having a huge impact when applied to billions of citizens.The next target is the numerous GRAS, but thinly-proven and barely-known developmental augmentations. One can barely make a browser query concerning any factor (e.g., “near-IR light therapy and pregnancy”; “D3 + K2 for teens”) without quasi-learning that we maybe could/should be doing better job. Again, at least some of the to-be-sorted outcome improvements would have a huge impact when applied to the billions, but fine intervention details, combination effects, and that patronizing glare from your skeptical pediatrician - who might merely be stuck in the past – put the conscientious parent in a tough spot.…Then comes pre-adult interventions that have had remarkable results for animals or adults/elders, but have never been proven for pre-adults. Herein lie some of the most promising elements of UP. For example, there exist a GRAS nutraceutical, HMB; used to accelerate healing by sports enthusiasts and wound-care specialists. A recent experiment with our embarrassingly-close porcine relatives revealed that pregnant sows who had HMB added to their feed delivered piglets that were reliably better developed. The enhancement was evident at birth and throughout their lives (unfortunately, artificially-truncated), in all of numerous measured parameters (e.g., ~20% [!!] increased birth weight). This is attributed to the high-damage environment of gestation being mitigated by HMB’s healing enhancement feature. Imagine broad spectrum, double-digit improvement of neuro-physical characteristics for human babies around the globe, and you will see the unmatched humanitarian and fiscal potential of UP! …And this is just one of many promising and yet-undiscovered pre-adult interventions that will be derived from adult and veterinary health, aesthetics, performance and longevity medicine.UP is dedicated to establishing, through assertive advocacy, well-funded discovery, optimization, and highly-inclusive implementation of known and pending pre-adult interventions that enhance post-pubescent bio-physical potential for health, happiness, unity, productivity and longevity. Due to the decades required for so-upgraded bairns to significantly enter the adult population, and the mounting existential threats to society, implementation of the Uberkinder Program is profoundly urgent, and merits the highest level of advocacy and financial support by the Foresight Institute. A monograph that includes deeper consideration, and numerous supportive references is available upon request. ""SENthetic LethalitySynthetic lethality is the biological phenomenon whereby inactivation of either gene A or B is tolerable, yet the loss of both is lethal. First described in 1922 (for Bar and Glass genes in drosophila), it wasn't until over 70 years later that Hartwell, Friend et al postulated that synthetic lethality could be used to target cancer, whereby one gene is a cancer-specific mutation and the second gene product acts as the therapeutic target. This approach has seen great success, for example the development of PARP inhibitors for treating BRCA-deficient cancers. Whilst early attempts relied on RNA interference methods, the advent of CRISPR-based gene editing has made full genome screening more tangible/scalable to the point where numerous big pharma companies have synthetic lethality cancer drug discovery programmes.I now propose that this method should be applied to longevity research to target senescent cells.The approach would be to make KO cell lines of genes downregulated in senescence (e.g Lamin B1, SIRT1 etc) and then perform full genome CRISPR KO dropout screens to identify new ""SENthetic lethal"" drug targets.Furthermore, a variation on the theme is Synthetic Dosage Lethality (SDL) whereby the downregulation of gene A coupled with the overexpression of gene B results in cell death. For this, overexpression cell lines of genes upregulated during senescence (e.g p16, p21 etc) would be used. An additional approach would be to utilise full genome CRISPR activation screens to allow the discovery of SDL targets whose overexpression in senescent cells leads to death - which could be exploited using RNA therapeutics.""Hormone replacement therapy (HRT) involves supplementing certain hormones that decline in production with age. The main focus of HRT is on growth hormone (GH) and testosterone, as they are both associated with age-related diseases and have been shown to decline with age. By replacing these hormones, it is thought that they can help to slow down or even reverse the effects of aging.Although this area of research has become increasingly popular in recent years, it is still not widely accepted. This is mainly due to the fact that there are still some major risks associated with hormone replacement therapy. These include side effects such as increased risk of cancer, stroke and heart attack; decreased fertility; and increased risk of blood clots. Additionally, there are concerns about long-term safety as well as potential for drug interactions when combined with other medications.Despite the potential risks, many researchers believe that hormone replacement therapy could be a viable treatment for aging-related diseases and conditions. For instance, animal studies have shown promising results when using growth hormone in the treatment of osteoporosis, type 2 diabetes and cardiovascular disease. Similarly, testosterone has been studied for its potential to improve strength, muscle mass, libido and overall quality of life.In conclusion, although hormone replacement therapy holds great promise as a way to extend lifespan or improve healthspan, it is still an underappreciated area of longevity research due to its associated risks. However, if further studies can provide evidence of safety and efficacy, then this approach could become an important tool in delaying aging-related diseases and ultimately extending the human lifespan."4a:T42fbe,

Longevity Prize - Hypothesis Prize winners and submissions

An overview of the Longevity Prize - Hypothesis Prize winners and submissions

“What is the most promising but underappreciated or obscure area of longevity science we should pursue?

Prize Description: "Over a century ago, many questionable experiments and surgical procedures were performed – heterochronic parabiosis (HCP) being one of them. This technique was revived by Irina Conboy at Stanford/Berkeley in the 200’s. Today, it’s one of the hottest (and most difficult to perform) models of aging research.

Over a century of all the world’s biological knowledge is available to anyone taking the time to read the literature. There are cases where key discoveries are made in the past, but forgotten for long periods of time – only to be rediscovered. The hypothesis prize aims to resurface such discoveries and research areas, focusing our attention on the most promising directions.”

Winners

#1 Carlos Galicia (USA), galiciaa@usc.edu, University of Southern California / Buck Institute https://www.linkedin.com/in/cargalicia

GPT-Summary: “The article proposes studying the rejuvenation process that occurs during embryogenesis as a promising opportunity against age-related decline. The study would involve identifying age-related changes in gametes, tracking those hallmarks of aging during embryogenesis, and harnessing the rejuvenation strategies used by the embryo. The article suggests using deep phenotyping, SMRT DNA sequencing, and multi-omic data analysis to gain insights into the rejuvenation processes. The article also discusses the potential clinical applications of the study's findings.”

In 1930 Tracy Sonneborn published his observations on the reproduction of the flatworm Stenostonum Incaudatum. This flatworm reproduces asexually by fission and produces two daughter organisms. Sonneborn noticed that the daughter piece of the worm that requires more cell divisions to become a full-size worm, lives longer than the one that undergoes less cell multiplication (Sonneborn, 1930). This observation suggested an interplay between cell division and rejuvenation during development. Some species of planarian flatworms and hydra, thought to be immortal, undergo a similar process in which old cells are constantly being removed and replaced by new ones (Muller, 1996; Sahu et al., 2017). One of the reasons why cell division might be necessary for rejuvenation to occur is that the molecular components of a cell might not be able to be repaired indefinitely. In E. coli, after cell division occurs, the cell that inherits the old pole has a higher incidence of death (Stewart et al., 2005). Similarly, in budding yeast, the mother cell accumulates damage that is not passed down to the daughter cells and leads to the detriment of the mother cell (Egilmez & Jazwinski, 1989).

In mammals, the major rejuvenation event that takes advantage of cell division occurs during embryogenesis, when gametes from adult individuals fuse to form a zygote that divides billions of times. Through this process, molecular damage and other age-related changes that occur to gametes are erased as new organisms that come from old parents are young. The rejuvenation processes utilized during embryogenesis present the most promising opportunity against age related decline, as they are the solution developed by evolution and the programs are already encoded in our DNA.

To understand and harness the rejuvenation process that occurs during embryogenesis I propose three phases. The first phase would focus on identifying age-related changes in oocytes and sperm. Before we can study the mechanisms that drive rejuvenation during embryogenesis, we need to understand the molecular damage that accumulates with age in the cells that give rise to the embryo. This can be accomplished with deep phenotyping of oocytes and sperm collected across the lifespan of individuals.

To study age related changes to the genome, one would focus on studying covalent modifications that occur to DNA as those have a higher chance of being passed down to the embryo. SMRT DNA sequencing and modular enzymatic labeling paired with nanopore sequencing would be able to detect 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, thymine dimers, uracil incorporation, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine at a single base resolution (Clark et al., 2011; Wang et al., 2017). These would make it possible to map these covalent modifications to specific sites in the genome and explore their dynamics during aging, similar to what has been accomplished with methylation clocks.

For oocytes additional characterization is needed because they provide most of the cytoplasm for the embryo. Characterization of mitochondrial function and dynamics, mass spectrometry of soluble and insoluble protein fractions, RNA sequencing, unbiased metabolic and lipidomic profiling as well as quantification of products of oxidation and lipofuscin would provide useful insights. These data would allow us to understand the age-related changes that embryogenesis is equipped to reverse as well as provide valuable insight into age related changes in the quality of oocytes which is indispensable for the area of reproductive aging.

Once age related changes have been identified in gametes, the second phase would focus on tracking those hallmarks of aging during embryogenesis to understand at what point they are rejuvenated. Transcriptomic, proteomic and metabolomic characterization of the embryos would also be obtained to gain deeper understanding of the mechanistic networks driving the rejuvenation processes.

The third phase of the study would make use of these data to harness the rejuvenation strategies used by the embryo. The multi-omic data could provide candidate genes, peptides or molecules that correlated with the rejuvenation event. This would allow for planning of screens or other mechanistic studies specific to the rejuvenation event.

FAQ:

Isn’t reprogramming with Yamanaka factors already trying to harness the power of development?

Yamanaka factors are believed to rejuvenate in a similar way to development. Nevertheless, induced pluripotent stem cells (IPSCs) generated with Yamanaka factors differ from pluripotent stem cells derived from embryos, suggesting different mechanisms. For instance, IPSCs are more susceptible to genomic instability than their embryonic counter parts (Zhang et al., 2018). It is possible that the embryo utilizes other rejuvenation strategies, and reprogramming resembles one of them but until a systematic study of embryogenesis is done, we don’t have assurances that Yamanaka factors are enough to produce a rejuvenation event equal to that of embryogenesis. By focusing our attention on the naturally occurring way of reversing aging, we could find new and better rejuvenation strategies.

How is this a promising and obscure area of longevity science?

The Gladyshev lab has shown the great potential of studying embryogenesis from an ageing perspective by showing methylation age reversal around gastrulation (Kerepesi et al., 2021). However, despite its great potential, very little effort is being directed towards understanding germline rejuvenation. A search on PubMed for germline rejuvenation produces 17 results while the amount of literature for other forms of aging research is vast.

What would be the timeframe and budget required for this study?

The timeframe and budget to complete this proposal would be highly flexible. For example, using Zebrafish or Xenopus, phase 1 and 2 could be completed in a year because collection of germ cells and embryos is relatively simple and older animals could be sourced from labs studying these organisms. However, obtaining germ cells and embryos at different stages from mice would be a more laborious process that could double the time to completion. The cost for data generation is equally flexible. With a small budget, exploratory experiments at very young and old ages could be performed to see what types of data are more promising and then increase temporal resolution on those. Significant progress could be made by an individual with $60,000-100,000 in funding in one year.

How could these discoveries be taken to the clinic?

The specifics of how to translate this newly gained understanding of embryonic rejuvenation would vary on a case-by-case basis. For example, if a well-defined transcriptional program is found to be correlated with rejuvenation of a specific hallmark of aging, one could attempt to modulate transcriptional networks through small molecules as has been done for drug repurposing with data from the L1000 project. If a hallmark can be measured at the single cell level and occurs at a stage of embryonic development where ESCs can be obtained, CRISPR screens could be used to identify targets able to modulate the program. Another important step would be to identify hallmarks that cannot be rejuvenated without cell division, this would create valuable insight into what form of rejuvenation would require more engineering.

References:

Clark, T. A., Spittle, K. E., Turner, S. W., & Korlach, J. (2011). Direct detection and sequencing of damaged DNA bases. Genome Integr, 2, 10. https://doi.org/10.1186/2041-9414-2-10

Egilmez, N. K., & Jazwinski, S. M. (1989). Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae. J Bacteriol, 171(1), 37-42. https://doi.org/10.1128/jb.171.1.37-42.1989

Muller, W. A. (1996). Pattern formation in the immortal Hydra. Trends Genet, 12(3), 91-96. https://doi.org/10.1016/0168-9525(96)81419-3

Sahu, S., Dattani, A., & Aboobaker, A. A. (2017). Secrets from immortal worms: What can we learn about biological ageing from the planarian model system? Semin Cell Dev Biol, 70, 108-121. https://doi.org/10.1016/j.semcdb.2017.08.028

Sonneborn, T. M. (1930). Genetic studies on stenostomum incaudatum (Nov. spec.). I. The nature and origin of differences among individuals formed during vegetative reproduction [https://doi.org/10.1002/jez.1400570104]. Journal of Experimental Zoology, 57(1), 57-108. https://doi.org/https://doi.org/10.1002/jez.1400570104

Stewart, E. J., Madden, R., Paul, G., & Taddei, F. (2005). Aging and death in an organism that reproduces by morphologically symmetric division. PLoS Biol, 3(2), e45. https://doi.org/10.1371/journal.pbio.0030045

Wang, F., Zahid, O. K., Swain, B. E., Parsonage, D., Hollis, T., Harvey, S., Perrino, F. W., Kohli, R. M., Taylor, E. W., & Hall, A. R. (2017). Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process. Nano Lett, 17(11), 7110-7116. https://doi.org/10.1021/acs.nanolett.7b03911

Zhang, M., Wang, L., An, K., Cai, J., Li, G., Yang, C., Liu, H., Du, F., Han, X., Zhang, Z., Zhao, Z., Pei, D., Long, Y., Xie, X., Zhou, Q., & Sun, Y. (2018). Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining. Cancer Commun (Lond), 38(1), 49. https://doi.org/10.1186/s40880-018-0313-0

Kerepesi C, Zhang B, Lee SG, Trapp A, Gladyshev VN. Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging. Sci Adv. 2021 Jun 25;7(26):eabg6082. doi: 10.1126/sciadv.abg6082. PMID: 34172448; PMCID: PMC8232908.

#2 Rakhan Aimbetov (Kazakhstan), r.aimbetov@gmail.com, Lead @ LabDAO and Hack-Age

GPT-Summary: Maintaining accurate conversion of genetic information into protein structure is crucial for cellular function, but errors in transcription and translation can disrupt proteostasis and contribute to aging and age-related pathologies. Methylglyoxal (MGO) is a byproduct of glycolysis that can modify amino acids and impair protein function, and its production has been implicated in diabetic complications and organ decline. Additionally, the stiffening of the extracellular matrix (ECM) can lead to a loss of proteostasis due to increased glycolysis and subsequent MGO generation, potentially leading to ribosomal dysfunction and an increase in mistranslation rate. Shedding light on these mechanisms could pave the way for interventions to extend healthy life.

Accurate conversion of genetic information into protein tertiary structure is one of the pillars of proper cellular function, and disruption of protein homeostasis (proteostasis) that leads to intracellular accumulation of misfolded proteins is a hallmark of aging and a contributor to age-related pathologies.

The mechanisms of the aforementioned conversion are somewhat “noisy” – for transcription, the error rate is 10-5-10-4 (1/100,000 ribonucleotides are mismatched with the transcribed DNA template); for translation, the rate is 10-4-10-3 (1/10,000 mRNA codons are paired with a wrong amino acid). In addition, erroneous tRNA aminoacylation (when tRNA is coupled with an amino acid that does not match the anticodon) presents an extra source of mistakes in the flow of information from DNA to protein.

Any deviation from the background error rate has its effect on the aging dynamics. It has been shown that certain long-lived organisms inherently benefit from a relatively more faithful translation (1–3), whereas experimental elevation of translation error rate in animal models shortens lifespan (4). It is therefore important to deconstruct the mechanisms that alter the fidelity of protein synthesis in order to devise strategies and interventions for extending healthy life.

Methylglyoxal (MGO) is a byproduct of glycolysis with reactivity towards amines – arginine guanidino groups, lysine amino groups, and exposed protein N-termini. Non-enzymatic modification of amino acid residues by MGO leads to the formation of various chemical adducts and crosslinks (collectively known as advanced glycation endproducts (AGEs)) that may affect the susceptible proteins’ three-dimensional conformations (in other words, how they are spatially folded) and proper functioning (5). There are numerous reports that implicate MGO production in diabetic complications – cardiomyopathy, nephropathy, peripheral neuropathy, retinopathy, etc. – and age-related organ decline.

Unfolded protein response (UPR) is a mechanism to circumvent the accumulation of misfolded proteins either in the lumen of the endoplasmic reticulum (ER stress) or in the cytosol (cytosolic UPR). It has been shown that cells grown in presence of high glucose display ER stress mediated by MGO (6). The authors conclude that the UPR is brought about by non-enzymatic post-translational modification of existing proteins by MGO. I propose an additional hypothetical mechanism: MGO-mediated glycation of ribosomes – i.e. ribosomal proteins, rRNAs – increases the error rate at the level of translation resulting in enhanced synthesis of polypeptides prone to misfolding.

Extracellular matrix (ECM) is a heterogeneous but highly organized matter located in-between cells that provides structural support to tissues. Cells and their environment, represented by the ECM, communicate reciprocally to drive organogenesis during development and remodeling in response to insult. ECM composition and ensuing mechanical properties greatly affect cellular behavior. For example, mesenchymal stem cells commit to either bone or muscle lineages depending on substrate elasticity. Moreover, the accumulation of AGE-crosslinks (including but not limited to MGO-derived crosslinks) in matrix collagen has been shown to increase ECM stiffness and contribute to cardiovascular pathology.

Cells continuously monitor the state of their environment, including its mechanical properties, via cell-surface receptors. Transduction of mechanical cues (mechanotransduction) is mediated by integrin receptors that probe the ECM elasticity and initiate a cascade of intracellular responses to fine-tune cellular behavior to the surrounding mechanical context. Increased ECM rigidity (stiffness) leads to translocation of the transcription coactivator YAP/TAZ from the cytoplasm into the nucleus where it binds to the transcription factor TEAD, driving expression of genes involved in tumorigenesis: those promoting growth, proliferation, survival, glucose uptake, glycolysis, epithelial-to-mesenchymal transition, apoptosis inhibition. Concurrently, I propose that enhanced glycolysis (and subsequent MGO generation) in response to the stiffened ECM leads to the loss of proteostasis at least partially due to ribosome glycation and an ensuant increase in mistranslation rate.

The proposed mechanism of proteostasis disruption might have broader implications for the ECM organization. For instance, the substitution of key amino acid residues in tropoelastin has been shown to impede monomer coacervation and lead to aberrant elastic fiber formation (7).

The hypothetical mechanism of ribosomal dysfunction has not yet been described in the literature. Shedding light on novel aspects of translation fidelity, especially in relation to metabolism, will draw a link between the tightly regulated proteostasis maintenance machinery and the stochastic nature of glycation, possibly paving the way to a new class of life-extending interventions.

For the full project details, including preliminary data and further plans, please visit -- https://docs.google.com/document/d/1QLzipbn6umWGg2MfyyxEROxdzsWMWvXoiG_WBVFHzPQ/edit?usp=sharing

#3 Shahaf Peleg (Germany), Group leader Leibniz Institute for Farm Animal Biology, shahafpeleg3@gmail.com, https://www.linkedin.com/in/shahaf-peleg-72790263/?originalSubdomain=de

GPT-Summary: ”The article discusses how mitochondrial dysfunction is a central hallmark of aging and current interventions aim to reduce mitochondrial activity to extend lifespan and health span. However, this may impair the animal's ability to respond to stress and it is questionable whether such approaches could be translated into human longevity therapy. The article proposes a novel approach called external energy replacement, which involves using an engineered light-sensitive proton pump called mtON to generate ATP in the absence of oxidative phosphorylation. The authors hypothesize that using mtON technology in mammals will attenuate aging in the eye and skin by compensating for lack of ATP, reducing metabolic associated damage, and enabling people to eat less while maintaining physiological ATP levels. The authors believe this approach has great promise in attenuating human aging in a novel manner.”

FBN "Mitochondrial dysfunction is a central hallmark of aging and many of the current interventions to extend lifespan and health span target the mitochondrial metabolism. For example, dietary interventions, genetic interventions (e.g., AMPK) and drugs (e.g., metformin and rapamycin) extends lifespan by, at least partially, targeting mitochondria. Generally, many of the interventions aim, quite surprisingly, towards a reduction of mitochondrial activity. A prominent example is the induction of mitochondrial unfolded protein response (mtUPR) during development, where reducing components of oxidative phosphorylation results in increased lifespan. However, it is noteworthy that such intervention reduces the physical activity of the animal in question and likely impairs the ability of the animal to respond to other type of stress. Therefore it is questionable whether such approaches could be translated into human longevity therapy.

Here we hypothesize a novel and radical approach termed external energy replacement. What does it mean?

It is established that plants and bacteria can capture the energy of light and translate it into energy in their cells, while animals must consume other organisms as food, in order to generate energy. Thus conventionally, evolution made a clear distinction between autotroph and heterotroph organisms. However, the idea of bringing proteins from evolutionarily distant organisms such as bacteria into eukaryotes can result in a leap in molecular biology. One well-known example is the bacterial CRISPR/Cas9 system, which was adapted to edit eukaryotic target genes. Another very recent example is introducing bacterial/fungal light-sensitive protein that generates proton gradients across in eukaryote mitochondria to generate chemical energy, which is a massive leap in molecular biology.

In this hypothesis, we propose a major breakthrough in the field of metabolic aging. Specifically, we propose to test if mtON, an engineered light sensitive proton pump, will act as metabolic rescue for mammalian age-related mitochondrial dysfunction in the eye and the skin, where light is easily accessible. When activated by light, mtON pumps protons across the inner membrane of the mitochondria, thus contributing to the proton gradient that is essential to generate ATP. This genetic intervention practically enables a heterotroph to harness the energy of light to generate ATP in the absence of oxidative phosphorylation.

We recently were the first and only scientists to show that the usage of mtON technology can increase lifespan in worms. Here, we hypothesize that using mtON technology in mammals will attenuate aging in the eye and skin. We hypothesize that using mtON will have several benefits. First, in an advanced form of aging, where mitochondria are dysfunctional and cannot generate enough ATP, that mtON treatment coupled with light can enable the aged eye and skin cells to generate youthful levels of ATP. Essentially, the light energy transformation will compensate for lack of ATP. Secondly, mtON technology may reduce the accumulation of metabolic associated damage. For example, we hypothesize that as ATP is now being partially generated by light, the cell would adapt and slow down its upstream metabolic (e.g., TCA cycle, glycolysis). Therefore, less biochemical reactions would take place, thus reducing the production of cellular waste, such as advanced glycation end-products, which contribute to the progression of aging. Also, as oxidative phosphorylation is less required, we hypothesize a reduction in the levels of reactive oxygen species (ROS), which may also benefit to generate less cellular damage during aging. Thirdly, it is possible that mtON would enable people to eat less while maintaining physiological ATP levels. As such, people may enjoy the benefits caloric restriction, which is believe to improved health span, while avoiding jeopardizing dysregulated ATP levels. This would be even more important once mtON technology would be upgraded to be used in inner organs beyond the skin and the eye.

Taken together, our hypothesis of using light as an external energy replacement is quite revolutionary and show great promise of success in attenuating human aging in a novel manner.

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Other interesting submissions

John Hemming: “The Gompertz-Makeham law of mortality predicts mortality, with the Gompertz part relating to gradual health deterioration. Stem cell failure to differentiate may cause this, with a feedback loop involving Interleukin-10, which inhibits Nuclear Factor-κB and reduces inflammation from senescent cells. This loop may drive much health deterioration, fitting the Disposable Soma theory. Mitochondrial function is important in aging, and Heterochronic Parabiosis can explain its benefits.”

Clarice Demarchi Aiello “The hypothesis proposes using tailored magnetic fields to control quantum signaling in cells for longevity's advantage. Weak magnetic fields, via the electron quantum property of 'spin,' have been shown to control many disease markers and cell activities. By designing novel instrumentation to directly measure and control spin states and their biological consequences, it may be possible to alter quantum signaling in cells to promote longevity. The long-term goal is to develop drugs and therapeutic devices that heal the human body via quantum signaling. The proposed instrumentation includes microscopes, electrophysiology setups, and scanning tunneling microscopes coupled to coils and radio-frequency microchips. These tools will allow for the visualization and manipulation of regeneration by weak magnetic fields, among other applications. Ultimately, the hypothesis seeks to control native electromagnetic-responsive pathways for personalized medicine and improved longevity.”

John Jackson “Studies have found that certain animals that live a slower paced life have "protective traits" that help to extend lifespan. Can we study these protective traits more and implement them permanently within the human body?”

Alexei Goraltchouk “Remedium Bio believes that Growth Factors can extend healthspan and lifespan, and are a promising solution to age-related diseases. Growth Factors have shown remarkable results in clinical trials, with repeat intra-articular injections of FGF18 reversing cartilage loss in Osteoarthritis patients. Remedium is developing a gene therapy based on FGF18 to restore a more youthful and healthy phenotype to arthritic joints.”

Dian Ginsberg MD “The study aims to evaluate the safety and efficacy of administering young Fresh Frozen Plasma (yFFP) to aged subjects for therapeutic uses. The study will compare epigenetic, proteomic, laboratory, and functional results taken from before the initial treatment to results taken one month after the last treatment. The study will involve 30 patients, and each participant's starting condition will be identified using TruDiagnostic and Seer tests, laboratory markers, and functional markers. The primary outcome is the change in the epigenetic age or genome pattern, laboratory, and functional assessments of the yFFP treated patients. The study will take place at multiple sites in the State of Texas.”

Kourosh Hojjati: “The writer believes that in the process of cell division, copying the ribosome is the most important factor, which becomes impaired with age. Finding an agent or stimulus that minimizes the problems of copying or prevents cells from reproducing with defects could lead to a longer life.”

Daniel Bar “Epigenetic clocks measure DNA methylation at specific sites and offer an accurate measurement of biological and chronological age. Interventions that prolong healthspan and lifespan also slow the biological clocks. Identifying the direct mechanism behind the epigenetic clock reset is important to exploring the causality of epigenetic clocks. A list of candidate genes can be generated by integrating single-cell expression data, which can be tested individually or in combinations in cell culture for epigenetic reset.”

Dmitry Dzhagarov (Dimitri Jagarovi) “The review proposes a new approach to treating age-related diseases using a combination of senolytics to remove aged cells and ROCK inhibitors and 5-LOX inhibitors to stimulate the regeneration of new cells. The review suggests that this combination of drugs can conditionally reprogram cells in situ, leading to proliferation of progenitor cells that can make up for losses. The review discusses the importance of senolytics and explains the difficulties in using mesenchymal stem cells for replacement therapy. The review explores the use of Conditionally Reprogrammed Cells (CRC) as a potential treatment for age-related diseases. The combination of ROCK and 5-LOX inhibitors may promote cell proliferation without promoting oncogenesis. The correct choice of Senolytic can significantly increase the effectiveness of such treatment. Treatment with such a combination of drugs should be short-term and repeated several times a year.”

Suslova Alexandra: “The article explores the idea that the human mind has the potential to heal the body, citing examples such as the placebo effect and personal experiences. It suggests that the science of neurobiology should be studied further to understand this connection. The article also discusses studies on the influence of thoughts and emotions on the body, including experiments with DNA molecules. It highlights the importance of a positive mindset for a healthy and happy life, and the potential of changing one's brain to achieve this. The article concludes by emphasizing the need to study the structure of the brain and the influence of thoughts and emotions on the body for optimal health and well-being.”

Varun Dwaraka: “The article discusses how epigenetic regulation through changes in gene expression can cause long-term effects on aging phenotypes, and how the stability of methylation marks on imprint control regions (ICRs) throughout life makes them ideal long-term records of early exposures. The article highlights the recent development of a custom Infinium methylation array to quantify DNA methylation patterns of the human imprintome and investigate any links between ICRs and age-related phenotypes, using DNA and multi-omic aging data previously collected from a collaboration with Partners Biobank and Brigham Women’s hospital. The article proposes three aims to investigate the relationship between ICRs, aging, and AD development, and to identify new aging biomarkers and possible therapeutic pathways.”

Muneera Fayyad “This project aims to isolate mesenchymal stem cells from plucked hair follicles, which have high proliferation, differentiation ability, and efficient reprogramming potential. These stem cells will be differentiated into various lineages and employed in pre-clinical and clinical models to treat aging and degenerative diseases such as osteoarthritis, cartilage degeneration, and spinal cord injury. The use of an easily obtainable, autologous stem cell source like the hair follicle alleviates safety concerns associated with most stem cell-based therapies.”

Albert Cheong: “The study aims to characterize the gut microbiome of centenarians and investigate the potential of using their microbiome profile as a method to increase human longevity. The study will involve sequencing the gut microbiome of 200 centenarians and comparing it to the gut microbiome of younger individuals. The study will also investigate the feasibility of developing interventions to modify the microbiome to promote healthy aging.”

Alexandra Suslova: “The article explores the power of the mind to affect the body's healing process and the promising areas of study for longevity science. It discusses how thoughts and emotions can influence matter, including DNA molecules, and how the mind and body are in constant sync. The article presents scientific experiments that show the relationship between a person's mood and their health and longevity. It concludes that a person can heal themselves and destroy themselves, and that our diseases are mainly caused by stress hormones rather than genetics. The article emphasizes the importance of studying the structure of the brain and the influence of thoughts and feelings on the body for a happy and healthy life.”

Ziwei Li: “The author is researching longevity by combining traditional Chinese medical theories, Taoism, and modern technological means of testing. They focus on the concept of inner alchemy, which is a Taoist practice of using the human body as a furnace and refining vital essence and Qi to reach immortality. The author believes that scientific tests can be used to quantify and record the changes that occur in the human body during the stages of inner alchemy. By combining Taoism, traditional Chinese medicine, and modern medical tests, the author hopes to find a natural way to enhance and transform human life, ultimately leading to a longer and healthier life.”

Steven Fowkes: “Cellular hydration, which drops from 90% in the early stages of embryonic development to 50% in adults, is proposed to be a foundational aspect of development and aging. Potassium utilization, the ratio of cellular potassium to serum potassium, is suggested as a surrogate marker for cellular hydration, which can be easily and minimally invasively measured, making it a viable option for assessing this underappreciated aging-associated biomarker.”

Denis Odinokov: “The weight loss industry is worth over $5.5 billion in the EU and muscle mass decreases while fat mass increases with age. A promising approach to weight management is changing the energy metabolism of adipose cells or supporting white-to-brown-fat conversion. Cell-free DNA fragments from adipose cells in the bloodstream can be used to track the ratio of white to brown fat cells as a novel metric for obesity research and healthy aging. A non-invasive laboratory method to measure this ratio is currently being developed.”

Mikhail Batin: “Age-related changes in the extracellular matrix (ECM) are an important but underestimated area in life extension research. The accumulation of crosslinks in proteins, such as collagen and elastin, contributes to ECM rigidity, cell dysfunction, and chronic inflammation. The slow renewal of these proteins and the absence of reliable antiglycation mechanisms make glycation of long-lived proteins almost inevitable. Age-related changes in ECM proteins are associated with most other signs of aging and contribute to the formation of mechanisms involved in aging and age-related diseases. The structure of the ECM also contributes to neurodegenerative processes in aging. Thus, age-related changes in the ECM structure play a large but understudied role in aging.”

Denis Odinokov: Stochastic damages to molecular structures lead to incomplete digestion of proteins and accumulation of indigested fragments, resulting in increased stiffness and malicious nano topography. Enzymatic activity against cross-linked collagen is limited, and targeting indigested fragments with ferromagnetic nano-tags and inducing local hyperthermia may be a better research direction.

Every submission (above the threshold votes for participation)

"Wolbachia is an alpha -proteobacterium, an intracellular symbiont of invertebrates - insects, isopods, arachnids and filaria. Its effects include cytoplasmic incompatibility, androcide, male feminization, and parthenogenesis. So far, Wolbachia has not been found in vertebrates, except for cases of infection with filariae, the obligate hosts of Wolbachia. Only one work indicated a positive analysis of the presence of Wolbachia DNA in biomaterial samples taken from a reptile, in the presence of a negative test for the presence of the obligate hosts of Wolbachia - filariae. The same work showed a high degree of Wolbachia infestation of ticks parasitizing reptiles. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397688/
We show that Wolbachia successfully passed from ticks to reptiles and subsequently to mammals, with the preservation of most of its effects, to which one more was added - slowing down age-related changes, puberty and aging.
https://www.facebook.com/valeriy.golub"
"Non-coding RNAs and aging

Main efforts in aging studies are devoted to processes associated with protein-coding genes. But as we know, there are less than two percent of such genes in the human genome. The rest of the genome related to aging remains little-studied. It applies to a great extent to non-coding RNAs.
Usually, all non-coding RNAs are divided into two large classes: microRNAs and long non-coding RNAs (lncRNAs).
MicroRNAs are a class of small non-coding RNA molecules 18-25 nucleotides long, which are actively involved in the regulation of gene expression. The action of microRNAs is very diverse and appears to be closely related to many processes occurring in the body, including maintenance of the genome stability, immune responses, differentiation, proliferation, and apoptosis of cells.
Numerous studies have shown a close link between changing microRNA levels and cardiovascular, cancer, and neurodegenerative diseases (i.e., major age-related diseases), as well as a direct role of microRNAs in the life span regulation.
At the same time, according to current estimates, the expression of about 60% of human genes is directly related to the action of microRNAs, and the functions of most of them still remain undetermined.
https://academic.oup.com/clinchem/article/55/11/1944/5629329
https://pubmed.ncbi.nlm.nih.gov/27192016/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655276/
https://pubmed.ncbi.nlm.nih.gov/28934394/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954352/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463915/
In a number of studies, long non-coding RNAs (lncRNAs longer than 200 nucleotides) have also been shown to be involved in the processes associated with aging and longevity. Depending on their location and orientation in the genome, all lncRNAs are divided into seven different groups: sense, antisense, bidirectional, intergenic, intronic, enhancer, and promoter lncRNAs. They play an important role in many biological processes, such as transcription, post-transcriptional processing, and chromatin modification. Mechanisms used by lncRNAs to perform their function include interaction with other types of RNA or DNA, creation of a framework of subcellular domains or complexes, and regulation of protein activity or amount. In addition to being functional, the lncRNA transcript itself can also influence the structure of the nucleus, the epigenetic landscape, or the expression of nearby genes.
Researchers describe the direct involvement of lncRNA in such aging-related processes as genome instability, cell aging, telomere shortening, chromatin remodeling, and development of main age-related pathologies.
https://pubmed.ncbi.nlm.nih.gov/32565330/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880696/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641121/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140545/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509469/
RNA editing (epitranscriptomics) is another understudied area closely related to aging. Nowadays, more than one hundred different RNA modifications are known.
https://www.nature.com/articles/s43587-021-00058-y
Circular RNAs (circRNAs) are another little-studied type of non-coding RNAs associated with aging and life span regulation. They are widely expressed in eukaryotes and have multiple functions. Recent studies show that levels of circRNAs change with age in various tissues in many species, from nematodes to mammals. It is no coincidence that a recent study proposed that changes in circRNA transcripts are another sign of aging.
https://www.cell.com/trends/genetics/fulltext/S0168-9525(21)00126-8
https://pubmed.ncbi.nlm.nih.gov/29753875/#:~:text=Recent%20profiling%20of%20circRNAs%20genome,role%20in%20the%20aging%20process%3F
With all that said, non-coding RNAs are still less studied by researches exploring protein-coding genes and aging.
And as João Pedro de Magalhães, a well-known researcher of aging, notes in his work, the aging-related changes in the transcriptome, which encodes proteins, have been characterized in detail. But much less attention has been given to the non-coding part of the human genome, especially long non-coding RNAs. As a result, only a small number of known lncRNAs have been functionally characterized.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961210/"
"Pseudogenes. Mysterious Elements of the Genome, Regulation of Gene Expression, and Much More

The very name of these genomic structures - pseudogenes - implies that something is wrong with them and that they are very different from the regular protein-coding genes. This is indeed true. Pseudogenes used to be functional genes that subsequently lost the ability to code for a protein due to mutations that arose in their sequence.

At this point a careful reader may immediately ask why such damaged genes were not selected by purge selection? Scientists have three answers to this question. In one case, a pseudogene can be a result of gene duplication (doubling), i. e. when one copy remained functional, and the second one became a non-functioning pseudogene due to damage. Since one copy of a regular functional gene proved to be sufficient for the life of an organism, the transformation of the second copy into a pseudogene did not go through the purge selection. It is estimated that 3,391 fully functional parental genes are associated with pseudogenes in the human genome. At the same time, almost two-thirds of the parental genes gave rise to only one pseudogene, and only a small part of the parental genes gave rise to dozens of pseudogenes each.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491395/

In the second case, many such duplications are created by reverse transcription using retrotransposon enzymes. The result is intron-poor fossils devoid of promoters that are called ""processed pseudogenes."" In the human genome, processed pseudogenes are the most common type of pseudogenes due to a surge in retrotransposition activity in primate ancestors about 40 million years ago. The total number of such retrocopies in the human genome is estimated at more than 8,000 units.
https://pubmed.ncbi.nlm.nih.gov/17424906/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC329124/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314676/

According to the third scenario, a pseudogene could flap away from the selection if the loss of its function became not critical for the organism. A typical example of this setup is the pseudogene GULO, typical of higher primates. For millions of years, it synthesized vitamin C in the organisms of our distant ancestors. Mutation that disrupted GULO’s functioning coincided with our ancestors’ transition to a plant-based diet rich in vitamin C. Thus, the internal synthesis of the necessary vitamin was replaced by external intake. The GULO gene, having ceased to be critically important, successfully turned into a pseudogene. Remarkably, dogs and cats have processed GULO pseudogenes, being the result of retrotransposons’ activity. In humans, the GULO pseudogene is also reliably blocked epigenetically, to prevent any possibly functional part of this former gene from affecting the expression of other genes - e. g. the Clu gene, which is next to GULO and is associated with exceptional longevity in humans. By evolution, GULO is completely inactivated and no longer bothers anyone.
https://link.springer.com/article/10.1007/s10528-013-9574-0

Note that a changed diet has repeatedly contributed to the creation of pseudogenes. Thus, our very distant ancestors used to actively eat insects. For their digestion they had special enzymes that break down chitin and chitinases. After the extinction of the dinosaurs, our ancestors began to grow larger and move on to more high-calorie foods, refusing or greatly reducing eating of insects. As a result, some of the genes encoding chitinases successfully turned into pseudogenes.
https://www.science.org/doi/10.1126/sciadv.aar6478

However, not all pseudogenes became genetic fossils. Some of them can be quite active.

First, they can regulate the expression of their parent gene, reducing the stability of its mRNA. For example, the MYLKP1 pseudogene, which is activated in cancer cells, generates non-coding RNA (ncRNA), which in turn inhibits mRNA expression of its functional parental gene, MYLK.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137860/

Studies in Drosophila and mice have shown that small interfering RNAs (siRNAs) derived from processed pseudogenes can regulate gene expression through one of the epigenetic mechanisms - RNA interference.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206313/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981145/
https://pubmed.ncbi.nlm.nih.gov/18404146/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708354/

It has also been described that pseudogenes with a high degree of sequence homology to their parental genes can regulate their expression through the formation of antisense transcripts, such as the antisense long non-coding RNA (lncRNA) of the Oct4 pseudogene, which suppressed the expression of both the pseudogene itself and its parental gene.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2999937/

On top of all this, pseudogenes can compete with their parent genes for microRNA (miRNA) binding, thereby modulating the repression of a functional gene via microRNA. For example, the PTEN pseudogene regulates the expression of its parent gene, a key tumor suppressor, through this mechanism of action. A part of the transcript derived from the pseudogene, PTENP1, acts as a miRNA bait that represses the parent gene. It is estimated that this mechanism can have a significant impact in this pathology.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206313/
https://pubmed.ncbi.nlm.nih.gov/18455982/

Another example from the same field is the DUXAP8 pseudogene. It generates a long non-coding RNA that promotes tissue degeneration by suppressing two suppressors, p21 and PTEN.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544748/

Do pseudogenes play a role in aging? All available data indicates that they do, and their role is not unimportant. But, unfortunately, they have been little studied so far. For example, there is a study of lncRNA pseudogenes’ involvement in cellular aging.
https://www.sciencedirect.com/science/article/pii/S1568163715300362

Above, we have already described several examples of the participation of pseudogenes in the processes associated with mutagenesis. In addition, pseudogenes are involved in the formation of neurodegenerative pathologies, one of the main age-related diseases. Thus, the analysis showed that some pseudogenes increase their expression in Alzheimer's, Parkinson's and Huntington's diseases. At the same time, these pseudogenes shared about 80% of the common microRNA binding sites with their parental genes. According to scientists, these highly expressed but untranslated transcripts may act as regulators of the expression of their parental genes. And contribute to the development of neuropathology.

The recently described T04B2.1 pseudogene plays a regulatory role and modulates the aggregation of α-synuclein and β-amyloid proteins, two key factors in neurodegeneration. Another pseudogene, ACTBP2, increases the permeability of the blood-brain barrier and exacerbates neuroinflammation and neurodegeneration in Alzheimer's disease.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484327/
https://www.sciencedirect.com/science/article/abs/pii/S0006291X20322063
https://www.nature.com/articles/s41420-021-00531-y

It is noteworthy that in long-lived naked mole rats, a very small number of pseudogenes, only 244 pseudogenes, were found in the genome after its full reading by Vadim Gladyshev's team. Same as transposons, 25%, compared to 37% in the mouse genome. As for pseudogenes, in naked mole rats predominate genes associated with the loss of functions that these animals apparently do not need underground, such as vision. Interestingly, there are pseudogenes associated with protein ubiquitination. Proteins are labeled with ubiquitin as being subject to degradation in the proteasome. For some reason, this pathway of proteostasis in mole rats is significantly reduced compared to mice.
https://www.nature.com/articles/nature10533

In the case of pseudogenes, one can clearly see how the diversity created in the course of evolution can have some ""side effects"".

When gene duplication, one of the main mechanisms in the evolution of species, in addition to new functional proteins also creates their ""corrupted"" copies, aka pseudogenes, which can later actively participate in pathological processes. It was the case in duplication of the Notch2 gene involved in brain development of embryos. The first duplication of this gene occurred in a common ancestor of humans, gorillas and chimpanzees. However, the resulting copy of the Notch2 gene turned out to be non-functional. It has remained so in monkeys, but not in humans. About three million years ago, a new gene in the genome of our ancestors, the australopithecines, became functional. There were three more subsequent cases of its duplication. As a result, the Notch2NL gene family was formed. The products of these genes are critical for the formation of a large ""human"" brain with a more developed cortex.
https://pubmed.ncbi.nlm.nih.gov/29856954/
https://pubmed.ncbi.nlm.nih.gov/29856955/

In general, it is safe to say that the definition of pseudogenes as ""genetic fossils"" is long outdated. Some of them may have certain functions important for the organism, functions related to aging, age-related pathologies and many other things
"
"The Role of Organs in Aging
Modern aging research focuses largely on aging at the molecular and cellular level, however integrative approaches establishing the contributions of tissues and whole organs to aging and longevity are not as well studied. Current data suggests organs may age at different rates and it is well established in transplantations that different organs have different donor age effects [1-3]. It is therefore hypothesized that organs may exhibit varying degrees of influence on overall organismal aging rate and lifespan. The most straightforward method to test this hypothesis, and provide a potential future aging therapeutic path, would be the evaluation of heterochronic transplantations. Experiments involving heterochronic organ transplants have previously been performed with several organs including ovary, kidney, and thymus [4-6]. Additionally, heterochronic BMD and blood exchange have been well studied [7,8]. There is some suggestion that such transplants can impact lifespan, specifically ovaries, however extensive studies for many organs have not been performed [4]. Notably, while there is evidence heterochronic ovary transplantation increases lifespan, a study of heterochronic BMD transplantation failed to observe an effect on lifespan, providing further evidence for the hypothesis that some tissues and/or organs may exert greater influence on organismal aging and lifespan than others, while some may have no effect [4,7]. In addition to the organs and tissues previously mentioned, heart, liver, and pancreas transplants have also been performed in mice with many organ transplants having well established methods with high degrees of transplant success [9-11]. To test this hypothesis groups of aged mice (or rats) would be assigned to receive a heterochronic organ transplant from young animals for each individual organ to be evaluated, and potentially combinations as well, before being followed up for a battery of health measures, such as the frailty index, and lifespan compared to a sham operated group. Such a study would establish the capacity of each tested organ to influence the aging rate and lifespan of a whole organism, would provide translatable insights into the effects of donor age on transplantation, and potentially even future avenues for aging therapeutics. Additionally, it could establish the effects of an aged organismal environment on youthful organs, furthering our understanding of the role of systemic factors in aging.

Citations:
[1] Nie, Chao et al. “Distinct biological ages of organs and systems identified from a multi-omics study.” Cell reports vol. 38,10 (2022): 110459. doi:10.1016/j.celrep.2022.110459
[2] Dayoub, Jose Carlos et al. “The effects of donor age on organ transplants: A review and implications for aging research.” Experimental gerontology vol. 110 (2018): 230-240. doi:10.1016/j.exger.2018.06.019
[3] Lau, Ashley et al. “Mixing old and young: enhancing rejuvenation and accelerating aging.” The Journal of clinical investigation vol. 129,1 (2019): 4-11. doi:10.1172/JCI123946
[4] Mason, Jeffrey B et al. “Transplantation of young ovaries to old mice increased life span in transplant recipients.” The journals of gerontology. Series A, Biological sciences and medical sciences vol. 64,12 (2009): 1207-11. doi:10.1093/gerona/glp134
[5] Li D, Zhao D, Zhang W, et al. Identification of proteins potentially associated with renal aging in rats. Aging. 2018 Jun;10(6):1192-1205. DOI: 10.18632/aging.101460. PMID: 29907735; PMCID: PMC6046247.
[6] Kim, Mi-Jeong et al. “Young, proliferative thymic epithelial cells engraft and function in aging thymuses.” Journal of immunology (Baltimore, Md. : 1950) vol. 194,10 (2015): 4784-95. doi:10.4049/jimmunol.1403158
[7] Jazbec, Katerina et al. “The Influence of Heterochronic Non-Myeloablative Bone Marrow Transplantation on the Immune System, Frailty, General Health, and Longevity of Aged Murine Recipients.” Biomolecules vol. 12,4 595. 18 Apr. 2022, doi:10.3390/biom12040595
[8] Conboy, Michael J et al. “Heterochronic parabiosis: historical perspective and methodological considerations for studies of aging and longevity.” Aging cell vol. 12,3 (2013): 525-30. doi:10.1111/acel.12065
[9] Oberhuber, Rupert et al. “Murine cervical heart transplantation model using a modified cuff technique.” Journal of visualized experiments : JoVE ,92 e50753. 12 Oct. 2014, doi:10.3791/50753
[10] Yokota, Shinichiro et al. “Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.” Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society vol. 22,4 (2016): 536-46. doi:10.1002/lt.24394
[11] Cardini, Benno et al. “Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique.” Journal of visualized experiments : JoVE ,130 54998. 16 Dec. 2017, doi:10.3791/54998
"
There is a powerful need to include cellular hydration in the list of aging-associated biomarkers. Cellular hydration drops from 90% in the early stages of the developing embryo to 50% in adults. This makes it a foundational aspect of development, the ongoing, post-maturation continuance of which is hypothesized to be a mechanism of aging (The Neuroendocrine Theory of Aging and Degenerative Disease, Dilman and Dean, 1992). Due to the severity of the challenge of direct measurement of hydration, a surrogate marker is desired. Potassium utilization is claimed to be such a marker (Revici, 1961). Potassium utilization (the ratio of cellular potassium to serum potassium) is (1) cheap to measure with serum-testing equipment, (2) highly precise due to sequential measurment of two samples in the same device, thus eliminating independent calibration issues, and (3) minimally invasive (a single blood draw, split into two tubes, one of which is lysed by an exact ten-fold dilution with water). This makes it a perfect option for re-evaluating an old finding with new scientific and analytic methods for assessing an underappreciated but likely foundational aspect of aging.
"Devloping small climate controlled labs where effects of temperature/humidity/whether on aging & regular metabolism may be seen in case of human cells as well as rats. The inspiration came from the book by David P. D. Munns: ""Engineering the Environment: Phytotrons and the Quest for Climate Control in the Cold War"", and I propose these experiments to also include plant life

Let us assume this experiment on surrounding climatic conditions on the ability to influence everyday metabolism takes places in two controlled chambers for a period of 17 months.

Imagine two trons (A & B): having the exact same ecosystems and resources (just like a mini Earth ecosystem. However the average temperature and the atmospheric concentrations of different gases are made to alter differently. In tron A, the temperature is so programmed to increase at a steady rate from 20 to 30 degrees at the end of the experiment and the oxygen levels are made to also reduce from 21% to 15%. In tron B, the same changes might be made to occur except than the entire time would be of 34 months; so in an ideal scenario, the metabolism rates of rats at 8.5 months of experiment in tron A would be equal to the metabolism rate in tron B at 17 months.

The experiment would help understand the process of climate change in case of plants as well which is the most vital in our zeal to survive eternally and would also give an accurate understanding of the metabolic changes with climate so that we may understand what are the best concentration of specific ingredients which might help reduce the process of natural aging.

An alternative to this would be to remotely monitor hibernating organisms who are capable of reducing their metabolic rates and the specific techniques which might help humans achieve similar feat. This research proposal focuses more on the lifestyle and the environmental factors (external) rather than the internal unique constituents of each human which seems to be the primary research goals presently. External factor control would also be important but we would need to know whether cold climatic conditions really help humans (as is intuitively predicted by many) and the oxygen levels ideal of humans to optimize their lifespan"
"# Pauling’s Folly: Reactivation of Endogenous Vitamin C Biosynthesis
Josh McNamara

### Introduction
Anyone who has hung around a biochemistry lab for long enough has probably heard Linus Pauling invoked as shorthand for someone who veered toward quackery on a late career dementia quest. Running on the fumes of his eminence, Pauling is said to have bet it all chasing something to do with vitamin C. Here, I argue that Pauling’s vitamin C idea was well-founded in evolutionary logic, has accrued several decades worth of supporting evidence, and could increase physiological stress resistance and prolong lifespan. We can now, for the first time, really test the idea by genome-editing guinea pigs.

### Vitamin C biosynthesis
Up until roughly 40 million years ago, our proto-primate ancestors had the ability to synthesize vitamin C (also called ascorbic acid). This ability is preserved in most modern animals except for humans and their ape and monkey cousins, guinea pigs, and several bird and bat species. Humans and guinea pigs both independently lost the capacity for vitamin C biosynthesis due to inactivating mutations in the GULO gene, which produces the last enzyme in the vitamin C synthesis pathway. We and other species that have lost this ability must obtain vitamin C in our diets to avoid an eventually lethal case of scurvy.

### Pauling’s orthomolecular hypothesis
Around 1970 Pauling began to expound his theory that the recommended dietary vitamin C levels were high enough to prevent the acute effects of scurvy, yet were insufficient to promote optimal health [1]. He contended that the recommended amount was lower than what a traditional pre-modern diet would provide and ~100 times lower than what rats, horses, dogs, pigs, and our presumed proto-primate ancestors produce each day, adjusted for bodyweight. In contrast to contemporary recommendations of ~40 milligrams, Pauling recommended supplementing with several *grams* of vitamin C per day to keep up with this evolved deficiency in our physiology. The search for the optimal balance of nutrients became the founding principle of his Institute for Orthomolecular Medicine (ortho- = “straight” or “correct”).

### A qualitative evolutionary rationale
The implicit idea in this line of thinking is that genes evolve over millions of years like parameters in an optimization problem, tuning to each other and the environment through the influence of selective pressure. Random mutations de-tune these parameters and are much more likely to diminish overall fitness than increase it. Furthermore, deleterious mutations can fix (become universal) in a population in a “bottleneck” event, in which only a few individuals pass on their genes and only the maladaptive gene variant survives. Evolution exhibits substantial hysteresis / path-dependence, and re-tuning the rest of the genome to accommodate a newly-fixed maladaptive variant (“suppressor mutations”) might take millions of years or more. The fastest way to make up for a fitness loss would be to restore the ancestral wild-type gene or find a way to recapitulate its phenotype using diet, drugs, or other means.

GULO inactivation might have been either a beneficial adaptation or a deleterious mutation that fixed in an archaic population consuming enough dietary vitamin C for it not to have mattered. If the latter, it follows that adjusting physiological vitamin C concentrations to the level that the rest of the genome had been tuned to for hundreds of millions of years may increase an organism’s overall fitness. Given that GULO inactivation likely occurred tens of millions of years ago, it is possible that suppressor mutations have since adapted humans and guinea pigs to any deficits that the mutation produced. Despite this, humans are thought to have only recently (on an evolutionary timescale) switched from a vitamin C rich diet to a vitamin C poor modern diet. High vitamin C consumption would weaken selective pressure for the evolution of GULO-null suppressors, and any fitness loss arising from this dietary change itself has had little time to select for suppressor mutations.

### Vitamin C megadoses haven’t been medically useful
Pauling co-authored a few vitamin C megadose studies that appeared to show promise in cancer, but nothing came of these protocols. One potential reason is that eating several grams of vitamin C per day has little impact on the active concentration in the blood and within cells because such super-doses are rapidly excreted in the urine. You simply can’t absorb vitamin C efficiently enough to get the active concentration that your dog enjoys. More recent studies have shown that intravenous dosing can achieve vitamin C levels much higher than the oral route. Some have even shown that intravenous vitamin C enhances checkpoint inhibitor immunotherapy efficacy in mouse cancer models and imparts a survival benefit [2].

### Genetic intervention
Aside from eating or injecting vitamin C, we can now test the spirit of Pauling’s proposal more directly with new technologies such as CRISPR that enable us to edit mammalian genomes. Thanks to genome sequencing, we can also infer by comparison to other species what a functional GULO gene should look like for a given organism and mutate its genome to resurrect the long-dead gene.

Some experimental results suggest that genetic delivery of vitamin C via repair of the GULO gene may provide benefits beyond what is possible with intravenous dosing. Budding yeast, a common model organism used in genetics research, can be mutated such that it can no longer synthesize several essential metabolites. These strains grow proficiently when the missing nutrients are supplied in their growth medium. However, restriction of the essential nutrient causes the strains to have shorter lifespans [3]. Experiments in mice have yielded similarly suggestive results. Researchers inactivated the GULO gene in mice and found that the animals quickly developed scurvy [4]. After supplementing their chow with vitamin C, the mice were able to grow and reproduce. Reducing the dietary vitamin C maintained viable mice but caused defects in the aortic wall. Would genetic rescue of GULO in a GULO-null animal such as humans or guinea pigs improve healthspan?

### GULO rescue longevity experiment
Several investigators have proposed genetic rescue of GULO-deficient animals, yet none have made a direct genomic repair of the gene. See [5] for elaboration. I propose to genome-edit the germ line of a guinea pig to restore native GULO function. Next, observe the animals and track their metabolic profiles and lifespans (~6 years on average) relative to control animals. One could give away 100 GULO+ guinea pigs and matched wild-type animals as pets on the condition of visits for regular exams and an autopsy. Perhaps there will be a difference in longevity. On a shorter timescale, one could measure a few correlates of lifespan such as time to sexual maturity, middle-age fecundity, resilience of cultured cellular explants, starvation resistance, strength and endurance, rates of tumorigenesis or vascular disease progression, and response to cancer immunotherapy. Similar experiments could be conducted in cultured human tissues with a repaired GULO gene. Even if these experiments do not produce a longevity boost, many investigators will be curious about the effects of reverting a DNA copying error that occurred in our lineage millions of years ago. Researchers have been pondering this experiment for decades, and we owe it to them to give it a try now that it is technically feasible.

### Bonus: Phylogeny as version control
From this course we can generalize to an even more fanciful idea- using phylogeny as genetic version control. As discussed above, bottlenecks have been frequent in our evolutionary history, and we can infer that many fitness-reducing mutations have fixed in the human population. These mutations, like GULO-null, are universal and all functional alleles are on the other side of a species barrier. With genome sequencing, we can see over that barrier and infer “weakly perfect” ancestral genotypes from a composite of our phylogenetic relatives. See Kondrashov for a more thorough discussion of mutation, imperfect alleles, sign epistasis, and Bateson–Dobzhansky–Muller interspecies incompatibility [6].

Many bacteria have found a solution to this problem through horizontal gene transfer. These organisms can achieve huge fitness gains by acquiring genes from distantly related species, as is commonly seen in the evolution of antibiotic resistance. Humans do not yet engage in gene transfer with other species, though genome editing enables it. Because random mutations are more likely to be deleterious than beneficial, it follows that reversion of fixed mutations is more likely to increase fitness than decrease it. As with GULO, we can use composites from other species to infer the sequence of potentially fitter human alleles and test whether they increase vitality and prolong lifespan in relevant model systems.

### References
1. Pauling, Linus. Evolution and the need for ascorbic acid.
https://doi.org/10.1073%2Fpnas.67.4.1643
2. Magrì et al. High-dose vitamin C enhances cancer immunotherapy.
https://doi.org/10.1126/scitranslmed.aay8707
3. Gomes et al. Low auxotrophy-complementing amino acid concentrations reduce yeast chronological life span.
https://doi.org/10.1016/j.mad.2007.04.003
4. Maeda et al. Aortic wall damage in mice unable to synthesize ascorbic acid.
https://doi.org/10.1073/pnas.97.2.841
5. Cao et al. Ectopic Gene Expression to Restore the Ascorbate Biosynthesis Pathway in Vertebrates Lacking Functional Gulo.
https://doi.org/10.23880/ggtij-16000108
6. Alexey Kondrashov. Crumbling Genome.
https://doi.org/10.1002/9781118952146"
"Title: Ageing as run-on development

Hypothesis Summary

Ageing is widely thought to occur due to the accumulation of various forms of molecular damage [1]. What if, however, ageing changes are not primarily a result of a build-up of stochastic damage but are rather a product of regulated processes? Multiple facets of mammalian ageing follow predetermined patterns encoded in the genome as part of developmental processes. One provocative hypothesis is therefore that the developmental program, optimized for reproduction, inadvertently regulates ageing [2]. More precisely, my hypothesis is that gene regulatory programs during development trigger changes in cells and tissues that result in human ageing because, after reproduction, developmental programs have no evolutionary reason to change their predetermined trajectories and hence become detrimental.

The hypothesis that ageing may be an extension of development dates back to the 19th century [3], and the notion that developmental factors can impact ageing has been debated for over a century [4], including as part of a program. In line with these largely unexplored concepts, I hypothesize that the majority, albeit not all, changes causing the functional decline that characterizes human ageing are triggered by a developmental program optimized for reproduction.

The implications of seeing human ageing as at least partly deriving from developmental programs are multiple and far-reaching. This hypothesis challenges current paradigms in the ageing research field that view stochastic damage as the primary driver of ageing. Focusing on this very promising but largely unexplored hypothesis could lead to a potential paradigm shift in the field. According to my hypothesis, ageing is an information problem, and if we could instruct cells to reset the developmental programs this would result in cellular rejuvenation, as observed already in cellular reprogramming. Therefore, we need better models of gene regulation, of development and more broadly of how the genome and epigenome operate in space and time. As such this hypothesis also opens several promising directions and has important implications for developing interventions.

In conclusion, I would like to resurface and focus on the hypothesis that the developmental program inadvertently regulates ageing.

References:

[1] López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013). The hallmarks of aging. Cell 153:1194-1217.

[2] de Magalhaes JP (2012). Programmatic features of aging originating in development: aging mechanisms beyond molecular damage? Faseb J. 26, 4821-4826.

[3] de Magalhães JP, Church GM (2005) “Genomes optimize reproduction: aging as a consequence of the developmental program.” Physiology 20:252-259.

[4] Loeb J, Northrop JH (1917). What Determines the Duration of Life in Metazoa? Proc Natl Acad Sci U S A 3:382-386."
"The Neuroendocrine Theory of Aging by V. Dilman.
In 1960-70, Russian biologist Vladimir Dilman published a series of scientific papers in which he outlined his vision on the nature of aging in organisms.
https://pubmed.ncbi.nlm.nih.gov/13816764/
https://pubmed.ncbi.nlm.nih.gov/5686906/
https://pubmed.ncbi.nlm.nih.gov/4425748/
https://pubmed.ncbi.nlm.nih.gov/1218271/
https://pubmed.ncbi.nlm.nih.gov/225186/
In the Lancet in 1971, his article ""Age-associated elevation of hypothalamic threshold to feedback control, and its role in development, ageing, and disease"" was published. In it he detailed his hypothesis of the formation of aging and age-related pathologies, called by him the elevation theory of aging, and later renamed the neuroendocrine theory of aging. The basis of the hypothesis is the relationship between violation of homeostasis during the development of the body and aging, and an increase in the sensitivity threshold of the hypothalamus to feedback mechanisms.
https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(71)91721-1/fulltext
The hypothalamus is the central organ in Dilman's theory of aging. Dilman suggested the key role of the hypothalamus in aging due to its important regulatory role at the junction of two body systems - endocrine and nervous. In this organ, control, regulation, communication, integration and homeostasis of metabolic and reproductive functions are carried out. The most important general principle by which this integration is achieved is the mechanism of negative feedback. As Dilman described in his hypothesis, maintaining a stable balance (homeostasis) is a key requirement for the life of an organism, but the development of the organism after its birth requires a violation of homeostasis, otherwise development is impossible.
As a typical example, Dilman cites changes in the body during pregnancy. Cholesterol is critical for fetal development. You need a lot of cholesterol. To do this, a special genetic program is launched with the participation of several hormones. such as placental growth hormone. That suppresses the use of glucose by cells, resulting in the formation of the necessary conditions for increasing cholesterol levels. The placenta forms this evasion from homeostasis during pregnancy, because it is not included in the general negative feedback mechanism, when an increase in one hormone or substance will cause a compensatory response that dampens such an increase.
After birth, the formation of an adult organism also requires a violation of homeostasis. Here, Dilman assigned a key role to the gradual increase in the hypothalamic sensitivity threshold to negative feedback mechanisms. A baby has only a small amount of sex hormones, so that they have an inhibitory effect on the hypothalamus. But in the process of development, the hypothalamus gradually increases its threshold of sensitivity to suppression by sex hormones, increasing its activity and stimulating the pituitary gland to further increase the activity of the gonads.
But with aging, this increase in the threshold of sensitivity of the hypothalamus to negative feedback mechanisms leads to negative effects. Among which he primarily singled out a violation of energy homeostasis (insulin resistance and hyperinsulinemia, increased blood glucose, excess free fatty acids and cholesterol), which in turn leads to age-related pathologies, cardiovascular, neurodegenerative, etc. In his hypothesis, Dilman did not separate aging from age-related pathologies, considering them to be an interconnected process.
What, in his opinion, could cause age-related changes in the hypothalamus? Among these reasons, he singled out:
- Decrease in the level of hypothalamic neurotransmitters (in particular, catecholamines and serotonin); - Decreased number of hormone receptors in the hypothalamus;
- Decreased secretion of pineal hormones (melatonin and polypeptide hormones of the pineal gland);
- Accumulation of fat; decreased glucose utilization;
- Accumulation of neuronal lesions caused by chronically elevated levels of cortisol due to prolonged stress;
- Accumulation of cholesterol in the plasma membranes of neurons.
The well-known gerontologist M. Blagosplonny in his theory of hyperfunction as a key factor in aging (based on the activity of the mTOR signaling pathway) emphasized its continuity with the theory of V. Dilman, which, in his opinion, is undeservedly forgotten today. And it is not even mentioned in works on the relationship between the hypothalamus and aging: “Vladimir Dilman suggested that aging is due to a progressive loss of sensitivity of the hypothalamus. This causes a progressive change in homeostasis, metabolic disorders leading to age-related diseases. There is an age-related loss of sensitivity of the hypothalamus to the negative feedback of certain hormones, such as estrogens and glucocorticoids. This explains the development of age-related diseases, including metabolic disorders and menopause. Unfortunately, the hypothalamic theory was far ahead of its time and too medical to be attractive to gerontologists. This theory, supported by excellent experiments, went unnoticed, only to be rediscovered recently.”
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765577/
In their brilliant work, the team of B. Cai et al., who described the systemic influence of the hypothalamus on aging, did not even mention the previous work of V. Dilman. B. Cai et al. revealed yet another yet unknown side associated with aging and the hypothalamus – microinflammation of the hypothalamus caused by an increase in the activity of the NF-kb signaling pathway.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756938/
Today, no one doubts the very large influence of the violation of regulatory functions by the hypothalamus on aging.
Аccording to V. Dilman, prophylactic and therapeutic measures should be directed towards the preservation of rhythmic function of the basic homoeostatic systems so that metabolic processes can be maintained at the level reached when development of the organism was complete. We need, therefore, the means to restore rhythmic activity of the homoeostatic systems by lowering the hypothalamic threshold to different types of homceostatic inhibition (i.e., by counteracting the process of the self-development of homreostatic systems).
Means of this sort are not yet available, but from what we already know we may presume that the pineal gland can exert such influence (i.e., it can directly inhibit some of the functions of the hypothalamus and increase its sensitivity to intrinsic regulating factors), and we may anticipate that other pharmacological agents possessing these properties will eventually be found. In the meantime research should be directed towards the use of those prophylactic and therapeutic measures that may act on different stages of the elevating mechanism of ageing. In this respect studies should be made of: oestrogens; pituitary inhibitors, especially sigetin (meso-3,4-bis-(p-sulphophenyl)-hexane)’; androsterone ; D and L thyroxine; rational diet; aminoglutethimide, which can suppress both steroidogenesis and gonadotrophin secretion a5; phenytoin, capable of inhibiting adrenal function and gonadotrophic secretion and, possibly, of influencing the synthesis of R.N.A. ; and phenobarbitone and other antiepileptic and neurotropic preparations. More attention should be paid to phenformin, which eliminates compensatory hyperinsulinism and thereby reduces the cholesterol and triglyceride levels, and reduces excess body-weight in aged patients. With this in view, phenformin should be investigated with regard to its ability to suppress the development not only of atherosclerosis, but also of tumour formation. Anahormones, with their ability to induce antibodies, and to suppress the secretion of protein hormones or to compete with them at the level of target tissue, may be interesting tools for the study of age and pathological processes.
The idea of using labelled immunoserum for selective retention of radioactive antihormonal antibodies in the hypophysis and hypothalamus should also be taken into consideration. More emphasis should be placed on the study of isohormones (e.g., those of stiocholanolone type) and of other ways which, by affecting the hypothalamic threshold to inhibition or by reducing compensatory reaction, might slow down the realisation of the genetic programme of ageing and related pathology.
"
"Cellular senescence has recently became very famous hallmark of aging. Pro-inflammatory and matrix-degrading factors that are secreted by senescent cells are detrimental to health and participate in pathogenesis of various age-related pathologies. However, there still is no proven theory which explains why senescent cells accumulate with advancing age.

In our work [1] I proposed a hypothesis that logically explains the accumulation of senescent cells as a result of non-enzymatic changes to the long-living proteins of the ECM: there's a growing body of evidence that cellular senescence (at least partly) is an anti-fibrotic cellular program. Cells stop to proliferate (thereby reducing the synthesis of collagen), secrete matrix-degrading enzymes that clear ECM debris and secrete pro-inflammatory cytokines that attract immune cells that clear degraded fragments of the ECM.
My point is that the aged (due to cross-links accumulation) ECM has very similar properties as fibrotic ECM which in turn erroneously activates anti-fibrotic cellular program - cellular senescence. If this hypothesis is true then it has profound implications for the understanding biology of cellular senescence and could lead to a new class of drugs that prevent excessive accumulation of senescent cells without hampering the anti-fibrotics mechanisms.

1. https://www.sciencedirect.com/science/article/pii/S1568163720302324"
"Proposal to test the Selective Destruction Theory of Ageing
In 1956 Harman proposed the free radical theory suggesting that molecular damage could accumulate to cause ageing (Harman, 1956). Then Kirkwood (1977) observed that evolution might allow damage to accumulate if energy spent on maintenance could be better utilised in other processes with a higher impact on fitness. These ‘common sense’ theories have dominated gerontology ever since; however, as our understanding of the mechanisms of ageing grows, it is becoming increasingly difficult to reconcile all aspects of ageing with accumulating damage. Long-lived worms and flies that accumulate more damage than their short-lived counterparts have caused some to ask if the free radical theory is dead (Pérez et al., 2009), while others have suggested that ageing via accumulating damage is contradicted by the rejuvenation potential of parabiosis and the ability of Yamanaka factors to return heavily damaged senescent and old cells to a youthful state (Katcher, 2015).
We recently published a new ‘selective destruction theory’ (Wordsworth et al., 2022), which is not only consistent with these rejuvenation revelations but indicates the underlying mechanism. The concept is simple: cells in multicellular organisms undergo mutations or epimutations which cause them to grow at different rates. Importantly, these changes can result from point mutations in genes and promoters as part of replication without need for damage accumulation or oxidative stress. Mutants that grow and replicate faster than wildtype cells will have a selective advantage, eventually allowing them to overtake the tissue if they are not controlled. It has been suggested that this alone might be sufficient to make ageing and death inevitable in multicellular mitotic organisms (Nelson and Masel, 2017); however, these authors did not consider that organisms could employ a mechanism of counterselection against fast cells. As we modelled in our paper, when slower cells are capable of epigenetically slowing down fast cells or killing them, termed selective destruction, it prevents their takeover and even allows the spread of slower cells (Wordsworth et al., 2022). The result would delay multiple diseases such as cancer and fibrosis that become more probable with accelerated metabolism, but at the same time would result in a gradual metabolic slowdown. Importantly, if the fitness costs of metabolic slowdown are lower than those associated with the spread of fast mutants, as our models suggested, then selective destruction could undergo positive selection despite the induced functional decline. Multiple lines of evidence suggest this is occurring, from the decline in cell proliferation rate (Tomasetti et al., 2019) and energy expenditure (Pontzer et al., 2021) to the hallmarks of transcriptomic ageing which include reduced growth factor signalling, protein synthesis, and mitochondrial respiration (Frenk and Houseley, 2018).
At the cellular level, the immediate consequence of metabolic slowdown will be a rise in ATP (due to reduced ATP hydrolysis). As ATP is tightly regulated to ensure constant availability, a decline in ATP usage necessitates a decline in ATP production to maintain ATP within the equilibrium range. Thus, a decline in cellular metabolism results in a decline in mitochondrial metabolism. However, our current modelling suggests that when mitochondria become sufficiently slowed, the shift in homeostasis becomes confused with the state of infection: infected cells slow ATP production to prevent energy utilisation by invading pathogens, inhibiting their replication and the spread of infection. We suggest that evolution has therefore linked reduced mitochondrial metabolism, which is frequently termed ‘mitochondrial dysfunction’, with inflammation by activating the NLRP3 inflammasome (Mishra et al., 2021). However, this mitochondrial dysfunction would not result from accumulated damage as is commonly believed, instead reflecting a homeostatic response. We therefore hypothesise that the pathway from selective destruction to inflammation is central to ageing and age-related disease.
Selective destruction is therefore one of the few theories that provides both an evolutionary and mechanistic explanation for ageing, perhaps the only plausible theory independent of damage accumulation. If ageing is induced by cell communication as we suggest, it explains the rejuvenating effects of parabiosis, and is consistent with Yamanaka factor rejuvenation by removing the epigenetic controls that have slowed cell growth. However, our idea is still theoretical: the notion of cells within tissues slowing each other down is highly likely given the emerging importance of somatic cell competition (Kakiuchi and Ogawa, 2021), but the regulation of neighbouring cell growth is novel to this theory.
Here we propose a series of simple cell culture experiments which could prove this communication is occurring, evidence its role in ageing, and elucidate the molecular pathways involved. Using labelled cells from old and young individuals with different growth rates, we will test if a low concentration of young cells grown in co-culture with old cells will be slowed compared to young cells in monoculture. If they fail to dominate despite an initially faster growth rate, then we can conclude that they have been slowed down or even killed by the older cells.
To elucidate the mechanism, we have identified Notch signalling as a regulator of cell growth by juxtacrine signalling. Notch signalling from the surrounding tissue controls tumour cells (Demehri et al., 2009) and knockout results in tumours (Kimura et al., 2019), as we predicted would result from loss of selective destruction. We will therefore knockout Notch in our cell lines and observe what effect this has on young cells in co-culture with old cells, predicting that this might abrogate selective destruction and allow the faster young cells to dominate the co-culture.
The prize money would be used to purchase the fluorescent and NICD knockout (to inhibit Notch) constructs and lentiviral agents as well as lab costs to carry out these experiments in our existing old and young dermal fibroblast and muscle stem cell lines, as shown in the following breakdown:
Media and plastic consumables for cell culture 5,500
Transfection reagent 850
Disposable plastics and gloves 4,500
General laboratory chemicals and media 2,000
shRNA/cDNA constructs 1,000
Bioimaging/cell sorting facilities charges 5,500
In addition to the prize money helping to fund these initial experiments, we also hope that the recognition accompanied by this prestigious prize might build awareness of selective destruction theory and encourage collaborators with epigenomic experience to help us identify the effects of selective destruction in these co-culturing experiments on epigenetic clocks. If young cells are in fact being aged rather than simply slowed then we predict this will be reflected by CpG methylation at the sites identified by Horvath (2013) and others.
If we can show evidence for selective destruction, the implications for ageing would be vast. Beyond providing a badly needed second theory, it would explain the limited efficacy of interventions such as calorie restriction and metformin after the regimens cease (Mair et al., 2003), as they are reducing basal inflammation while the underlying selective destruction is continuing unabated. Interventions aimed at altering cell communication and epigenetic regulation may prove significantly longer lasting, but must contend with the increased risk of overactivity disorders such as cancer and fibrosis.
NB: This work would be conducted in collaboration with Viktor Korolchuk (panel member).
"
"Quantum Biology for Longevity Research

1-page hypothesis found in the link below:

https://drive.google.com/file/d/1rfyAsx2cwGHQVjUwWbWftRZAFoOn2AP_/view?usp=sharing"
At Remedium Bio, we believe that Growth Factors are the most promising and near-term solution to meaningful extension of healthspan and lifespan. While significantly under-represented in therapeutic research, Growth Factors have delivered remarkable results in placebo controlled clinical trials for a number of age-related diseases. Most recently, repeat intra-articular injections of the FGF18 protein have demonstrated the ability to reverse cartilage loss in Osteoarthritis patients relative to placebo control - a never before seen result in Rheumatology. As a classical disease of aging, Osteoarthritis affects 1 in 7 US adults and presents with progressive erosion of cartilage, eventually leading to debilitating pain and loss of function. Remedium is developing a novel FGF18 gene therapy with the aim of reversing cartilage loss to restore a more youthful and healthy phenotype to arthritic joints.
"With the promising potential of hundreds of autologous cell therapies currently in the pipeline, there has been increasing interest in finding stem cell populations that can be obtained in a less invasive manner and yet exert similar therapeutic properties. Studies over the past two decades or so demonstrated success in isolating multipotent and pluripotent stem cell populations from plucked hair follicles. The easy accessibility, high proliferation, differentiation ability, and efficient reprogramming potential associated with this stem cell source make hair follicle an ideal candidate for autologous cell therapy and regenerative medicine.

In this project, we aim to isolate mesenchymal stem cells from the outer root sheath of the human plucked hair follicle. We will work towards differentiating them into osteogenic, adipogenic, chondrogenic, and neuronal lineages. Following validation and scale-up, we aim to employ them in various pre-clinical and clinical models to treat aging and degenerative diseases such as osteoarthritis, cartilage degeneration, and spinal cord injury. Use of a totally autologous stem cell source that is easily obtainable during adulthood, such as the hair follicle, alleviates the limitations and safety concerns associated with most stem cell-based therapies."
"What if we could effectively harness cancer to live longer?

Cancer is the immortal phenotype. If cancer can enable biological immortality, perhaps we should be more carefully looking at ways to harness cancer to promote longevity. This is a contrarian hypothesis, but the clinical framing around cancer as “the enemy” has likely prevented meaningful work exploring how cancer could potentially be leveraged from a longevity-enabling perspective. "
" Antiaging research
Theoretical Framework: Genomic stability
The novelty in this project is that it focuses on the quiescent1 stage of the cell cycle (fase G0), what we intend is to increase the period of time in which the cell is in the quiescence stage, with the premise that the quiescent period can be affected by epigenetic2, 5 factors. We do not intend in this case that the cell reproduces itself a greater number of times, or indefinitely, but that it increases the amount of time it works efficiently without dividing.
In this project we agree with the theory of genetic damage3, considering aging as: a multifactorial process of generalized cellular degeneration, which has its origin in the damages that our genome experiences, having as a consequence one of three possibilities: the cells enter senescence, they become malignant or massive cell apoptosis; manifesting itself in a structural and functional insufficiency that finally leads to death.
VISION:
The health and longevity of the human species does not depend on a gene, a specific protein or a biomolecule “x”, but on the total genome integrity, both of coding genes and regulatory sequences.
GENERAL OBJECTIVE
Stop Genetic Damage, achieving a radical increase in life expectancy in individuals under treatment and in their offspring, since it is genetic to transmit this benefit to succeeding generations increasing results.

SPECIFIC OBJECTIVES.
a) Achieve genomic stability4.
b) Prevention of degenerative chronic illnesses with genetic component.
c) To exceed with much the known longevity levels.

HYPOTHESIS:
By eliminating harmful element Carbon 14 of our genome we will be eliminating a major source of mutations and senescence3, which will give the genome greater stability and increase cell life.

Our genome has adapted evolutionarily over the course of millions of years to the levels of natural radiation that exist in the environment, however, sometimes fast and drastic changes occur on our planet that cannot be assimilated as quickly by our genome, such is the case of the last glaciation, implied a drastic change in the terrestrial climate and consequently in the concentrations of C14 present in the atmosphere. In this project we intend to drastically reduce the levels of C14 present in the genome and in general throughout the organism.

This project presupposes that the cell division cycle can be affected by stress factors of the genome (genetic damage) and that if we reduce C14 (stress factor) we can slow down the cell division process, obtaining longer-lived healthy cells and increase health, strength and longevity of the human species. There are multiple stressors of the genome, external, internal, genetic, epigenetic, but in this case we will focus specifically on the C14 element.

Expected results:
In vitro we expect to observe that the cell division process (human cells) is slowed down compared to control cultures; and that although slow, the cell division process in cultures with reduced C14 substantially exceeds the survival of the control culture.
In vivo we expect a substantial increase in the strength, health and longevity of the species with which we will work, fruit fly and laboratory mice.
We will observe results in successive generations, up to the tenth generation, and in the case of human cells, the age of the donor (newborn, young adult and elderly) will be taken into account due to the load of genetic damage.

In this project we consider that if the stability of the DNA nuclear and mitochondrial is not achieved, all the tissues, organs and systems at the human body will end up failing. Our goal is to prevent the occurrence of mutations or any damage to our genetic code.

1.- https://www.nature.com/subjects/quiescence

2.- https://www.genome.gov/genetics-glossary/Epigenetics

3.-https://www.semanticscholar.org/paper/The-essential-mechanisms-of-aging%3A-Irreparable-of-Yin-Chen/7bf085463e44d3da5a6e2a95ed1f8976d2ab2f73

4.-https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/genetic-stability

5.- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3481136/

Kindly

Professor Ruben Lopez Cortes.
Biology teacher.

"
"link to text file in case copy/paste does not work:
https://docs.google.com/document/d/1ehCXArbfy5CmuJ6KwWY_baxSh69nsANL4WQ93GX03dA/edit?usp=sharing

Carbon Disulfide and Aging

Hypothesis: Endogenous carbon disulfide production changes during aging and these changes are associated with alterations in neurological and immune function.

Rhyothemis princeps
30 December 2022

Carbon disulfide (CS2) is a volatile liquid at room temperature; its vapors are heavier than air [1]. Although CS2 is naturally produced by some anaerobic bacteria it is best known as an industrial solvent used in the production of rayon (also known as viscose) and as feedstock for the production of carbon tetrachloride and dithiocarbamate fungicides [1,2].

Neurotoxic effects of CS2 inhalation were first observed from its initial use in rubber product manufacturing in the mid 19th century [3]. Intoxication from acute exposure, which Charcot termed “carbon disulphide hysteria”, can result in dizziness, nausea, headache, numbness, autonomic dysfunction, anorexia, loss of consciousness, and neuropsychiatric symptoms including anxiety, hallucinations, delusions, paranoia, hypersexuality, extreme emotional lability, and suicidal ideation [1,3,4]. Severe cases can result in death from respiratory arrest [4]. Chronic occupational exposure to CS2 has been linked to cardiovascular disease, kidney disease, loss of sexual function, menstrual cycle irregularities, early menopause, polyneuropathy, retinopathy, encephalopathy, parkinsonism and multiple system atrophy [1,3,4,5,6,7,8,9,10].

Given the preceding litany of harms, it may be surprising to learn that CS2 is present in the breath of rodents [11]. In rats CS2 is appetitive and mediates social learning of food preferences [11,12]. CS2 can be detected in the breath of humans and elevated CS2 levels have been found in the exhaled breath of schizophrenia and cystic fibrosis patients; this may be the result of either higher rate of production or reduced rate of metabolism and excretion via the urine [13,14,15]. CS2 levels in urine have been found to fluctuate over the course of the menstrual cycle [16]. In humans, CS2 is present in the feces and its increase after supplementation with a synbiotic indicates production by gut microbiota [17,18]; bacteria in the lung may be another source of CS2 [15].

Endogenous production of CS2 might not be so unexpected given that nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (HS2) are all toxic gasses that are also endogenously produced and, additionally, function as small molecule bioregulators (SMB) (also termed gasotransmitters) [19]. DeMartino et al. [19] have proposed that CS2 could be an SMB based on its similarities to NO, CO, and HS2 and that like these SMBs, the toxic effects of exposure to CS2 may be due to relatively high doses overwhelming regulatory or signaling processes in which it hypothetically normally participates. DeMartino et al. [19] suggest that in addition to the gut and/or lung microbiome “one should also consider the possibility of a dedicated, [human] enzymatic source.”

The acute neurotoxic effects of CS2 and its dithiocarbamate (DTC) metabolites are mainly attributed to catecholamine disruption; following acute inhalation exposure rats show decreased brain levels of noradrenaline (NA) and increased dopamine (DA) [20]. Noradrenaline can be inactivated by protein cross-linking induced by DTCs, and CS2 and DTCs can inhibit dopamine beta hydroxylase (DBH), which catalyzes the conversion of dopamine to noradrenaline, leading to increase in the former and decrease in the latter [20,21]. Inhibition of DBH is the result of chelation of its cofactor, copper, by DTCs formed from the reaction of CS2 with amino acids and/or catecholamines [22]. Chronic CS2 exposure was reported to be associated with decreases in serum levels of both noradrenaline and dopamine, as well as copper and zinc [23]. DTCs also inhibit carbonic anhydrases and aldehyde dehydrogenases, which are also important to neurological processes [24,25]. The aldehyde dehydrogenase ALDH1A1 is strongly expressed in brain dopaminergic neurons where it functions to oxidize 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of DA, preventing the conversion of DOPAL to even more toxic compounds such as tetrahydropapaveroline [26]. ALDH1A1 inhibition could explain parkinsonian symptoms of chronic occupational CS2 exposure. An excess in endogenous CS2 levels above normal physiological levels caused by an increase in production by the microbiome or by a decrease in its rate of metabolism and excretion could exert harmful neurological effects through inhibition of ALDH1A1.

De Martino et al. [19] propose that DTC and CS2 are in labile equilibria under physiological conditions and that CS2, which is lipophilic, can enter into membrane-bound catecholamine storage granules where it converts to DTC (a polar molecule unable to traverse membranes) and inhibits DBH - thereby exerting a regulatory influence on the production of NA from DA. It is difficult to predict if this would affect levels of these neurotransmitters in normative aging since their levels decline overall due to an age-dependent increase in activity of monoamine oxidases [27].

It is worth noting, however, that while CS2 exposure is associated with parkinsonism and has been implicated in multiple system atrophy, large epidemiological studies have not found an association with increased risk of Parkinson disease [1,8,9,10,28]. People with ulcerative colitis, an inflammatory bowel disease, have increased risk of developing Parkinson disease [29]. In recent years there has been growing recognition of the role of the gut microbiome in both UC and PD, as well as other age-dependent neurodegenerative diseases [30,31]. As mentioned above, CS2 in feces can be increased by supplementation with a synbiotic [18]. Garner et al. [17] found CS2 present in 100% of fecal samples of healthy volunteers (n=30), but only in 61% in those of people with UC (n=18); CS2 was also reduced in Clostridium difficile patients, with only 41% (n=22) producing CS2. These results suggest that CS2 production may be an indicator of a healthy gut microbiome.

DTCs inhibit the nuclear factor kappa beta (NF-κB) inflammatory signaling pathway [32]. DeMartino et al. [19] have proposed multiple mechanisms through which CS2 might also inhibit NF-κB. The NF-κB signaling pathway is thought to play an important role in the pathogenesis of UC and also PD and other neurodegenerative diseases [33,34]. NF-κB is a major signaling pathway involved in ‘inflammaging’ [35]. Chronic activation of NF-κB can induce cellular senescence and activation of longevity genes such as SIRT1, SIRT6, and FOXOs can suppress NF-κB signaling [35]. It is possible CS2 produced by the gut microbiome decreases intestinal inflammation through inhibition of NF-κB, reducing risk of UC, PD and perhaps even extending healthspan and lifespan. Interestingly, CS2 is present in cigarette smoke and smoking is associated with reduced risk of both UC and PD, though nicotine and carbon monoxide are usually named as the most likely active constituents [36,37].

Occupational CS2 exposure is associated with endocrine and reproductive changes which include alterations in hormone levels, increase in low density lipoprotein, menstrual cycle irregularities, loss of libido, sexual dysfunction, changes to sperm morphology, and earlier onset of menopause [1,6,7,38]. Based on evidence from animal model studies, Printemps et al. [21] concluded that thyroid hormone disruption from CS2 exposure is most likely due to central nervous system toxicity rather than by a direct endocrine disruption mechanism.

However, women chronically exposed to CS2 were found to have higher serum levels of dehydroepiandrosterone sulfate (DHEA-S) than controls [7]. Another study of rayon factory workers by Djuric et al. [39] found CS2 exposure may have caused an irreversible disturbance in sulfur metabolism [1,39]. Sulfation and desulfation are key mechanisms for regulating the availability and activity of steroid and thyroid hormones as well as neurotransmitters such as DA [40,41]. Sulfation of hormones renders them soluble for transport via the circulatory system whereas desulfation within cells converts them to their active form in most cases [40]. Sulfation and desulfation are affected by activities of sulfotransferases and sulfatases, respectively; availability of sulfur donor adenosine 3 ́-phosphate 5 ́-phosphosulfate (PAPS) is rate-limiting for sulfation [42]. Not surprisingly, some known endocrine disruptors such as acetaminophen and phthalates act through altering sulfation levels [42,43,44]. Since CS2 increases sulfation of DHEA, it may be considered an endocrine disruptor at exposures similar to those of rayon factory workers.

If the deleterious effects of an SMB at toxic exposure levels implicate its bioregulatory activities at physiological concentrations, then modulation of sulfation by CS2 could be a particularly important aspect of its hypothetical regulatory roles in neurological function and aging. Reduction in DHEA-S is associated with Alzheimer disease (AD), whereas DHEA is not associated with AD [45]. Pérez-Jiménez et al. [46] demonstrated that inhibition of sulfatase increases lifespan and protects against age-related proteotoxicity in Caenorhabditis elegans, and also reduces β-amyloid aggregation and cognitive behavioral deficits in an Alzheimer disease mouse model. Interestingly, steroid sulfatase expression is upregulated by NF-κB [47], which leads to speculation that increase in DHEA-S from CS2 exposure is the result of decreased sulfatase expression through suppression of NF-κB. Note that NF-κB is Janus faced in neurodegeneration, having neuroprotective or harmful effects depending upon signal strength and type of NF–κB dimers activated [48].

Sulfation also impacts the function of perineuronal nets (PNNs), which are specialized extracellular matrix structures that surround neurons [49]. PNNs are composed of chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, glycoproteins and glycosaminoglycans, such as hyaluronan [50]. PNNs function as molecular scaffolding that stabilizes and regulates the synapses they surround; they also protect neurons from oxidative stress and toxins such as β-amyloid (1-42) [50]. Aging is correlated with changes in sulfation patterns of PNNs which result in lowering of neuroplasticity and decreases in memory-forming ability [49,51]. Unfortunately, there appear to be no published studies on the effects of CS2 on perineuronal nets at the present time.

In summary, CS2 is a product of the human microbiome which could plausibly impact aging and age-related neurodegeneration through the mechanisms discussed above. The hypothesis put forth here is that endogenous CS2 production changes during aging and these changes are associated with alterations in neurological and immune function. To test this hypothesis, a cohort study could be conducted with participants ranging in age from early 20s to late 80s that measures CS2 presence in feces and CS2 levels in exhaled breath. The study would also measure biomarkers of neurological aging and inflammation, as well as serum DHEA-S. Serum neurofilament light chain (Nfl) would serve as a biomarker of neurological aging; Nfl is a dynamic, robust biomarker of neuroaxonal damage that increases with age across the lifespan [52]. Biomarkers of inflammation would include high sensitivity C-reactive protein, interleukin 1β, interleukin 6, and tumor necrosis factor α.

If one or more associations between CS2 levels and the listed biomarkers are found, this would not serve as evidence for a causal relationship. However, a positive finding would indicate whether further investigation into the potential impact of endogenous CS2 on aging is warranted and in that case, studies in animal models would likely be the next step. It should be noted that rodents and humans have an important difference with respect to CS2 signaling - humans lack the guanylyl cyclase D (GC-D) receptor [53]. In rodents, the GC-D receptor is required for CS2-mediated social learning of food preferences [11]. This could be an important point to consider in designing mouse or rat model experiments on CS2 and aging since the social environment affects the aging process [54].

References

1 - Abadin, H., & Liccione, J. J. (1996). Toxicological profile for carbon disulfide. U.S. Department of Health and Human Services. https://www.atsdr.cdc.gov/ToxProfiles/tp82.pdf

2 - Australian Government, & Department of Climate Change, Energy, the Environment and Water. (2022, June 30). Carbon disulfide—DCCEEW. https://www.dcceew.gov.au/environment/protection/npi/substances/fact-sheets/carbon-disulfide

3 - Blanc, P. D. (2016). Fake Silk: The Lethal History of Viscose Rayon (1st edition). Yale University Press.

4 - National Research Council (US) Committee on Acute Exposure Guideline Levels; National Research Council (US) Committee on Toxicology. (2009). Acute Exposure Guideline Levels for Selected Airborne Chemicals: Volume 7. In Carbon Disulfide Acute Exposure Guideline Levels (Vol. 7). National Academies Press (US). https://www.ncbi.nlm.nih.gov/books/NBK214898/

5 - Yan, Y., Wang, C., Zheng, Z., Qu, L., Zeng, D., & Li, M. (2019). Renal injury following long-term exposure to carbon disulfide: Analysis of a case series. BMC Nephrology, 20(1), 377. https://doi.org/10.1186/s12882-019-1553-1

6 - Krstev, S., Peruničić, B., Farkić, B., & Banićević, R. (2003). Neuropsychiatric Effects in Workers with Occupational Exposure to Carbon Disulfide. Journal of Occupational Health, 45(2), 81–87. https://doi.org/10.1539/joh.45.81

7 - Pieleszek, A. (1997). [The effect of carbon disulphide on menopause, concentration of monoamines, gonadotropins, estrogens and androgens in women]. Annales Academiae Medicae Stetinensis, 43, 255–267. https://pubmed.ncbi.nlm.nih.gov/9471921/

8 - Huang, C.-C., Yen, T.-C., Shih, T.-S., Chang, H.-Y., & Chu, N.-S. (2004). Dopamine transporter binding study in differentiating carbon disulfide induced parkinsonism from idiopathic parkinsonism. Neurotoxicology, 25(3), 341–347. https://doi.org/10.1016/S0161-813X(03)00147-5

9 - Frumkin, H. (1998). Multiple system atrophy following chronic carbon disulfide exposure. Environmental Health Perspectives, 106(9), 611–613. https://doi.org/10.1289/ehp.98106611

10 - Frumkin, H. (2000). Carbon Disulfide: Frumkin’s Response. Environmental Health Perspectives, 108(3), A110–A112. https://doi.org/10.1289/ehp.108-a110b

11 - Munger, S., Leinders-Zufall, T., McDougall, L., Cockerham, R., Schmid, A., Wandernoth, P., Wennemuth, G., Biel, M., Zufall, F., & Kelliher, K. (2010). An Olfactory Subsystem that Detects Carbon Disulfide and Mediates Food-Related Social Learning. Current Biology : CB, 20, 1438–1444. https://doi.org/10.1016/j.cub.2010.06.021

12 - Wang, T., & Chen, H. (2014). Carbon Disulfide Mediates Socially-Acquired Nicotine Self-Administration. PLOS ONE, 9(12), e115222. https://doi.org/10.1371/journal.pone.0115222

13 - Phillips, M. (1992). Detection of carbon disulfide in breath and air: A possible new risk factor for coronary artery disease. International Archives of Occupational and Environmental Health, 64(2), 119–123. https://doi.org/10.1007/BF00381479

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"
"This study will employ Proteomic and Epigenomic Methylation testing to identify proteome changes, biologic age shifts, blood inflammatory and aging changes, as well as functional markers of health that might be attributable to administering 2 liters of yFFP within 30 days into aged subjects for therapeutic uses across a wide spectrum of conditions.
Plasma exchanges that first remove old plasma and then replace the volume removed with yFFP have been proven to be safe and effective. Total plasma exchanges of 1 liter saline/albumin and 2 liters of yFFP have been successfully performed, as have two 1-liter exchanges of yFFP.
This study will compare epigenetic, proteomic, laboratory and functional results taken from just before the initial treatment to results taken one month after the last treatment. The comparative results from each study participant that has received 70% or more of a total of 2 liters of yFFP over a period not to exceed one month through intravenous or exchange methodologies, will be analyzed and reported.
Results from all individuals will be correlated and compared, which is expected to allow the categorization of distinctive disease states to identify improvements derived from yFFP therapy that could apply to broad demographic and sex identified populations.

The study is a 30-patient epigenetic, proteomic, laboratory and functional outcomes investigation designed to evaluate both the safety and efficacy of intravenous and exchange administered young Fresh Frozen Plasma at 25 ml/kg in no less than two doses over no more than thirty days.
Each participant's starting condition will be identified by:
- a TruDiagnostic test of the level of epigenetic methylation
- a Seer test of blood proteomics -laboratory markers:
- Leptin(an obesity signaling marker) -HbA1c(average blood sugar over 3 months)
-Adiponectin(modulates glucose regulation and fatty acid catabolism
-Troponin-(heart structural integrity) -NT-ProBNP(Heart function)
-TNF alpha (assesses inflammatory responses in a large range of diseases)
-Cystatin C (marker for kidney function)
-IL6 a marker of aging, frailty, and disease -Functional Markers
- standing balance testing
-body composition (BMI)
-waist circumference
-Grip strength
Just prior to the commencement of their yFFP treatment(s), each participants starting condition will be identified by the above markers, with the same markers taken one month after the conclusion of their yFFP treatment(s).
The primary outcome is the change, if any, in the epigenetic age or genome pattern as well as the laboratory and functional assessments of the yFFP treated patients.
There will be multiple study sites participating throughout the State of Texas."
Epigenetic clocks, based on measuring DNA methylation at specific sites, offer the most accurate measurement of biological and chronological age of any molecular-biology method known today. While causality for epigenetic clocks has not been established, interventions that prolong healthspan and lifespan also slow the biological clocks. Moreover, restoration of cell youth by partial reprogramming with a subset of Yamanaka factors resets the epigenetic age. However, these factors are unlikely to be the natural direct drivers of epigenetic clock reset. In mouse embryonic development, all factors are expressed until implantation (E4.5), and only a subset is expressed and only in specific cells past this point. However, epigenetic reset occurs predominantly in gastrulation (E5.5-E7.5) and in all cells. Thus, identifying the direct mechanism behind the epigenetic clock reset is of fundamental importance to exploring the causality of epigenetic clocks. By integrating single-cell expression data before, during and after the reset with extensive gene annotation, a list of candidate genes can be generated. These can be tested individually or in combinations in cell culture, for epigenetic reset without complete or partial reprogramming.
"Age-related changes in the structures of the extracellular matrix.

One of the most underestimated and important areas in life extension research is age-related change in the extracellular matrix. As early as 1942, Johan Bjorksten formulated his “The crosslinking theory of aging”. It is based on the assertion that aging is the result of the accumulation of intermolecular covalent bonds (crosslinks) in proteins, which increase very slowly. Examples of these proteins in the extracellular matrix are collagen and elastin.
https://www.sciencedirect.com/science/article/abs/pii/0531556590900395

Crosslinks are formed non-enzymatically (stochastically) and have a great influence on the physical properties of the extracellular matrix (ECM). In turn, the increased rigidity of the ECM will contribute to cell dysfunction through mechanosensory perception.
https://www.nature.com/articles/s41586-019-1484-9

In addition, advanced glycation end products can generate inflammatory responses through activation of the advanced glycation end products (RAGE) receptor, thus contributing to the formation of age-related chronic inflammation and age-related pathologies associated with inflammation.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661616/

The extracellular matrix is mainly composed of collagen and elastin. Both proteins renew very slowly. The half-life of collagen in the skin, muscles and intestines is 74, 45 and 244 days, respectively. In human intervertebral discs, the half-life of collagen has been estimated to be at least 95 years.
https://www.sciencedirect.com/science/article/pii/S002192582052841X
https://www.sciencedirect.com/science/article/abs/pii/030441659290095C

Elastin is another very highly stable molecule. Its functional non-renewable period in the human body is about 80 years and is comparable to the duration of human life. Therefore, any non-enzymatic modifications of elastin (such as glycation and carbamylation) will have serious consequences.
https://www.jci.org/articles/view/115204

The wide participation of sugars and their derivatives (such as methylglyoxal) in metabolic processes and the absence of absolutely reliable antiglycation mechanisms make glycation of long-lived proteins almost inevitable. Age-related decrease in elastin vessels in the ECM with simultaneous accumulation of collagen will contribute to the development of cardiovascular pathologies and accelerated aging. As expected, the decrease in elastin content imposes an upper limit on the functioning of the cardiovascular and pulmonary systems, which is estimated at about 100-120 years for humans.
https://link.springer.com/article/10.1007/s10522-007-9122-6

In general, according to the latest data, age-related changes in ECM proteins associated with non-enzymatic modifications are somehow associated with most other signs of aging. Often acting as a driver in the formation of mechanisms involved in aging and the development of age-related diseases. ECM aging forms ""vicious cycles"", positive feedback loops with other mechanisms of aging. such as inflammation and oxidative stress.
https://www.sciencedirect.com/science/article/pii/S1568163720302324?via%3Dihub#bib1115

In addition to non-enzymatic modifications of long-lived ECM proteins, its other structures also make a significant contribution to aging. Especially in the neurodegenerative processes characteristic of human aging. This applies to changes in the levels of hyaluronan, chondroitin sulfates, etc.
https://www.nature.com/articles/s41380-021-01208-9?fbclid=IwAR0TET-rdro2p-Fm8Bdr9sP1tgYMLK665RpZWHlCYfNpItYsYTGIhZv92zs

Therefore, it can be clearly stated that age-related changes in the ECM structure play a very large, but so far underestimated and poorly studied role in aging."
"The substantial body of existing literature supports the leading role of the accumulation of stochastic damages of long-lived molecular structures in the aging process (https://pubmed.ncbi.nlm.nih.gov/32540391/).

These damages (namely, Maillard reaction - non-enzymatic cross-linking of the nucleophilic amino group of the amino acids and carbonyl group of sugars, spontaneous protein-protein cross-linking (https://pubmed.ncbi.nlm.nih.gov/31794011/), racemization of aspartic acid residues) lead to incomplete digestion of proteins that compose the extracellular matrix, the nuclear pore, and other molecular structures with a slow or absent renewal.

In the case of the extracellular matrix, probably, these indigested fragments accumulate over time and, due to a lack of specific enzymes, remain incorporated into the medium. Such accumulation fortifies the extracellular matrix and results in its increased stiffness and malicious nano topography (where needle-like fragments are poking out the collagen and elastin fibrils).

The enzymatic activity of known collagenases against cross-linked collagen is limited (https://pubmed.ncbi.nlm.nih.gov/23957394/), and the efficacy of specific artificial enzymes or abzymes (https://diabetes.diabetesjournals.org/content/67/Supplement_1/1229-P) might be very low as they may not be able to penetrate the narrow space between cross-linked fiber due to their size.

I may speculate that the development of ferromagnetic nano-tags exclusively targeting these indigested fragments might be a better research direction.

Such nano-tags would stick to their targets (for example, to glucosepane-containing fragments), while the rest of them would shortly excrete from the body. Subsequently, an applied alternating magnetic field would induce local hyperthermia and destruction of fragments, the debris of which, along with used nano-tags, likely can be removed by macrophages."
"Our proposal to treat age-related chronic degenerative diseases involves a cell-based gene therapy approach using genetically-matched differentiated embryonic stem cells (from somatic cell nuclear transfer; SCNT) for mitochondrial transfer therapy. See these publications from our group for details:
https://pubmed.ncbi.nlm.nih.gov/35243258/
https://pubmed.ncbi.nlm.nih.gov/23683578/"
"DNA methylation is currently the most promising molecular marker for monitoring aging and predicting life expectancy.

However, the mechanisms underlying age-related DNA methylation changes remain mostly undiscovered, but scientists believe drug intervention could reverse epigenetic mechanisms that serve as regulators in diseases of aging. .

Numerous studies over the past decade have strongly implicated epigenetic mechanisms in regulating the expression of genes involved in the regulation of several aging-related diseases, such as cancer and fibrotic disorders ((Ropero and Esteller 2007, Pang and Zhuang 2010), heart failure, neurodegenerative diseases such as Alzheimer's or Parkinson's, artheriosclerosis and other aging processes in different tissues (Hoeksema and de Winther 2016).
(Lovrečić et al. 2013; Pagiatakis et al. 2019).

Drugs that interfere with DNA methylation and histone deacetylation have been approved for clinical use by the Food and Drug Administration (FDA) (Jones et al. 2016).

Epigenetics and therapeutic approaches
Experiments in animal models of neurodegenerative disorders, diabetes or cataracts have confirmed the possible role of epigenetic drugs, as well as inhibitors of histone deacetylases and methyl donor compounds (Adwan and Zawia 2013).

Drugs such as histone deacetylase (HDACi) inhibitors can cross the blood-brain barrier (BBB), thereby slowing the initiation and development of symptoms in animal models of neurodegenerative diseases (Coppedè 2014).

Several structural classes of HDACi are under clinical trial for various diseases, including hydroxamic acids (vorinostat), cyclic peptides (depsipeptide or romidepsin), benzamides, and aliphatic acids (valproic acid (VA)) (Adwan et Zawia 2013).

Nutrition
Nutrition and food compounds, for example vitamin B, folate and methionine, are also known to affect epigenetic regulation and mechanisms.

- Polyphenols, for example, highlight the unique cooperation between the genome and the environment, especially at physiological concentrations (Remely et al. 2015).
They only induce a significant effect if they are consumed in large quantities or if the levels of methyl donors are limited; however, possible toxicity resulting from the oxidation of polyphenols may occur and care should be taken when consuming these compounds (Fang et al. 2007).

- Folate supplementation, for example, can inhibit the adverse effects of aging, such as uracil malincorporation, DNA methylation, protein methylation, mitochondrial deletion, and critical gene expression, and it It has been observed to protect against colon cancer (Jang et al. 2005; Kim 2005).
The impact of folate deficiency on DNA methylation could be target site and gene specific and it is proposed that the progression of genomic and site specific DNA methylation due to deficiency in folate may vary (Mierzecki et al. 2015).

5-Azacytidine or azacitidine (AzaC) is one of the most studied DNMT (DNMTi) inhibitors and is primarily used in the management of myelodysplastic syndrome (MDS), in which it acts as a methyl donor eliminator in vitro to weaken the effect of gene silencing on methylation.
A 67-year-old man with high-risk MDS showed hematologic improvement three months after the first course of AzaC treatment (Takaoka et al. 2014).

Conclusion
Epigenetic changes play an important role in the progression of aging.
Therefore we propose to develop and improve techniques to understand epigenetic regulation in normal aging. Studies should be carried out to restore chromatin dynamics and therefore proper gene expression, which could provide new therapeutic strategies if applied early and in combination with other therapies addressing all aspects of aging."
"The Uberkinder Program: Spawning globally superior humans through repurposing of adult longevity and sports interventions to foetal and pre-adult phases of life.

There are demonstrated and speculative reasons that suggest application of adult health and longevity interventions in the earliest phases of life can dramatically improve physiological function, including longevity and cognitive capacity. Many such interventions are highly accessible and GRAS, but efficacy and dosing have not been established for uberkinder application. The list of possibly effective interventions is long, and center around management of biological aging rate, genetic expression, and resilience enhancement.

Uberkinder interventions will enhance overall physiology, and be extremely important as 'preventive' enhancements of longevity and disease resistance and recovery. Perhaps more importantly (and more speculatively) in our time of self-generated existential threats, it is likely that the interventions will enhance cognitive capabilities; spanning intellect, emotions and perception.

Existing non-human mammal data points to dramatic results with simple interventions. Importantly, most envisioned initial interventions are very inexpensive; economically accessible to the vast majority of humanity. Research can begin almost immediately upon gaining IRB approval, with volunteer mothers.

Although uberkinder interventions will not save my generation, they will be essential in the evolution of a safer, healthier, more creative, happier future, and likely go down in future history as amoung the most effective humanitarian use of resources, ever.
"
"The Longevity Prize

Hypothesis Prize

Human Very Small Embryonic-Like (hVSEL) Stem Cells and Longevity

VSEL stem cells were first identified in murine bone marrow in 2006 and subsequently in human bone marrow1. Our own research has clearly shown the presence of CXCR4+, SSEA4+, Oct 3/4+, CD34-, CD133-, CD45-, Lin- hVSEL pluripotent stem cells in Platelet Rich Plasma (PRP) derived from human peripheral blood2. Other authors also describe hVSEL stem cells being present in human peripheral blood3 whilst some suggest that the hVSEL stem cells found in umbilical cord blood are an ‘aberrant and inactive’ population of cells4. It is evident that there are conflicting reports in the scientific and medical literature about the existence, viability and biological activity of hVSEL stem cells and more research is required to clarify this situation. Nevertheless, it is also clear that PRP contains hVSEL stem cells and that PRP is therefore a readily available source of pluripotent stem cells which is currently ignored by most people working in stem cell technology. The biological and therapeutic action of PRP does not currently include discussions on the importance of hVSEL stem cells in PRP in the overall efficacy of PRP treatments. The standardisation of the production of PRP is required to ensure homogeneity, safety and efficacy and carefully controlled clinic trials are needed using standardised preparations and methods to fully understand the full potential of PRP treatment5.
The persistence of hVSEL stem cells throughout life, from young to old, has been reported6 suggesting a potential homeostatic mechanism which maintains the hVSEL stem cell pool throughout life. This is supported by our unpublished observations of there being a constant availability of hVSEL stem cells in the PRP of patients who have undergone multiple PRP collections. The bone marrow is the likely source of the hVSEL stem cells to maintain the peripheral blood hVSEL stem cell pool. This is supported by the observation of the mobilisation of hVSEL stem cells into the peripheral blood following acute myocardial infarction7. In further support of this concept, hVSEL stem cells have been shown to be present in human bone marrow itself and also in human leukapheresis products8. This supports the hypothesis of migration of hVSEL stem cells from the bone marrow to the peripheral blood during physiological homeostasis and during pathological stimuli. Similar studies in vitro have suggested that hVSEL stem cells are the ‘original embryonic stem cell’ highlighting the critical importance of hVSEL stem cells in normal embryonic development and subsequent physiological homeostasis9. All stem cell types are subject to both intrinsic and extrinsic stress, including the aging process, during normal physiology and in pathological states. Such stress can have detrimental effects, especially on rapidly dividing stem cells10.
Hypothesis
Our hypothesis is as follows:
• hVSEL stem cells appear to be biologically quiescent in normal physiology.
• The QiLaser (formerly known as the SONG modulated laser) can activate hVSEL stem cells in PRP thus re-activating their biological role as pluripotent stem cells.
• Aging is a multifactorial process but the stem cell pool reduction, and the stem cell niche degeneration, are assumed to play a critical role. This results in a significant depletion of the stem cell pool over time, and we propose that this is a key factor in the aging process.
• If hVSEL stem cells are quiescent in normal physiology (which at the moment seems to be a reasonable conclusion from the evidence available), then QiLaser activated hVSEL stem cells (with pluripotent biological activity restored) could make an important contribution to replacing the depleted total stem cell pools and also to the repair of the age damaged total stem cell niches following aging.
• If hVSEL stem cells are quiescent in normal physiology, then they represent pluripotent stem cells which have not aged since the time of embryonic development when they arose from the Primordial Germ Cell (PGC). This means that QiLaser activated hVSEL stem cells could repopulate all of the stem cell compartments and niches with stem cells and stem cell niche cells which have undergone no aging at all.
• Telomere length in hVSEL stem cells in vivo may be conserved if hVSEL stem cells are truly biologically inactive in vivo. If this proves to be correct, then this will also enhance the longevity producing potential of pluripotent QiLaser activated hVSEL stem cells which will produce somatic cells with relatively long telomeres.

Our hypothesis will be tested by carrying out a placebo controlled preliminary clinical study to assess the effect of QiLaser activated hVSEL stem cells on C-G DNA methylation and telomere length. The study will take place at Qigenix which is the Medical Clinic of Dr Ovokaitys. DNA methylation and telomere length studies will be carried out by a third-party collaboration.

Professor Peter Hollands PhD (Cantab), Chief Scientific Officer, QiGeneration, Carlsbad, California, USA
Dr Todd Ovokaitys MD, Chief Executive Officer, QiGeneration, Carlsbad, California, USA

References

1. Ratajczak MZ, Zuba-Surma EK, Machalinski B, Kucia M. Bone-marrow-derived stem cells--our key to longevity? J Appl Genet. 2007; 48: 307-19. doi: 10.1007/BF03195227. PMID: 17998587.
2. Hollands P, Aboyeji DR, Ovokaitys T. The action of modulated laser light on Human Very Small Embryonic-Like (hVSEL) stem cells in Platelet Rich Plasma (PRP). CellR4 2020; 8: e2990 DOI: 10.32113/cellr4_202012_2990
3. Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Ratajczak J, Kucia M. Very small embryonic-like stem cells: characterization, developmental origin, and biological significance. Exp Hematol. 2008; 36: 742-751. doi:10.1016/j.exphem.2008.03.010
4. Danova-Alt R, Heider A, Egger D, Cross M, Alt R. Very small embryonic-like stem cells purified from umbilical cord blood lack stem cell characteristics. PLoS One. 2012; 7: e34899. doi:10.1371/journal.pone.0034899
5. Everts P, Onishi K, Jayaram P, Lana JF, Mautner K. Platelet-Rich Plasma: New Performance Understandings and Therapeutic Considerations in 2020. Int J Mol Sci. 2020; 21: 7794. doi:10.3390/ijms21207794
6. Sovalat H, Scrofani M, Eidenschenk A, Hénon P. Human Very Small Embryonic-Like Stem Cells Are Present in Normal Peripheral Blood of Young, Middle-Aged, and Aged Subjects. Stem Cells Int. 2016; 2016: 7651645. doi: 10.1155/2016/7651645. PMID: 26633977; PMCID: PMC4655065.
7. Zuba-Surma EK, Kucia M, Dawn B, Guo Y, Ratajczak MZ, Bolli R. Bone marrow-derived pluripotent very small embryonic-like stem cells (VSELs) are mobilized after acute myocardial infarction. J Mol Cell Cardiol. 2008; 44: 865-873. doi: 10.1016/j.yjmcc.2008.02.279. PMID: 18430437; PMCID: PMC2692386
8. Sovalat H, Scrofani M, Eidenschenk A, Pasquet S, Rimelen V, Hénon P. Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells. Exp Hematol. 2011; 39: 495-505. doi: 10.1016/j.exphem.2011.01.003. PMID: 21238532.
9. Virant-Klun I, Skerl P, Novakovic S, Vrtacnik-Bokal E, Smrkolj S. Similar Population of CD133+ and DDX4+ VSEL-Like Stem Cells Sorted from Human Embryonic Stem Cell, Ovarian, and Ovarian Cancer Ascites Cell Cultures: The Real Embryonic Stem Cells? Cells. 2019; 8: 706. doi: 10.3390/cells8070706. PMID: 31336813; PMCID: PMC6678667.
10. Mierzejewska K, Heo J, Kang JW, Kang H, Ratajczak J, Ratajczak MZ, Kucia M, Shin DM. Genome-wide analysis of murine bone marrow derived very small embryonic-like stem cells reveals that mitogenic growth factor signaling pathways play a crucial role in the quiescence and ageing of these cells. Int J Mol Med. 2013; 32: 281-90. doi: 10.3892/ijmm.2013.1389. PMID: 23708325; PMCID: PMC3776718.

"
Testing and using Unpatentable Natural Remedies will certainly be overlooked. I've written and tweeted this repeatedly. I would like to build a gym facility to record the efforts of people learning to breath better. I'm using cardio machines and full gym to increase muscularity and VO2max. I have a GS 1712 = Doctorate in Education, I beat cardio vascular disease in 7.5 years I got a 'clear/normal' heart reading. Nearly impossible but I'm gifted, autistic and with my Phd in Edu I know what I did to fix my heart, and the 'cardio' element of living longer. At 63 I'm doing 12 minutes of Bruce Protocol with bad lumbar, I'm injured - it's incredible. I used Mg, Turmeric, ALA, Potassium, Amino Acids, Collagen Peptides, B vitamins, now using Ashwaganda and Fenugreek for testosterone boost. I want to build a center in Southern Arizona [outside Tucson 30 min, 85602 zip] to partner with my local gym, we need to add a pool, sauna and not much else to begin. I want to charge $1200 year/$100 month to work with people on treadmill, stepper, versa climber, bikes and exercise 6 days/week to reach MAX VO2. We track progress and take vitals weekly. I'll write a report weekly. When I was a professor at the phone company they always said, 'don't ever bet against Anderson' - I've always had big ideas. I'm a world class troubleshooter - very open minded - the problem can be anything - the solution anything is possible. I also want to ADD CBD for telomer and Magnesium for telomer - testing. I need sauna because we rub on Mg in the sauna, pool and the rest is available at the gym next door. They will do my membership and I'll let them have swimming/sauna that they lack. Anyway CBD and Magnesium won't be tested by the BIGS. Thank you -Neo- Oren Anderson
"Apolipoprotein A-1 Milano is a missense naturally occurring variant (rs28931573) of the apolipoprotein A1, discovered in a small population of Valerio Dagnoli of Limone Sul Garda, a small village in northern Italy, which causes a reduction in the risk of cardiovascular disease.

Despite promising pre-clinical results, a clinical trial of a synthetic ApoA-1 Milano protein in patients with atherosclerosis failed to produce significant results in 2016. Since then, there have been no new clinical trials conducted.

However, the prophylactic use of ApoA-1 Milano-based drugs hasn't been studied yet, probably due to the intravenous route of their administration.

In light of that, the reported positive effects of ApoA-1 Milano full-length proteins produced by generically modified rice and delivered via oral administration (https://pubmed.ncbi.nlm.nih.gov/29907443/) raise the question of whether it is possible to construct vectors for inducible and constitutive ApoA-1 Milano expression in Lactobacillus."
"RAPID REDUCTION OF EPIGENETIC AGE

Hypothesis:
Given that viable stem cell numbers decline dramatically throughout life even while cellular replacement needs increase, the endogenous proliferation of stem cells should provide the most direct and comprehensive approach to restoring cellular maintenance and reducing epigenetic age.

It is known that stem cells are quiescent due to high expression of uncoupling protein 2 (UCP2), which allow protons to dissipate through the inner mitochondrial membrane without producing ATP. It is also known that mitochondrial fusion will bias stem cell division to proliferation. Thus by simultaneously supplying a fusion supplement and a blocker for UCP2, quiescent stem cells should awaken and begin proliferating, subsequently replacing senescent cells as needed.

A proposed method can be divided into three parts:

A. Filling stem cell niches: Blocking proton leakage through UCP2 pores with C60* activates stem cells while mito fusion directs them to proliferation.
B. Replacement of senescent cells: Mitochondrial fission/apoptosis using senolytics removes senescent cells, which are replaced by cells derived from proliferated stem cells of part A, at the direction of natural paracrine signaling.
C. Maximizing epigenetic age reversal: Supplying a demethylase promoter during parts A and/or B further reduces aberrant methylation and epigenetic age.

Proof of Concept:
1. A 68 year old individual who had previously used a fullerene C60/oil supplement over several years showed a baseline epigenetic age of 0.5 years above his chronological age, suggesting that C60 used alone had no long-term epigenetic benefit.
2. After treatments employing C60 according to Hypothesis parts A and B, his epigenetic age dropped 12 years over a period of about 2 years. And after treatment with parts A-C, further declines in epigenetic age were noted, reaching a maximum decline of 28 years. His appearance and general sense of physical well-being improved in accordance with the measured epigenetic age reduction.

*The fullerene C60 dissolved in oil is proposed herein as a UCP2 blocker. The use of C60 to extend rodent lifespans was the subject of three papers. The first (in 2012, PMID 22498298) showed that feeding rats C60 in oil increased rat lifespans by 90% over controls, and attributed the increase to the antioxidant properties of C60. The next two (in 2021, PMID 33123847 and PMID 33849306) attempted to replicate this result in mice, but found no increase in lifespan. The discrepancy can be resolved by postulating a different MOA for C60 — UCP2 blocking rather than ROS quenching — and a different feeding regime for the three experiments. Although none of the papers precisely described how their test animals were fed, it is likely the research group of the first paper fasted their rats overnight (as they did in a previous C60 toxicology study), while research groups of the next two papers likely did not, as there was no suggestion to do so in the first paper. Rodents have a metabolic rate about six times that of humans, so a state of fasting-induced mitochondrial fusion would have been easily achieved in the first study, but no fusion would have occurred in next two. As noted in the proof of concept trial, C60 sans fusion produced no epigenetic reversal in the human subject, and likewise no increase in rodent lifespan.
"
"RESTORATION OF MITOCHONDRIAL FUNCTION

Hypothesis:
Methylation of mitochondrial DNA loops (mtDNA) can be expected to produce an energetic deficiency that is difficult to treat, as normal cellular QC does not remove methylation, while methyltransferase insures its persistence. Herein it is proposed that cellular QC can be tricked into removing methylated and sub-functional mtDNA by briefly forcing ATP output to zero.

Background: Mitochondrial quality control involves PINK1/Parkin labeling of fissioned mitochondria with zero surface potential (ΔΨm). Labeled mitochondria are then removed by recycling in lysosomes (mitophagy). Mitochondria with methylated mtDNA may have lower than normal ATP output due to deficient enzyme synthesis, but are not removed as surface potential doesn’t go to zero. Such sub-functional mtDNA may have a survival advantage over unmethylated mtDNA due to fewer ROS and a lower mutation rate, and thus may come to dominate the cell.

Proposed treatment method: Fissioned mitochondria having a single loop of sub-functional mtDNA will also have a smaller reserve of enzymes. If enzyme synthesis stops during biogenesis, they may run out faster than normally functioning mitochondria. ATP production will briefly cease and surface potential will go to zero. PINK1/Parkin will then label them for mitophagy. Contracting and expanding the cellular mtDNA population by cycling fission/biogenesis with fusion/biogenesis should remove sub-functional mtDNA in an iterative fashion, in which the mtDNA removed per cycle is limited by lysosomal capacity. Adding a demethylase promoter should accelerate the process.

Oral supplements for promoting fission, fusion, biogenesis and demethylase are readily available.

Proof of Concept:
The cyclic method proposed above was trialed by a subject who suffered persistent mitochondrial damage from statin use years before. Fission/biogenesis/demethylase was alternated with fusion/biogenesis/demethylase on a daily basis (using a “reps to failure” exercise to monitor progress). Reps to failure during fission were initially 43 percent lower than during fusion*, but as cycles were completed, fission reps rose and ultimately matched fusion reps after three weeks, suggesting that sub-functional mtDNA had been removed.

*Fusion reps were expected to be initially higher than fission reps due to enzyme sharing.
"
"Protein glycation is a non-enzymatic post translational modification in which sugars or reactive carbonyls react with amino acids to form Advanced Glycation End products (AGEs). AGEs constitute a structurally diverse group of molecules linked to malignant ageing and age-related diseases. The human body can eliminate only early glycation intermediates, but not AGEs. As a result, AGEs accumulate in our body as we age and exert their pathophysiological effects via interaction with cell-surface receptors and by altering the structure and function of proteins. AGEs are ligands for the receptor for AGEs (RAGE), a multi-ligand receptor associated with oxidative stress, cellular damage and chronic inflammation. Despite implications of AGEs in age-related pathologies, mechanistic studies are limited by the lack of precision molecular tools to reverse AGE modifications.
In this project, we aim to expand the ageing research toolbox by engineering enzymes with the ‘new-to-nature’ capacity to catalyse AGE reversal. Enzymes are perfectly suited for this application, as they can enhance challenging transformations with exacting selectivity and deliver these reactions in cells. Developing AGE-repairing biocatalysts remains a significant challenge due to the structural diversity and site-specificity of AGE formation. Although there are currently no naturally occurring enzymes known to repair AGEs as their native function, protein engineering can expand their catalytic repertoire and tailor them to biotechnological and clinical applications.
We will create precision molecular tools for ageing research by addressing the following aims:
(1) Biocatalytic reversal of AGEs. We will develop novel AGE-repairing enzymes by repurposing promiscuous biocatalysts and extending their substrate scope by rational design.
(2) Engineering of AGE-reversing biocatalysts. Using a hybrid experimental and computational approach, based on machine learning-guided directed evolution and droplet microfluidics, we will dramatically improve the activity, stability and biocompatibility of the AGE-repairing enzymes to meet high biotechnological standards. Additionally, structural and functional characterisation of enzyme variants will be performed in order to understand the basis of improvements along the evolutionary trajectory.
(3) Cellular impact of biocatalytic AGE reversal. Last, we will extend the functionality of the enzymes to AGE reversal in cells, and will determine the impact of AGE reversal on RAGE signalling and inflammation.
The availability of precision molecular tools with the ability to repair AGEs is envisioned to revolutionise the fields of ageing and glycation damage, by allowing researchers to uncover causal relationships that drive ageing and diseases via accumulation of AGEs. Finally, the superior biocatalysts developed in the study could pave the way towards enzyme therapeutics for the treatment of AGEs and related pathologies.

"
"Hepatocyte growth factor for aging and age-related diseases

Persistent low level injury is the underlying cause of modern society’s most prevalent and costly diseases (1). Pulmonary disease, liver disease, kidney disease, diabetes, intestinal disorders, neurological disease, atherosclerosis, autoimmune disease, even aging itself, all have the common denominator of persistent low level injury. While there are many routes to each individual malady (obesity, chemicals, autoimmunity) the response to ongoing low level organ damage is very consistent. Non-epithelial cells are activated and proliferate to repair and limit the damage. Cells of the immune system, macrophages, and myofibroblasts are all recruited to the site. In the face of ongoing damage, the repair process never completes and the functional cells of the organ are slowly replaced by extracellular matrix resulting in fibrosis. There is an urgent need for a mechanism to limit inflammation, stimulate regeneration and reverse existing fibrosis.

The response to tissue damage is complex but two cytokines play a central role in initiating repair and then terminating the response. TGFβ is released from the surface of cells in response to injury. This initiates the repair process, acting through a series of TGFβ family receptors, mobilizing events to reestablish the epithelial barriers(2). HGF and its receptor, c-met, are subsequently induced, stimulating division and differentiation of new epithelial cells and eventually shutting down the TGFβ response(3). In the face of ongoing damage, TGFβ signaling becomes dominant, suppressing transcription and activation of HGF and leading to replacement of functional tissue with fibrous extracellular matrix leading to fibrosis and aging.

A major transcriptional target for TGFβ is plasminogen activator inhibitor (PAI1) (4). PAI1 inhibits both urokinase type plasminogen activator (uPA) and tissue plasminogen activator (TPA). Both uPA and TPA are able to convert the HGF precursor, proHGF, into the two chain active HGF (aHGF) (5, 6). PAI1 inhibits activation of proHGF. Defective activation of proHGF due to high PAI1 has been demonstrated in bleomycin induced pulmonary fibrosis, kidney and liver fibrosis, and muscle regeneration (7–11).

Generalizing the idea that poor HGF synthesis and activation is detrimental to the organism, HGF may have been overlooked in relation to aging itself. HGF is implicated in three interesting age-related examples. The most startling is the finding of an Old Order Amish cohort in Berne, Indiana, who carry a defect in SERPINE1, the gene coding for PAI1 (12). Homozygotes have a mild hemophilia-like disorder due to overactive uPA and TPA. The heterozygotes have about half the normal level of PAI1, a reduced incidence of cardiovascular disease, a lower circulating insulin level and live, on average, about ten years longer than their normal matched controls. HGF is known to reduce cardiovascular disease and plays a role in insulin and glucose homeostasis (3, 13). It is possible, but unproven, that the reduction in PAI1 produces a somewhat higher level of HGF activation, resulting in the improved phenotype.

A second instance for a possible role for HGF in aging is found in Werner syndrome (14). This is a late onset progeria caused by a mutation in the WRN gene, coding for a DNA helicase. Tu, et al, used CRISPR to correct the gene defect in induced pluripotent stem cells derived from Werner syndrome patients (14). When these cells were differentiated into mesenchymal stem cells, they were found to produce dramatically higher levels of HGF, resulting in improved angiogenesis and wound healing. The authors suggest that at least some of the aging phenotype in Werner syndrome is due to poor synthesis of HGF and thus poor stromal/epithelial communication. Werner syndrome patients most commonly succumb to cardiovascular disease (15). HGF is known to reduce cardiovascular disease (3).

The third example comes from a recent paper demonstrating high levels of PAI1 synthesis in fibroblasts derived from patients with Hutchinson-Gilford progeria (16). The authors showed that these fibroblasts had a myofibroblast phenotype with high synthesis of extracellular matrix proteins, defective mitochondria and poor cell cycle progression. Inhibition of PAI1 with a small molecule inhibitor restored a normal phenotype, reducing synthesis of fibronectin and collagen, restoring the mitochondrial structure and stimulating DNA synthesis. The authors suggest that PAI1 inhibition could relieve some of the symptoms of Hutchinson Gilford progeria.

Hypothesis: Aging and age related diseases are caused by poor synthesis and activation of HGF subsequent to elevated TGFβ signaling. HGF, or an HGF mimetic, could cure age related diseases, increase longevity and relieve the symptoms of both Werner syndrome and Hutchinson Gilford progeria.

Experiment – A simple experiment to test this idea could be done using the same Hutchinson Gilford progeria derived fibroblasts described by Catarinella, et al (16). These cells and their controls are available from the Progeria Research Foundation and the Coriell Institute. Fibroblasts and myofibroblasts are a main source of HGF. As determined by SDS gel electrophoresis and immunoblotting of serum free supernate from the cultures, normal fibroblasts will have primarily active HGF while the progeria derived fibroblasts will have primarily proHGF. Inhibition of PAI1 with TM5441, as done by Catarinella, et al. will demonstrate increased concentrations of active HGF. Addition of an antibody to HGF or to c-met will block the effect of TM5441 on the cells. Finally, addition of recombinant human HGF to the cells will have the same effect as TM5441.

References

1. Furman, D., Campisi, J., Verdin, E., Carrera-Bastos, P., Targ, S., Franceschi, C., Ferrucci, L., Gilroy, D. W., Fasano, A., Miller, G. W., Miller, A. H., Mantovani, A., Weyand, C. M., Barzilai, N., Goronzy, J. J., Rando, T. A., Effros, R. B., Lucia, A., Kleinstreuer, N., and Slavich, G. M. (2019) Chronic inflammation in the etiology of disease across the life span. Nat Med. 25, 1822–1832
2. Katsuno, Y., and Derynck, R. (2021) Epithelial plasticity, epithelial-mesenchymal transition, and the TGF-β family. Dev Cell. 56, 726–746
3. Gallo, S., Sala, V., Gatti, S., and Crepaldi, T. (2015) Cellular and molecular mechanisms of HGF/Met in the cardiovascular system. Clinical Science. 129, 1173–1193
4. Samarakoon, R., Overstreet, J. M., and Higgins, P. J. (2013) TGF-β signaling in tissue fibrosis: Redox controls, target genes and therapeutic opportunities. Cell Signal. 25, 264–268
5. Mars, W. M., Jo, M., and Gonias, S. L. (2005) Activation of hepatocyte growth factor by urokinase-type plasminogen activator is ionic strength-dependent. Biochem J. 390, 311–315
6. Naldini, L., Tamagnone, L., Vigna, E., Sachs, M., Hartmann, G., Birchmeier, W., Daikuhara, Y., Tsubouchi, H., Blasi, F., and Comoglio, P. M. (1992) Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. Embo J. 11, 4825–4833
7. Eitzman, D. T., McCoy, R. D., Zheng, X., Fay, W. P., Shen, T., Ginsburg, D., and Simon, R. H. (1996) Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest. 97, 232–237
8. Hattori, N., Mizuno, S., Yoshida, Y., Chin, K., Mishima, M., Sisson, T. H., Simon, R. H., Nakamura, T., and Miyake, M. (2004) The Plasminogen Activation System Reduces Fibrosis in the Lung by a Hepatocyte Growth Factor-Dependent Mechanism. Am J Pathology. 164, 1091–1098
9. Dworkin, L. D., Gong, R., Tolbert, E., Centracchio, J., Yano, N., Zanabli, A. R., Esparza, A., and Rifai, A. (2004) Hepatocyte growth factor ameliorates progression of interstitial fibrosis in rats with established renal injury. Kidney Int. 65, 409–419
10. Wang, H., Zhang, Y., and Heuckeroth, R. O. (2007) PAI‐1 deficiency reduces liver fibrosis after bile duct ligation in mice through activation of tPA. Febs Lett. 581, 3098–3104
11. Sisson, T. H., Nguyen, M.-H., Yu, B., Novak, M. L., Simon, R. H., and Koh, T. J. (2009) Urokinase-type plasminogen activator increases hepatocyte growth factor activity required for skeletal muscle regeneration. Blood. 114, 5052–5061
12. Khan, S. S., Shah, S. J., Klyachko, E., Baldridge, A. S., Eren, M., Place, A. T., Aviv, A., Puterman, E., Lloyd-Jones, D. M., Heiman, M., Miyata, T., Gupta, S., Shapiro, A. D., and Vaughan, D. E. (2017) A null mutation in SERPINE1 protects against biological aging in humans. Sci Adv. 3, eaao1617
13. Fafalios, A., Ma, J., Tan, X., Stoops, J., Luo, J., DeFrances, M. C., and Zarnegar, R. (2011) A hepatocyte growth factor receptor (Met)−insulin receptor hybrid governs hepatic glucose metabolism. Nat Med. 17, 1577–1584
14. Tu, J., Wan, C., Zhang, F., Cao, L., Law, P. W. N., Tian, Y., Lu, G., Rennert, O. M., Chan, W. Y., and Cheung, H. H. (2020) Genetic correction of Werner syndrome gene reveals impaired pro‐angiogenic function and HGF insufficiency in mesenchymal stem cells. Aging Cell. 19, 13–20
15. Kato, H., and Maezawa, Y. (2022) Atherosclerosis and Cardiovascular Diseases in Progeroid Syndromes. J Atheroscler Thromb. 29, 439–447
16. Catarinella, G., Nicoletti, C., Bracaglia, A., Procopio, P., Salvatori, I., Taggi, M., Valle, C., Ferri, A., Canipari, R., Puri, P. L., and Latella, L. (2022) SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson–Gilford progeria syndrome. Cell Death Dis. 13, 737

"
"Isocaloric twice-a-day-feeding improves brain aging
Scientific background
As we age, our brain changes. Cognitive capabilities and motor control are detrimentally affected. At a microscopic level, our brain accumulates the age-pigment lipofuscin, leading to neuronal loss, and reactive gliosis with an accumulation of astrocytes and microglia1. Western diet, characterized by a high intake of saturated fats and refined carbohydrates, induces overweight and metabolic changes, which exacerbate these aging features. Diet, neuronal loss, and gliosis have been linked to most of the known neurodegenerative diseases. Caloric restriction (CR) extends health and lifespan in multiple organisms2. Despite its remarkable benefits, humans adhere poorly to CR. Other strategies try to emulate CR, for instance, intermittent fasting or the isocaloric twice-a-day (ITAD) feeding, which benefits are mediated by autophagy stimulation. From those, ITAD-fed model mice consume the same food amount as ad libitum controls but at two short windows early and late in the diurnal cycle. This has proven metabolic and cognitive benefits. This is a strategy easier to implement in humans than CR or intermittent fasting. However, ITAD-fed benefits in brain aging have not been explored. We hypothesize that ITAD feeding ameliorates features of brain aging, particularly, those produced by western diets. We aim to test the improvement of aging markers, particularly neurodegenerative ones, using the ITAD strategy in high-fat diet (HFD) fed mice, that emulate the western diet.
Preliminary work
We have conducted a pilot study in a reduced group of mice to test that ITAD-feeding benefits metabolism, and behavioral and motor skills. 6-month-old C57BL6 mice were fed for 12 months with HFD, half with the ITAD-feeding strategy and the other half ad libitum. ITAD-feeding strategy feed mice with the same calories as ad libitum but in just two meals (two time periods) separated by a long starvation period, 10-12 hours. ITAD-feeding reduces mice weight and triglycerides, insulin, and leptin in serum samples.
Thus, our model improves whole animal metabolism, as it has been described before3. Regarding behavior, we have found a motor improvement, analyzed by rotarod and grip strength test, less anxiety and more wiliness to explore, by open field, and a reduction of hindlimb clasping. All these tests showed a clear improvement of key aging behavioral features thanks to the ITAD diet.

Aims and work program
Aim 1: Test ITAD-feeding benefits with macroscopic analysis, including behavioral and motor skills.
6-month-old C57BL6 mice are fed for 12 months with HFD, half with the ITAD-feeding strategy and the other half ad libitum. Mice in a regular chow diet are kept in control, with ITAD and ad libitum diet. We are monitoring feed intake and weight. After the 12 months, we will conduct behavior analysis, e.g., open field, rotarod; and we analyze metabolic markers in serum, e.g., insulin, leptin to check that the ITAD-feeding is working.
We are performing a life expectancy test, so we will keep mice fed with all the strategies previously described until they die. Thus, we will clarify if the ITAD-feeding strategy prolongs life spam.
Aim 2: Decipher ITAD-feeding molecular mechanism to improve brain health.
Mice in ITAD-feeding, ad libitum, and controls will be divided into two groups: histological and for biochemical analysis. We will collect different tissues (brain, liver, muscle, adipose tissue) to evaluate the impact of ITAD-feeding benefits on them.
Histological analysis: to evaluate neurodegeneration we will use neuronal (MAP2, Tuj1) and apoptotic markers (TUNEL, Caspase3). To understand the causes of cell death, we will analyze lipofuscin accumulation, gliosis (GFAP and Iba1), microglial activation markers (CD86, CD206), and other disease markers (amyloid-β peptides).
Biochemical analysis: CR and other similar feeding strategies rely on autophagy activation to exert their benefits. Autophagy is the cellular clearing and recycling program that degrades cytoplasmic content in lysosomes. Autophagy decline is well known during aging, which contributes to waste accumulation. To analyze whether the ITAD-feeding system stimulates autophagy in the brain we will perform flux analysis in different brain areas (cortex, hippocampus, and hypothalamus).
Impaired autophagy contributes to neuroinflammation through the accumulation of damaged structures (Aβ, P-Tau, and mitochondria) and the activation of microglia. Then, we will analyze inflammation (NF-Kβ, TNF-α, interleukins, by western and ELISA) and microglia activation markers (CD86, CD206). Also, we will address how brain improvement benefits the whole of mice's well-being. Thus, specific pathologic, inflammatory, and autophagic markers will be evaluated in the other tissues (liver, adipose tissue…). Our design provides the proof-of-concept needed to establish ITAD-feeding as a plausible therapy to improve brain aging, particularly with western diets. As such, it would be of great relevance when ITAD-feeding is implanted in a future clinical trial.

Impact, including interdisciplinarity
The overall burden of disease is assessed using the disability-adjusted life year (DALY), which expresses the years of healthy life lost due to disability (YLDs) or lived in less than full health5. Age-related diseases are a very important factor causing DALYs, particularly neurological diseases, including dementias, which are affecting over 7 million people in Europe, costing approximately €130 billion per annum6. Caloric restriction has been proven effective to reduce the hallmarks of aging in mice and humans, including improving memory loss7. However, human adherence to these diets is very complicated. In our project, we will evaluate the benefits of a change of habits to brain aging. We maintain the same calories but are divided into just 2 meals. The intervention we are proposing to reduce aging brain symptoms won’t increase the cost to the health system, as it will not require any additional treatment. Therefore, this project has the potential to significantly reduce the impact of neurodegenerative diseases and improve the quality of life, particularly in elderly people, also reducing the neurodegenerative disease burden on the health system.

Timeline of activities
The mice are already under the ITAD-feeding strategy, so this project will be finished in 2023 year, as proposed in the Gantt chart. We will finish the first aim during the 3 first months of 2023 and the second aim will be performed during the following 8 months. The manuscript is expected to be sent at the end of November 2023."
"Mitochondrial dysfunction is a central hallmark of aging and many of the current interventions to extend lifespan and health span target the mitochondrial metabolism. For example, dietary interventions, genetic interventions (e.g., AMPK) and drugs (e.g., metformin and rapamycin) extends lifespan by, at least partially, targeting mitochondria. Generally, many of the interventions aim, quite surprisingly, towards a reduction of mitochondrial activity. A prominent example is the induction of mitochondrial unfolded protein response (mtUPR) during development, where reducing components of oxidative phosphorylation results in increased lifespan. However, it is noteworthy that such intervention reduces the physical activity of the animal in question and likely impairs the ability of the animal to respond to other type of stress. Therefore it is questionable whether such approaches could be translated into human longevity therapy.

Here we hypothesize a novel and radical approach termed external energy replacement. What does it mean?

Taken together, our hypothesis of using light as an external energy replacement is quite revolutionary and show great promise of success in attenuating human aging in a novel manner.

"
"Background/Motivation of Study
A relevant term in this context is cellular energy homeostasis and by extension, energy metabolism. While different theories of aging consider different variables and factors – this is one that is recurrently observed in most cases(K. Jin & Rose, 1988). Complex biological constructs are open thermodynamic systems that are subjected to constant exchange of energy components with the environment(Flatt & Partridge, 2018; Kowald et al., 2020; López-Otín et al., 2013; Maklakov & Immler, 2016). Cellular homeostasis and survival in general are threatened with age due to the loss of capacity of organisms to maintain a balance in generation, usage and storage of biochemical energy. The energy component is seminal to this understanding because it links the aging process to a seemingly distant function – sex and reproduction. While aging is generally considered for somatic tissue maintenance, reproduction is another fundamental process that shares the same variables and objectives as aging – survival, proliferation and maintenance(da Costa et al., 2016; Gems, 2014; K. Jin & Rose, 1988; Kowald et al., 2020). Organisms need to allot a proportion of their system energies to reproduction, that impacts other metabolic processes – particularly fitness and aging. Therefore, in a dimorphic system where reproduction is handled heterogeneously, the aging process should be expected to be perturbed divergently as well. This notion is supported by a large body of existing literature where a significant bias is observed in mammals where female have longer maximum lifespans as well as average lifespans than males(Benayoun et al., 2020; Bonduriansky et al., n.d.; Garratt, 2019; Gems, 2014; Sampathkumar et al., 2020; Tower, 2006). However, this is not true in all cases with clusters of contradictory data spanning across different species and experimental conditions. Moreover, the underlying molecular processes behind aging are just as confounding as the sexual dimorphism behind them. Apart from the last few years of research, very little effort has been previously invested in studying the sexual disparities in senescence – further extending the gap of knowledge required to be addressed in the molecular and cellular levels.
Over decades of empirical research, the biological aging process in cellular and molecular terms have been illustrated through various inter-connected components known as the hallmarks of aging(López-Otín et al., 2013). While the hallmarks of aging themselves are often considered as driving forces in aging, there are certain molecular pathways and metabolic networks that act as intermediate regulators of these endpoints(Childs et al., 2015; Fahy et al., 2019; Pagiatakis et al., 2019; Ribarič, 2012; Serrano, 2017; Soto-Gamez et al., 2019). Among these, a clear energy metabolism associated pattern is observed – constructing a complex network of genes and gene products across carbohydrate and fat metabolism, oxidation, inflammation, stress response, immune-senescence, nutrient sensing, starvation and autophagy, apoptosis, cellular proliferation and epigenetic regulation. Among these gene products, some major regulators of energy homeostasis and aging have been broadly investigated as therapeutic targets. 5' AMP-activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR), insulin and insulin-like growth factor-1 (IGF-1), Sirtuin family proteins (SIRT1-SIRT7), forkhead box transcription factors (FOXOs) and different kinases construct an evolved survival-proliferation regulating network utilizing other aging associated processes(Arpón et al., 2019; Bonkowski & Sinclair, 2016; Imai & Guarente, 2014; J. Kim & Guan, 2019; Lewinska et al., 2020; Michan & Sinclair, 2007; C. Zhang et al., 2020). While literature provides substantial evidence on sexual bias in the expression and impact of these regulators, the nature and direction of the bias is often contradictory(Benayoun et al., 2020; de Arellano et al., 2019; Fagan et al., 2020; Garratt, 2019; McCartney et al., 2019; Regan et al., 2016; Sampathkumar et al., 2020; Tower, 2006; Tower et al., 2020).
The current project proposal takes these different aspects and notions into account in order to re-evaluate the current concept of a sexually homogenous molecular biological aging process. In order to fully understand the aging process, we must trace back the evolutionary mechanisms in aging and sexual dimorphism as well as the cellular, molecular, metabolic and epigenetic elements contributing to this divergence.
Genetic analysis supports the notion of sexually divergent expression patterns of aging associated alleles and transgenes. These examples hint at a female driven selection mechanism that influences males to inherit genes and by extension, traits that do not necessarily favor them in longevity and fitness (""Mother's Curse"" in mitochondrial functionality, X chromosome optimization for females, i.e. different forms of sexually antagonistic pleiotropy). Some genes and by extension, traits, asymmetrically evolved in this sex-specific manner in turn, evidently aid a selected sex in life extension. Other avenues of sexual dimorphism in longevity genes extend to nutrient sensing and fat metabolism in aging, adaptive homeostasis and hormesis, endocrine regulation of senescence and gonadectomy, and epigenetic regulation/markers(Salekeen et al., 2021). We have attempted to derive an evidence-based hypothesis on the sexually dimorphic relation of a key regulatory protein families relevant to human longevity.

Hypothesis
In order to develop a model that attempts to explain an integral aspect of the sexually dimorphic nature of the biological aging process, we may define some key considerations to be made as follows:
a) Should there be a sexual divergence of a particular outcome of inheritance, it must be either divergent from the beginning or the trait must be introduced to a population as a common trait until it is sexually and naturally selected to be sex-limited due to simultaneously advantageous and detrimental effects in a sex-specific manner.
b) Should there be an evolutionary event of sexual divergence in the core processes of biological aging, the role of at least one key survival-proliferation switch element must also be sexually divergent.
c) Should there be a divergence event in the aging associated machinery, the likelihood of it occurring in mitochondrial function and energy metabolism should be significantly higher.
d) One key junction of sexual dimorphism and biological aging mechanisms is the endocrine regulation of fitness and longevity. Therefore, should there be an underlying mechanism to this divergence, it should be linked to sexual endocrine dimorphisms and provide significant advantages to one sex while causing deleterious effects to the other.
e) Should there be a dimorphism and decoupling event, it should accommodate the evidence of post-pubescent/post-reproductive onset of benefits/detrimental effects in different sexes.
f) Should there be an element driving sexual dimorphism, it should be epigenetically responsive to stimulants, and be reproducibly tracked.
g) Finally, should there be an evolutionary divergence event in the aging metabolic circuit; it should be conserved through elements present in a wide range of genetically distant organisms.
Based on the evidence from previous investigations and these key considerations, we propose a model where key survival-proliferation protein families which are highly conserved and work as response element to epigenetic stimuli to be sexually dimorphic in their roles. While the presence of these genes/proteins are sex-nonspecifically beneficial to longevity and fitness, there is a clear disparity on how much the sexes depend on these proteins to maintain homeostasis and function. Therefore, the hypothetical model states that Survival-Proliferation switch proteins provide survival benefits in a sex-nonspecific manner but the dependence on these proteins in driving metabolic networks after reaching reproductive maturity is evolutionarily decoupled from male/female longevity.
Proposed Testing Model and Implications
Providing a testing model for a hypothesis like this is complicated – less because of complex features and more because of the sheer lifespans of test subjects or models. Using Yeast, Drosophila and C. elegans are particularly of interest due to their short lifespans. Regulatory protein homolog knockout models or inhibitors can be used and different stress conditions can easily be simulated because of their short lifespans(Taormina et al., 2019). On the other hand, their evolutionary distance from mammals raises issues on the applicability of the findings in more complex biological systems such as humans or rodents. Contrastingly, testing in human subjects require multiple generations of data to fully interpret and establish the evolutionary decoupling and its translational applications therewith. Nevertheless, to simplify the hypothesis testing, we may consider a simple model using murine variants.
One proposed testing model would be to simplify the hypothesis in a two-phase testing process. The first phase would be to disprove that selected regulatory switch proteins (eg: SIRTs, FOXOs, IGF, PARPs etc.) are sex-nonspecific in providing longevity benefits in model organisms under different stress conditions, respective of reproductive stage. A simple approach to this would be to take different aged groups of male and female model organisms with knockout variants and subject them to stress conditions and observe whether the benefits are sex-limited. In this context, not only lifespans of subjects should be considered – but also other biohorological markers of longevity including inflammation and senescence associated biomarkers. If the results are found to benefit both sexes but only cause detrimental effects on one after reproductive maturity, we can infer that the model is fundamentally correct.
The subsequent stage would be to establish which aging associated molecular pathways do the screened proteins/genes perturb in a sex-specific manner. Notably, decoupling from certain regulatory genes also imply the presence of a possible bypass mechanism that assists in sustaining a dimorphic system. Identification of such bypass mechanisms require complex systems-level approaches to identify hidden links or molecular associations among different protein pathways. While designing such models can be complicated, similar studies exploring the evolutionary, sexual and genetic prevalence of such associations and constructing metabolic networks for AMPK, FOXOs and SIRTs as well as aging-associated disease networks have been undertaken before(Mostafavi et al., 2018; Sharma et al., 2012; Soltow et al., 2010; Webb et al., 2016). Secondly, another consideration to be made is that not all tissue systems have the same epigenetic profile with regards to survival and proliferation(Basisty et al., 2020; Chen et al., 2016; Galkin et al., 2020; García-Giménez et al., 2017). Hence, the expression and regulation of this decoupling mechanism may not be pan-tissue. In order to address that possibility, samples from different tissue systems and organs need to be verified before the hypothesis can be proved or disproved. Based on available technology, high throughput analysis of single cell transcriptomics and metabolomics can be applicable in determining such tissue-specific biases. This would be refined according to a trade-off between the parallel development in sequencing, multi-omics, and imaging technologies Nevertheless, these are major factors to be considered when designing studies to test different aspects of this hypothesis. Detailed and integrated multi-disciplinary approaches exceeding cell biological techniques need to be developed in future undertakings to fully explore how and to what extent these sexual divergence may exist in model organisms.
In conclusion, a clear evidence of one statement can be inferred from the available literature, and that is the lack of necessary data in the context of sexual dimorphism in fundamentally understanding the biological aging process. While this area has only been interrogated in recent years, the available evidence already hints at significant prevalence of dimorphic mechanisms involved in human aging. Moreover, the roles of primary aging associated regulatory factors responsible for oxidation, inflammation, immune modulation, tissue homeostasis and proliferation deem it imperative to establish the sexually dimorphic roles and dependencies associated to develop effective, safe and sustainable anti-aging therapeutics and to revisit a majorly underexplored area in the field.

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" Key to solving the problem of aging is engaging more minds towards the problem. This means increasing efforts at educating potential researchers and thought leaders on the nature of the science, the current longevity technology strategies and the many open questions still remaining. This is true for undergraduate education, but it is even more essential to target a younger audience, high school and possibly earlier, so that an initial seed of curiosity for this field is planted and can compete with all the other interesting areas of study in biology and other fields. In our last five years of working with high school age students and engaging this age group in original life science and physical science research, we have found that this is true for a number of other fields untouched by secondary school education, including natural product total synthesis [organic chemistry], green materials science, quantum mechanics, all of which the typical high school student is unacquainted with, and may view as not only unfamiliar but also esoteric. While the Next Generation Science Standards (NGSS) have attempted to distill the fundamental first principles of science that undergird how any discipline operates, standard public school biology curriculum does not include any substantial unit on aging or, in general, central theories of disease or decay. It is astonishing that there are units on the creation of life, the different types of living beings, how it is sustained, the makeup and structure of organisms as they grow and function, and some, but very little, aspects of damage and disease but nothing substantial about why organisms fundamentally degrade and die. Generally speaking, most modern life science units tend to incorporate into teaching natural systems or first principles a “reversal foil” to “natural systems”, i.e. anthropogenic greenhouse gas pollution is a reversal foil to natural nutrient cycles in the environment, and likewise human autoimmune or endocrine disorders are presented as a reversal-foil subtheme in the cell signaling module. Notably, while there is an entire unit on ‘growth and development’, there is no such ‘reversal foil’ that exists for this unit. This vacuum in completing the circle between the closely interrelated disciplines of developmental biology and aging research is therefore an incomplete picture. This may be tied to hesitancy in standard curricula in presenting the world as filled with unsolved problems, which more often than not prefer to pedagogically establish a world of pure “facts” that aren’t further questioned or explored – despite the reality that is the very purpose of all research. This presentation of the world as solved is a greater fallacy that spans the teaching of many disciplines, but outside of solving this fundamental issue of education, we ask what can be done specifically about bringing the known “facts” of aging biology into high schools.
There is still much to understand about the process of aging, yet there have been major strides towards revealing the core aspects of how the aging process is measured, motivated and manifested which should still be discussed. Interventions of course remain somewhat controversial because of the general public’s stance on trying to alter the aging process, but ultimately not much time need be explicitly spent on discussing solutions. This is because simply characterizing the nature and manifestation of aging as just another tangible, biological process, not just a philosophical dogma, will naturally lead kids to question about ways to manipulate, alter or reverse aging. This may especially hit home because, sadly, they may be at a point in their lives where they are starting to see the ravages of age in their grandparents and possibly even their parents. In our experience educating kids for the last five years, we find that the instruction needn’t be heavy handed, just simply present what is known and what are the open problems and let the kids start questioning things themselves. Furthermore, the unit needn’t be particularly long, it can stretch from a couple weeks to maybe a month. All that is required is a teaser and then the natural demand from this exciting field will create a need for more extensive courses in colleges. As our organization works by giving high schoolers exposure to the latest fields of biological and biochemical investigation, we can appreciate how exciting the field of aging is among the many biotechnology initiatives around it, right here in the bay – some of whom we work with. When the concepts are explained the kids naturally gravitate towards it as a pressing and open problem and so we would like to propose a challenge to create a standardize and easily adoptable aging unit that can be incorporated into the high school curriculum in a manner that fits into our organization’s vision in training students in original hands-on research while also supplementing the core pillars of growth and developmental biology as presented in the NGSS framework.
In terms of the approach of introduction, though a chapter in the curriculum textbook might at first glance seem to be the most straightforward and canonical approach, our experience with the local schools shows this may be difficult given the amount of recent and historic politics over what goes into kid’s textbooks. Instead, we’ve found that a more digestible and tangible strategy would be to create a contained experiment module, that is cheap, consistent and effective at conveying the core modus operandi of aging research as a unifier of the chronic failure modes in the body. In addition, the module should fit well into the standard curriculum and one area we have seen that has been particularly emphasized in the NGSS curriculum as of late is cell signaling. This might present a good opportunity to get a foot in the door so to speak by branding one aspect of aging as a failure of systemic cell signaling. An example contained and reproducible experiment module we could propose would be one on learning about and deriving senescent cells. These can be simply achieved after growing cells for a long time or having an inducible cell line which the students can maintain and chart the population doubling to find when they no longer cycle. Additionally they could also then apply the very standardized pH dependent beta-galactosidase stain to confirm the cells are senescent or qPCR can be used to measure telomere length, if resources permit. Furthermore, a rudimentary cytokine measurement can be done to understand the senescence associated secretory phenotype or SASP which is the critical “aging signaling” component. Though these are often done in full multiplexed panels to show the global change in signaling, a couple cytokines can be chosen and evaluated with simple Elisa based tests that are cheap and usable like a standard COVID test. This would demonstrate how a fundamental process like cell division – widespread in a number of cell types - can lead to change in cell state and cell signaling with global consequences. This can then be used as a springboard for talking about other types of signaling in aging like the results of the parabiosis experiments or ways to intervene for senescent cells like senolytics. And then a further brief dive into just the literature on some of the other areas like the hallmarks, caloric restriction and epigenetic reprogramming can complete the unit. This achieves the concrete and reproducible example point, fits into the cell signaling curriculum and teases the larger field and purpose of the science, with the ultimate goal being an early introduction to the questions posed by aging research, its integration into other core disciplines in the life sciences, and its potential therapeutic and scientific impact on the lives and livelihoods of people in society. We therefore propose inquiry-driven, project-based research as an entrypoint of young people into the arena of aging biology, first conceptualized as completing the circle to an unfinished story of developmental biology, and further actualized through hands-on research experiences for high school age kids.

"
"Age-associated Neurodegeneration Induced by Mitochondrial Dysfunction, ROS Accumulation, Oxidative Stress, and Telomere Erosion

One of the most crucial and yet most obscure areas of longevity science is the study of age-associated neurodegeneration which contributes to debilitating conditions (such as Alzheimer's disease (AD), Parkinson's Disease (PD), Parkinson's Disease Dementia (PDD), Cerebrovascular Disease (CVD), Lewy Body Disease (LBD), Mild Cognitive Impairment (MCI), Progressive Supranuclear Palsy (PSP), and Amyotrophic Lateral Sclerosis (ALS)) that prevent one from aging with dignity. Achieving longevity without achieving an optimum quality of life is contrary to the aim.
Considering that most age-associated pathologies have been linked to Mitochondrial dysfunction, ROS accumulation, oxidative stress, and telomere erosion, it will be helpful to further analyze the factors that can induce the abnormal activity of these mechanisms in order to better understand the potential counteracting mechanisms that can help with inhibition of such dysfunctions.
Thus far, the unspecified/unidentified underlying factors contributing to the onset of these conditions have been making it far more challenging to investigate them in great detail. Moreover, the fact that the booming market of anti-aging products is mainly concentrated on the aesthetic aspects of aging, also contributes to overlooking the more detrimental conditions. However, what is the point of aging gracefully in appearance, if these neurodegenerative conditions are preventing mankind from aging with dignity?
Various limitations associated with the study of these neurodegenerative conditions prevent scientists from thoroughly investigating them in order to identify the precise underlying factors and to develop feasible and effective treatments. Therefore, it is important that the prospective research on these conditions begin by addressing the limitations that have prevented scientists from making advancements in these studies thus far."
"Most anti-aging strategies in geoscience currently focus on druggable targets and hallmarks of aging to address tissue damage. Since there is quite a diverse repertoire of macromolecular forms of damage, including some that are irreversible like DNA mutations, drugs and therapies aimed at targeting endogenous cellular repair and maintenance machinery are probably not enough to slow aging to the point of significantly extending maximal lifespan. An alternative approach to correcting existing forms of damage is to try and replace existing aged tissue with new tissue. One of the challenges to this approach is replacing brain tissue, since it is responsible for mediating self-identity and therefore not possible replace all at once without the loss of existing sense of self. However, it is possible that by gradually replacing discrete regions of the brain at a time, namely within the neocortex (which makes up the bulk of the brain and is the center of higher order processing functions), we can still manage to replace the aged brain tissue without significantly altering the activity related individual self-identity at any given point in time.

The Hébert lab is working to achieve this aim of neocortical replacement and has already demonstrated, along with other groups, that integrating new neocortical cells into existing brains is possible in mice. One of the major goals of the lab is to now take this further and construct neocortex from pluripotent stem cells at the tissue level with the correct architecture, all the right cell types, and supporting ECM environment. This should make for neocortical transplants more functional with greater replacement potential beyond simply dissociated neural precursor cells.

One of my projects is centered around characterizing the iPSC-derived neocortical cells we are generating through our directed differentiation protocols using scRNAseq. Part of this involves finding out which timepoint(s) in fetal development (as shown by comparison to fetal cortical scRNAseq datasets from different ages) they most resemble at different points in the differentiation protocol. This information is relevant to understanding both what fetal age to look at as a guide to reverse engineer the neocortical tissue transplants and to see how long it takes to differentiate our cells to particular fetal ages we want to test the transplant efficacy at. Beyond this, I am going to characterize the general transcriptional fidelity of our iPSC-derived cells to fetal cortical tissue cells as well as compare the pattern of trajectory using RNA velocity-based approaches.

My other project seeks to use CRISPR-Cas9 Gene editing to create hiPSC cell lines that express, specifically in neurons, inhibitory DREADD receptors that allow for selective silencing of the targeted cells through the administration of DREADD receptor ligand. This will be important to test the integrative capacity of the neurons within our reconstructed neocortical transplants. This is because if the transplant neurons encode useful behaviors then silencing them should result in the transient loss of those behaviors in our animal models.
"
"Each target was reduced individually using siRNA-mediated knockdown in the presence or absence of DOT1Li. Depletion of Fosl1 resulted in cell death with three different siRNAs (data not shown) and was not analyzed further. For each of the other five targets, siRNA-mediated depletion was robust and resulted in a similar fold change compared with that obtained by DOT1Li (Figure S5A). When siRNA for each target was combined with DOT1Li, mRNA levels were further reduced (Figure S5A). Hic1, Hoxd12, and Twist2 depletion did not enhance reprogramming in the presence or absence of DOT1Li (Figures S5B and S5C). Meox2 depletion alone or combined with DOT1Li slightly decreased NANOG+ colony formation (Figure S5B) but did not affect bona fide colonies that remained after doxycycline withdrawal (Figure S5C). Depletion of Nfix alone increased the occurrence of NANOG+ colonies 2-fold compared with a 3-fold increase with DOT1Li (Figure 5E, left). However, Nfix depletion did not lead to a robust formation of bona fide colonies, as DOT1Li treatment increased transgene-independent colonies 11-fold, whereas only a 4.8-fold increase was observed with siNfix (Figure 5E, right). When combined with DOT1Li, Nfix depletion enhanced transgene-independent colony formation (Figure 5E, right). Thus, Nfix depletion acts in conjunction with DOT1Li to promote reprogramming.

To interrogate whether these genes are barriers of the process, we overexpressed MEOX2 and NFIX individually in MEFs prior to the induction of reprogramming (Figure S5D). Exogenous MEOX2 expression inhibited reprogramming on its own but did not affect DOT1Li-mediated reprogramming (Figure S5E, left). Surprisingly, MEOX2 increased stable colony formation in the absence of DOT1Li (Figure S5E, right). These contrary effects of Meox2 depletion and overexpression on reprogramming may be related to its heterogeneous expression in the reprogramming population (Figure 5C) and the increase in proliferation in MEFs overexpressing MEOX2 (data not shown). Ectopic expression of NFIX during reprogramming reduced DOT1Li-mediated NANOG acquisition below control levels and prevented stable colony formation (Figure 5F), suggesting it is a potent reprogramming barrier independent of DOT1L activity. Thus, the downregulation of Nfix acts in an additive manner to increase pluripotency by contributing to the DOT1Li phenotype.

By comparing the results of the depletion experiments (Figures 5E, S5B, and S5C) with the pattern of expression from single-cell analysis (Figure 5C), we find that DOT1L acts in a collaborative manner with Nfix that is still expressed at later stages of reprogramming. Hic1, Fosl1, and Twist2 are downregulated in most cells by day 3 of reprogramming, and Meox2 is upregulated in only a portion of reprogramming cells (Figures 5C and 5D). Taken together with the temporal effectiveness of DOT1Li at the mid stages of reprogramming (Figure 1D), DOT1Li sets the stage for enhancing reprogramming efficiency along with the downregulation of persistently expressed genes such as Nfix.

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Discussion
DOT1L is crucial for mammalian development, yet its role in cell-fate determination is still unknown. Here, we find that DOT1L is a barrier to pluripotency acquisition of MEFs throughout reprogramming but acts most strongly during mid-reprogramming (Figure 1). Stages of reprogramming when starting from MEFs include an early inactivation of the somatic program, an important component of which is the mesenchymal genes (Li et al., 2010; Samavarchi-Tehrani et al., 2010). Inhibition of DOT1L was reported to enhance human fibroblast reprogramming by facilitating MET (Onder et al., 2012). The reverse process, epithelial to mesenchymal transition (EMT), is prevented in other systems, such as renal (Liu et al., 2019) and breast cancer cell lines (Cho et al., 2015), by DOT1Li. We demonstrate that DOT1L control of cell identity extends far beyond epithelial transitions. Although inhibition of DOT1L increases Cdh1 expression in our mouse reprogramming studies, it also enhances reprogramming of keratinocytes that do not have to undergo MET and MEF reprogramming post-MET (Figure 4). This corroborates our recent discovery that the epithelial and mesenchymal programs are independently regulated in the presence of DOT1Li during reprogramming using scRNA-seq (Tran et al., 2019).

In the course of assessing DOT1L transcriptional regulation, we identified contributions of the reprogramming barrier Nfix that maintain cellular identity with DOT1L (Figure 5). NFIX is a transcription factor important for neural (Pekarik and Belmonte, 2008) and muscle development (Pistocchi et al., 2013). In addition, NFIX is required to maintain murine hair follicle stem cell enhancer function (Adam et al., 2020), and its depletion increases the number of pluripotent colonies (Yang et al., 2011). From our previous analysis of reprogramming with scRNA-seq, we ordered single cells in a trajectory (Tran et al., 2019). We found a major branchpoint where cells stalled and did not complete the transition to iPSCs (Tran et al., 2019). Nfix was found to be a branchpoint gene such that downregulation was required to continue in the reprogramming trajectory, further suggesting that it may have a specific role in mid-reprogramming. Of the identified DOT1L targets, Nfix continues to be expressed in cells later in reprogramming and gains H3K79me2 after OSKM induction (Figures 5B and 5C), suggesting DOT1Li may facilitate its downregulation by preventing the gain in H3K79me2 enrichment at later reprogramming time points. This result is in contrast to previous findings that DOT1Li enhances pluripotency acquisition by downregulation of the established transcriptional program (Onder et al., 2012). It is important to note that depletion of Nfix does not reach the reprogramming efficiency of DOT1Li, and the effect is additive with DOT1Li (Figure 5E). Thus, downregulation of reprogramming-associated factors may contribute but is not solely causal of the DOT1L reprogramming phenotype.

We find that modulation of DOT1L-DE genes does not substitute for DOT1L catalytic inhibition (Figures 4 and and5),5), suggesting that H3K79me may have a role in reprogramming beyond transcriptional regulation of single genes. During development, only four genes were DE in Dot1l KO mouse c-kit+ cells sorted from embryonic day 10.5 (E10.5) yolk sacs, where profound phenotypic alterations in vascular morphology and erythrocyte maturation were observed (Feng et al., 2010). These small transcriptional changes in key factors like Nanog during reprogramming or Gata2 during erythrocyte maturation suggest that loss of H3K79me may alter global epigenetic profiles rather than local expression profiles. For example, loss of H3K79me2 allowed for spread of H3K27me3 on downregulated genes in leukemia cells (Deshpande et al., 2014). H3K79me2/3 are enriched at certain intronic enhancers in leukemia cell lines and regulate future deposition of H3K27ac (Godfrey et al., 2019), a modification associated with enhancer activity. In neural differentiation, DOT1Li decreased accessibility of a subset of enhancers (Ferrari et al., 2020). In support of further epigenetic alterations, both upregulated and downregulated genes can be decorated by H3K79me2 before DOT1L depletion in development of the cerebral cortex (Franz et al., 2019). Thus, H3K79me may affect histone modification, depending on chromatin context.

We observed that many more genes are upregulated rather than downregulated in their steady-state expression by DOT1Li during reprogramming (Figures 1 and and4).4). Many of the upregulated genes are expressed in other lineages and are not modified by H3K79me2 in MEFs or ESCs (Figures 3 and S3D). Alternatively, these genes may be regulated indirectly by the binding of DOT1Li-DE transcription factors, such as HOXD12 or HIC1. The aberrant expression of lineage-specific genes may promote reprogramming, a notion that can be further investigated by single-cell transcriptional analysis. Regardless, the boost to reprogramming cells by DOT1Li seems to outweigh the burden of spurious lineage gene expression. Taken together, our results demonstrate that DOT1L activity functions beyond steady-state alterations to the somatic transcriptome; rather it collaborates with reprogramming-associated factors to safeguard cell identity.

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Experimental procedures
Cell isolation and culture
Male and female MEFs were isolated on E13.5 from embryos that were homozygous for the Oct4-2A-Klf4-2A-IRES-Sox2-2A-c-Myc (OKSM) transgene at the Col1a1 locus and either heterozygous or homozygous for the reverse tetracycline transactivator (rtTA) allele at the Rosa26 locus, as previously described (Tran et al., 2019). MEFs were grown in DMEM, 10% fetal bovine serum (FBS), 1× non-essential amino acids, 1× GlutaMAX, 1× penicillin/streptomycin, and 2-mercaptoethanol (4 μL/500 mL). Feeder MEFs were maintained and isolated as above from DR4 mice genetically resistant to geneticin (G418), puromycin, hygromycin, and 6-thioguanine. Feeder cells were irradiated with 9,000 rad after three passages. ESCs V6.5 were grown on feeder MEFs in KO DMEM, 15% FBS, 1× non-essential amino acids, 1× GlutaMAX, 1× penicillin/streptomycin, 2-mercaptoethanol (4 μL/525 mL), and leukemia inhibitory factor. Keratinocytes were isolated from reprogrammable mice 4 days postnatal as previously described (Li et al., 2017) and cultured in EpiLife medium with 60 μM calcium (Thermo Fisher Scientific MEPI500CA) with the EpiLife defined growth supplement (Thermo Fisher Scientific S0125). 293T cells were acquired from ATCC and grown in DMEM and 10% FBS. Mice were maintained according to the UW-Madison institutional animal care and use committee (IACUC)-approved protocol.

A detailed description of all materials and methods is provided in the supplemental information.

Data and code availability
The accession number for all RNA-seq datasets reported in this paper is National Center for Biotechnology Information Gene Expression Omnibus (GEO): GSE160580.

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Author contributions
C.K.W. performed experiments and bioinformatic analysis, C.K.W. and R.S. wrote the manuscript and acquired funding, and R.S. conceived and directed the project.

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Conflict of interests
The authors declare no competing interests.

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Acknowledgments
This work was supported by a UW-Madison Stem Cell and Regenerative Medicine Center postdoctoral award to C.K.W., a UW-Madison Fall competition, and a Shaw Scientist award to the R.S. lab. We thank Dr. Roice Wille for R script design, Stefan Pietrzak and Brenton Halvorson for scRNA-seq analysis, Dr. Jason Tchieu for the NFIX vector, and Dr. Konstantinos Chronis and the R.S. lab members for critical reading of the manuscript."
"As the interest in longevity research increased in recent years, so did the numbers of publications on the subject that are available online. While results and conclusions from earlier publications are often referenced and discussed in later articles, the datasets underlying these results are usually analyzed only once, for the purposes of the original publication.
Thus, one of the most underappreciated areas of longevity research is not any single particular publication, but the opportunity of recycling available “single-use” datasets of previous publications. Generating aggregated datasets that are used to repurpose, reanalyze, and reinterpret data in a broader context can provide deeper and more robust insights than would be possible by analyzing a single dataset.

Current data availability policies of scientific journals require uploading and sharing of all relevant datasets in online repositories such as Gene Expression Omnibus (GEO). This includes datasets such as DNA methylation array data and RNA-seq gene expression data, which are typically generated using widely available methods and instruments (e.g. Illumina platforms) and analyzed using standardized, i.e. comparable, analysis workflows. This leads to the accumulation of extremely large datasets. However, although the datasets are available online for everyone, they are not easily accessible and usable.Many datasets, however, are standardized enough to be compared in principle, but not standardized enough to be easily analyzed in a meaningful way by the no-code research community.
Machine learning algorithms are especially suited to find patterns in datasets that 1) are large, 2) have many parameters, and for which 3) an interplay of a multitude of parameters shapes the outcome. As aging is characterized by the functional deterioration of highly complex intra- and intercellular networks, it is in particular suitable for machine learning applications. To enable meaningful aggregated data analysis driven by state-of-the-art machine learning applications, certain quality criteria must be defined and met for individual datasets that are to be included. In particular, a standardized format and annotation is required because quality control, preprocessing, and formatting of data is usually the most time- and resource-intensive step in bioinformatics analyses. Facilitating the analysis of aggregated data by establishing an accessible online tool for the no-code research community should provide the basis for a closer connection between bioinformatics and bench scientists. This online tool would make these aggregated datasets accessible so that they can either be analyzed online within the provided tool or easily fed into customized machine learning pipelines from the research community.

As aggregate data analysis provides more statistical and explanatory power to evaluate large integrated datasets, this will facilitate the identification of novel cellular and molecular candidate factors to generate new hypotheses about how these candidates influence and shape aging. To confirm promising drug targets and signaling pathways, these identified candidates should then be further characterized in vitro and in vivo.
An application example of this approach would be to aggregate datasets generated from numerous datasets that include data from healthy volunteers who are typically part of many studies as a control group, but for whom additional information such as age or another clinical age-related biomarker or outcome is available.
In addition, aggregated data could be used to generate regularly updated reference datasets that can be made available to the research community to improve contextualization of new findings by promoting comparability of results (e.g., through cross-validation of models).
In principle, the aggregate data analysis approach can be applied to data from all -omics fields, including RNAseq, proteomics, epigenetic clock research. Importantly, this approach is not limited to datasets originally generated for aging research, but can also incorporate datasets from other fields that can also be applied to aging because they contain relevant data such as age or a specific treatment method.
Importantly, input and suggestions from the broader longevity research community are critical to shaping this approach of aggregate data analysis to reach its full potential.

In summary, the rediscovery and reuse of previously published data currently requires considerable bioinformatics expertise and is not being pursued in a coordinated and standardized approach in the field of longevity. Therefore, data must be made available to laboratory scientists in a meaningful way.
Aggregate data analysis of previously published datasets combined with subsequent confirmation of the testable hypotheses derived from them in vitro, in vivo, and ultimately in clinical trials will help identify and validate biomarkers and mechanisms of aging and longevity.
"
"Accurate conversion of genetic information into protein tertiary structure is one of the pillars of proper cellular function, and disruption of protein homeostasis (proteostasis) that leads to intracellular accumulation of misfolded proteins is a hallmark of aging and a contributor to age-related pathologies.

For the full project details, including preliminary data and further plans, please visit -- https://docs.google.com/document/d/1QLzipbn6umWGg2MfyyxEROxdzsWMWvXoiG_WBVFHzPQ/edit?usp=sharing"
" Humanity's most important biological discoveries, of course, have had to do with the origins of life itself. Furthermore, if we are aiming to reverse aging and prevent death, it would make the most sense to solve this problem, with it anti-thesis, the origin of life. Via the discoveries of the egg cell, the sperm cell, and the ensuing zygote which forms during fertilization, and the further ensuing process of embryogenesis, capable of faithfully creating healthy cells of every type and form, from what were once ""biologically aged"" sex cells in adult human beings, humanity has already discovered the fundamental process for reversing aging and preserving immortality, which naturally has allowed for the healthy and immortal continuation of cellular lineages and the human species. Therefore, my hypothesis for the cure to aging and death is simply that we must recapture the potential of the processes at the origin of life, as it occurs between generations via fertilization. More specifically, my hypothesis is that the fundamental minimum combination of rejuvenation processes that the scientific community has discovered to occur during fertilization, is a combination of 1) Epigenetic reprogramming; 2) DNA repair; and 3) Autophagy/Mitophagy; And, that furthermore, therefore, an in-vitro rejuvenating processing of adult stem cells, screened for high initial genetic quality, should at least enact the simultaneous combination of all 3 fundamental processes of reproductive rejuvenation (""3FPRR""), which may thus successfully and fully rejuvenate a small selection of an adult's stem cells, which can then become the starting material for manufacturing large quantities of autologous, non-immunogenic, non-cancerous, cellular replacement therapies. It should be further noted that the 3FPRR are synergistic to the extent that DNA Repair and Epigenetic Reprogramming enhance each other, as do Autophagy/Mitophagy and Epigenetic Reprogramming, and therefore the potential of any single 3FPRR processes by itself is even further diminished compared to my proposal for the minimum 3FPRR.
In order for this vision to practically work to truly reverse aging and avoid biological death, these rejuvenated & reprogrammed stem cells (""RRSCs"") would need to be delivered in such a way and as part of a system capable of in-vivo cellular replacement. I propose that this initially could be done via a 2 step process of 1) initially tagging a random and very small subset (ie. .05%) of all cells, evenly distributed throughout the body, with dual aptamer homing ligands, for attracting both engineered macrophages, and RRSCs; 2) administering the RRSCs to home to the distributed tagged cells, whereby they could engraft, and if necessary, spontaneously differentiate, and become functional resident tissue, enacting cell replacement with single cell fidelity; and 3) administering engineered CAR macrophages to target and phagocytose the tagged old cells; thus, completing the cycle of fully removing aging and having a functional cell by cell level replacement process.
Furthermore, this vision requires the RRSCs to be able to be manufactured in an unlimited supply, which can be achieved by a combination of telomerase enforced immortalization, and periodic RRSC re-induction. A portion of RRSCs can be kept after early passaging, via cryopreservation, to mimic oocyte quiescence, and provide a more ideal source of future RRSC re-induction. Ideal culture conditions for the virtually infinite manufacturing of iPSCs has already been databased, and will work for this vision, and therefore insurmountable infinite manufacturing limitations are in no way a serious threat.
This vision furthermore requires the RRSCs to be primed for in-vivo applications, with non-cancerous regeneration of all lineages. This should ideally be done via the Division of Regenerative Labor (""DRL""), which is a novel concept I have coined, meaning the use of both a LT-HSC lineage derived from RRSCs, and the use of a multipotent MSC (""MMSC"") lineage derived from RRSCs. The LT-HSC lineage is easy to understand, as we have already proven over the last 30 years that cellular replacement, and therefore the immortalization and reversal of aging, for the hematopoietic lineage at least, is a viable model, as widely proven by successful bone marrow transplantations. The MMSC lineage approach is less understood but viable nonetheless, and should obviously be the approach taken given the proven success of LT-HSC cell replacement therapy which can be mimicked, to cover the remaining gap toward the end goal of Total Body Cell Replacement (""TBCR""). These MMSCs would have to have the ability to migrate, home, engraft, survive, spontaneously differentiate into all non HSC lineage cells throughout the body, and not form cancers. Thankfully, we can mimic another natural solution, which is an under-appreciated discovery, namely the discovery of SSEA3+ MSC subset cells, which have already proven in dozens of studies to have all of these exact properties. These SSEA3+ MSC cells, which have been dubbed by their founding research team and in the literature as ""MUSE"" cells, have been shown to be able to migrate effectively to areas of cell death, damage and inflammation, without getting trapped in the lungs like traditionally studied MSCs, and they can spontaneously differentiate via clues from the local niche, into functional tissue of every non-HSC lineage based organ and tissue type so far studied (brain, spinal cord, skin, lung, liver, kidney, pancreas, heart, endothelium, etc.), and they can persist for years, again, unlike traditionally studied MSCs. Therefore, we can differentiate RRSCs into MMSCs, which at the very least copy these traits of endogenously existing, regenerative SSEA3+ MSCs, but ideally which are over-engineered and evolved upon this basis, via specific culturing and priming as discussed, to optimize cell replacement goals, and therein be able to cover, alongside with the LT-HSCs, every type of cell needed to accomplish TBCR, and the only true, ethical, understandable, and currently viable form of fundamentally and totally reversing aging, and curing biological death. The endogenous SSEA3+ MSCs also provide us initial pathways to mimic to ensue that these undifferentiated stem cells do not cause tumors, unlike the direct use of iPSCs, while still maintaining a virtually pluripotent potential. The cancer preventing pathways discovered thus far may have to do mainly with the fact that these SSEA3+ MSCs have low levels of telomerase, low levels of OCT4 and Nanog, a low ratio Lin28 to Let7, and a high level of the tumor suppressor CDKN2A. Therefore in addition to priming the RRSC derived MMSCs for cell replacement, they should be primed to be non-cancerous, although it should be assumed, and not completely avoided, that a small cancer risk will inevitably be a part of regenerative therapies. By further understanding these stem cells, we will nevertheless continue to get a better grasp on targetting cancer stem cells, and curing cancer, and cancer is not a fundamental limit to reversing aging, especially if we already are working on a viable cell replacement therapy that could work to replace cancer cells as well as normally aged cells, again very similarly to copying the successful model for curing cancerous HSCs of the bone marrow, which are simply eliminated and replaced with healthy HSCs.
In order to enact the reprogramming of cells, I propose humanity should likely universalize the use of a universally accessible, viable, simple, cheap, and highly effective adult stem cell, which can be isolated from a voided human urine sample. Dozens of research groups have already shown the high efficiency of Urine Derived Stem Cells (""UDSCs"") to be reprogrammed into iPSCs. UDSCs express high levels of embryonic surface markers SSEA4 & PDX1, pluripotent marker OCT4, and even significant telomerase levels, unlike other adult stem cells. They express MSC markers such as CD44 and CD90, but are epithelial in identity, and are therefore logically, but are also already experimentally proven to, have higher reprogramming efficiencies, as it is known that the Mesenchymal to Epithelial (""MET"") transition is a bottleneck and barrier to iPSC induction from the low efficiency and more commonly used fibroblasts or MSCs. Furthermore the MET transition during traditional iPSC induction is a major source of genetic instability and raises cancerous risks of such a stem cell lineage. Therefore, collecting a simple urine sample from any human being on earth, and isolating these cells with an already high degree of stemness, as the starting source for RRSCs is logical and viable. This isolation could be further enhanced by using various means of further filtration to select a smaller UDSCs sub-population which have the highest degree of genetic quality, and the least damage. As we know from the immortal germ line and embryogenesis, even a single cell could be enough if handled correctly.
In order to enact the 3FPRR it is important to show sufficient knowledge and capacity to faithfully recreate the end result of a cell lineage capable of healthy immortality. I have done the research to create the initial concepts, and provide experimental starting grounds, to achieve this assurance.

1) Epigenetic reprogramming; While lauded as possibly the greatest discovery in cell biology, the nobel prize winning traditional process of iPSC induction using the 4 transcription factor cocktail ""OSKM"" is not ideal. I propose a more universal, simpler, cheaper, faster, more efficient(higher success rate), and safer method, which would pair well with UDSCs, towards achieving RRSCs for TBCR. In recent years, small molecule and chemical replacements to these genetic transcription factors have been disclosed. I have compiled them into the most exhaustive list to date, and have significant detailed backup research on their more precise reprogramming roles and potentially ideal media concentrations. With further high throughput combinatorial testing optimization for optimal synergy, concentrations and temporal requirements, I believe the compounds on this list to already suffice for the job needed. The list is currently; 0) Low Oxygen, 1) NaB > 1.5) VPA, 2) CHIR99021 > 2.5) 6-bromoindirubin-3-oxime, 3) 616452(TGF-B Inhibitor), 4) Tranylcypromine, 5) Forskolin, 6) DZNep, 7) TTNPB(RAR agonist), 8) Repsox > 8.5) Dasatinib > 8.5') PP1 >8.5'') iPYrazine, 9) cyclic pifithrin-α (a P53 inhibitor), 10) A-83–01, 11) thiazovivin, 12) Trametinib, 13) PS48 14) DHEA 15) Lactoferrin, 16) Spermine, 17) H2S, 18) Purmorphamine > 18.5) SAG, 19) Oxysterol, 20) 2-methyl-5-hydroxytryptamine, 21) D4476, 22) Melatonin , 23) Vitamin C + 24) Alpha KetoGlutarate, 25) Beta-Hydroxybutyrate, 26) SB43 +> 26.5) PD03 (SYNERGY), 27) JQ1 > 27.5) iBET-151, 28) Y-27632 > 28.5) Thiazovivin, 29) FGF2 / 30) Collagenase, 31) PD173074, 32) Kenpaullone, 33) SU5402(3i>2i), 34) OAC1, 35) SC1 ""Pluripotin"", 36) Bayk8644, 37) Parnate, 38) Suberoylanilide hydroxamic acid, 39) AMI-5, 40) 2, 4-Dinitrophenol, 41) Fructose 2, 6-bisphosphate, 42) N-oxalylglycine, 43) BIX-01294 44) AM580 > 44.5) CD437(RAR agonists), 45) 100mm NaCl(hyperosmolarity), 46) Rapamycin(autophagy), 47) SMER28(autophagy)

2) DNA Repair; Although the great discovery of DNA, the code and source of generating biological life, has been lauded as one of, if not the most important discovery in biology, and what furthermore literally defines us, not enough work has been done to focus on repairing DNA to reverse aging. It is obvious to any cell biologist, and should be easily explained to any human being, that DNA damage is the most fundamental damage to a cell, and therefore is a fundamental driver of aging. Although cells have DNA repair mechanisms, not all damage is repaired. In oocytes, which play the most important role in anti-aging processes though-out the human species, there is very clearly the most outsized upregulation of multiple mechanisms to ensure genetic quality, check for DNA damage, and enact DNA repair, which we would be wise to learn from, mimic and build upon when trying to solve the problem of aging. I have compiled many of the most important DNA repairing enzymes, molecules and pathways into an exhaustive list, and have significant detailed backup research on their more precise repair roles, interactions and modes of action. With further high throughput combinatorial testing optimization for optimal synergy, concentrations and temporal requiements, I believe this list to already suffice to prove a completely higher level of quality and possibly even the potential for infinite healthy cell replacement. The list is currently; 1) MGMT: (Direct Reversal of DNA Alkylation Damage), 2) AlkB: (Direct Reversal of DNA Alkylation Damage), 2.5) ALKC & ALKD, 3) CHK1 & CHK2, 4) NUDT1, 5) GADD45, 6) SSB1(SINGLE STRAND DNA DAMAGE REPAIR + Facilitates TERT Recruitment to Telomeres and Maintains Telomere G-Overhangs), 7) ATRX, 8) RAD51 (Synergy with iPSC induction), 9) XRCC1 (single strand breaks), 10) SMUG1 (Base excision repair enzyme that removes uracil and oxidized pyrimidines), 11) OGG1, 12) NTHL1, 13) DJ1, 14) Glutathione, 15) SIRT6-PARP-NAMPT, 16) PGP, 17) BRD4, 18) PRDM14(DNA demethylation + TET mediated BER) , 19) TDP1 (single strand breaks, and formaldehyde induced DNA cross links), 20) BRCA1, 21) 53BP1, 22) XRCC4 (NHEJ in double strand breaks).
Perhaps one of the most important discoveries related to the 3FPRR is that Histone Deactylase Inhibitors enhance DNA damage repair during epigenetic reprogramming, and may be attributed to histone acetylation at specific sites that enhance DNA repair. It is further known that the chromatin remodeling during epigenetic reprogramming also promotes DNA damage repair, and the general knowledge that cells with increased stemness such as in iPSCs and Zygotes have more open and relaxed chromatin, which is more accessible, not only to epigenetic modification, but surely at the same time, to DNA repair recruitment. This provides us with an ""opening"" in every sense of the word, a gift of an ideal opportunity to both enact epigenetic reprogramming and enhance DNA repair at the same time, which are both required for true rejuvenation. This process would make sense biologically as well, as this is the time in life when the window for true rejuvenation occurs. Enhancing DNA repair during this step is further synergistic because it helps to avoid cancer risks as well, which is necessary for the downstream use of RRSCs, and epigenetic reprogramming is known to cause genetic instability and DNA damage, which therefore logically requires us to enhance DNA repair with the addition of exogenous repair enzymes, molecules and repair pathway activators. Moreover, studies across long-lived species across the biological kingdoms show enhanced DNA repair to both underly low cancer rates, and reduced aging, as a conserved mechanism, and therefore at the very least, enhancing humanity's capacity to enact successful DNA repair, both in-vitro and possibly in-vivo, through the manufacturing and practical applicative expertise with DNA repair mediators, such as those listed above, will undoubtedly be a 100% surefire way to help us towards longer maximal lifespans.

3) Authophagy/Mitophagy; There has been much fanfare regarding fasting and caloric restriction in anti-aging research, and much of this research has delegated a large portion of the benefits to autophagy. Furthermore, there are other large groups of prominent and outspoken anti-aging thought leaders and researchers who are interested in mitochondrial health and related effects of oxidative stress. It is vital to note that mitochondria have their own DNA as well, and therefore mitochondrial DNA quality enhancement must be a part of the DNA repair process in the 3FPRR.
Mitochondria do share much of the same DNA repair pathways and machinery as the nucleus, which could be applied to them as well in the 3FPRR for adult stem cells, but there is another, and perhaps more fundamental mechanisms for enhancing mitochondrial DNA quality, which is through the process of mitophagy. Mitophagy is the process by which damaged mitochondria are removed and/or recycled from a cell, so as to dilute poor genetic quality copies, and allow the higher quality mitochondria to reproduce. (Mitophagy Flux goes hand in hand with mitochondrial Fusion and Fission Flux, which also allow for a dilutive effect and selective enhancement of Mitochondrial DNA quality.) Research has shown that Oocytes in specific, as well as Zygotes and Embryonic stem cells, have significantly enhanced pathways and mechanisms for Mitophagy, to ensure healthy and immortal mitochondrial lineages are also passed down generation to generation. ""Pink1 "" is an example of a mitophagy enhancer that also synergistically promotes epigenetic reprogramming, as are UCP2 and ""GAS6"". FZO1 effects are mediated by the mitophagy receptor Atg32, and another important mitophagy receptor is OPTN, which also enhances an embryonic identity. PHB2 is an inner mitochondrial membrane mitophagy receptor that also regulates embryonic identity. Nix is another selective autophagy freceptor for mitochondrial clearance. BNIP3 is another important pathway that mediates mitophagy. Moreover, studies have shown that fundamentally mitochondrial clearance is essential during the process of iPSC reprogramming, and more specifically ATG3 dependent autophagy has been shown to mediate mitochondrial quality control during the acquisition of induced Pluripotency.
As a single applicable intervention example, Melatonin is a molecule that has been shown to enhance epigenetic reprogramming, DNA repair, authophagy and mitophagy, and therefore represents the ideal of 3FPRR anti-aging. Unsurprisingly melatonin is perhaps one of the oldest and most fundamental molecules in biology and is severely decreased and dysregulated in the elderly, while its in-vivo therapeutic use has been shown to be beneficial in thousands of studies across nearly every disease model, including extension of lifespan.
During the 3FPRR enforcing and enhancing mitophagy could be done synergistically by at the least enhancing general cellular autophagy, which will provide other synergistic benefits and would be advisable. Autophagy is a logical and actual target for immortal achievements in RRSC not only because of its role in mitophagy and mitochondrial quality, but because it is able to clear an old and damaged proteome, which also paves the way for a new proteome to be developed as part of the epigenetic reprogramming and assumption of a new identity. Autophagy is a fundamental mechanism for protection against the otherwise would be damaging levels hypoxia as well, which is generally favored for conditions of epigenetic reprogramming and pluripotent cell culturing, and enhanced autophagy has been shown to maintain stemness and repress the development of senescence, or even reverse it, in otherwise would be senescent stem cells. Enhanced general autophagic flux, via the FOXO1 pathway related Ulk1, Beclin1, Atg5, Atg3, and LC3 has been shown to maintain Embryonic identity as well. Supporting this, knockdown of LC3 in ESC also shows to reduce pluripotency. Phosphatidylethanolamine could be a useful in-vitro treatment molecule, as it is structurally necessary for autophagy machinery, and has been shown to enhance autophagy and longevity, and is required at the early stage of epigenetic reprogramming anyway, providing another synergistic opportunity. Phosphatidylcholine(PC) is another essential molecule for embryonic identity and pluripotency, and plays a function in autophagosome formation. The primary lysosome biogenesis and autophagy transcription factor HLH-30/TFEB has also been shown to increase lifespan, and could be a helpful target for 3FPRR autophagy induction. Rab32 is another target which increases autophagy, and, in addition, synergistically the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, and improving the induction of iPSCs through the enhancement of lipid biosynthesis. TFEB is another fundamental target pathway linking enhanced autophagy to lysosomal biogenesis. Beclin 1 is also an autophagy inducer which is synergistcally essential for embryonic identity as well, and interestingly has shown to be able to reverse neurodegenerative accumulation or amyloid proteins in alzheimer's models, and generally support adult neurogenesis. Activation of the Sigma-1R receptor to induce autophagy and enhance proteostasis is another appealing pathway, and even reduces stress between the endoplasmic reticulum (ER) and mitochondria. SIRT6, which is involved in DNA repair also suppresses senescence and induces autophagy, as another attractive target.

Ultimately my hypothesis and supporting research is approachable and simple, but is the most sensible and reliable method for humanity to attain true reversal of aging. Total body cellular replacement with reprogrammed and rejuvenated stem cells via the 3 fundamental processes of reproductive rejuvenation, is what we should be investing in. We could always evolve upon the basis of this organic approach, but in my hypothesis this is the minimal requirement, and potentially fully sufficient approach to start with. In a sense, this hypothesis also would advise that support for other approaches to reverse aging are incomplete, and therefore it is important to be clear on one's goal of simply extending human lifespan and, or, healthspan, with “anti-aging” interventions, or fundamentally and finally attempting with experimental, practical, and engineering based advancements on a platform biotechnology to attain a fundamental and complete solution to aging itself. Comparing all other options to completely cure aging, there seems to be nothing that compares, as the greatest biological problem requires a total replacement of the problem with a solution, down to every last cell, throughout the entire body.
"
"We would like to submit our proposal. Please find a shared link below for reviewing a PDF format.
Thank you.

https://www.dropbox.com/s/08na5w5bpdqe95a/Proposal%20for%20the%20Longevity%20Hypothesis%20Prize_BioAtla%20Inc.pdf?dl=0"
"The Uberkinder Program (UP) aims to inclusively foster innately stronger, smarter, happier, healthier and more enduring adults, in an extremely cost-effective fashion, based upon two tenets:
1) Construction of an optimized adult requires maximization of neural and physical capacity and fitness, which are rooted in the bio-physical environment during early developmental
2) Proactive health and longevity measures reliably offer reduced need for, and superior results from reactive repair measures

Evolution never delivers an optimum possible set of features; rather, it reflects countless compromises between past environments, limitations of genetics, immunity, and bodily structure, developmental circumstances, and pure chance. Additionally, the genetic diversity that mitigates unpredictable existential threats also yields progeny that are poorly adapted to current circumstances. On the bright side, there are social and bio-physical factors that we increasingly can optimize. ‘Pre-adult’ bio-physical factors are the preponderant focus of UP, and include parental reproductive fitness at conception, plus bio-physical developmental environments during gestation and puberty. These factors significantly influence physical, neural and emotional capacities at maturity, and thus are strong determinates of adult limitations that can only be partially mediated by social factors (emotionally nurturing home life, good education, etc.).

Minus social influences, powerful evidence suggests that the most economical and humane approach to lasting health, comfort, social harmony and productivity is non-invasive optimization of pre-adult development, growth and repair factors that are the soil in which community behaviors are planted. Discovery, characterization, optimization, approval and universal implementation of these pre-adult factors is the sole goal of UP.

UP begins with vitalized and broad integration of known and easily accessible bio-physical boosters of pre-adulthood (e.g., avoiding alcohol during pregnancy; good sleep habits), but that only scratches the surface of what can be done. Indeed, even the cited, well-known boosters are imprecise, and have not been validated in detail. It might be that a single cc of 80-proof liquor every pregnant day is a net positive; that a 20-minute mid-morning and mid-afternoon nap, plus six hours sleep at night yields better outcomes than an eight-hour stretch at night. How can you know? Where is the data? What about a forty-minute nap after lunch that includes that one cc? Studies behind popular pediatric advice almost never offer fine detail, but the stakes are high, with seemingly minor outcome improvements (e.g., a 1-point average IQ increase) potentially having a huge impact when applied to billions of citizens.

The next target is the numerous GRAS, but thinly-proven and barely-known developmental augmentations. One can barely make a browser query concerning any factor (e.g., “near-IR light therapy and pregnancy”; “D3 + K2 for teens”) without quasi-learning that we maybe could/should be doing better job. Again, at least some of the to-be-sorted outcome improvements would have a huge impact when applied to the billions, but fine intervention details, combination effects, and that patronizing glare from your skeptical pediatrician - who might merely be stuck in the past – put the conscientious parent in a tough spot.

…Then comes pre-adult interventions that have had remarkable results for animals or adults/elders, but have never been proven for pre-adults. Herein lie some of the most promising elements of UP. For example, there exist a GRAS nutraceutical, HMB; used to accelerate healing by sports enthusiasts and wound-care specialists. A recent experiment with our embarrassingly-close porcine relatives revealed that pregnant sows who had HMB added to their feed delivered piglets that were reliably better developed. The enhancement was evident at birth and throughout their lives (unfortunately, artificially-truncated), in all of numerous measured parameters (e.g., ~20% [!!] increased birth weight). This is attributed to the high-damage environment of gestation being mitigated by HMB’s healing enhancement feature. Imagine broad spectrum, double-digit improvement of neuro-physical characteristics for human babies around the globe, and you will see the unmatched humanitarian and fiscal potential of UP! …And this is just one of many promising and yet-undiscovered pre-adult interventions that will be derived from adult and veterinary health, aesthetics, performance and longevity medicine.

UP is dedicated to establishing, through assertive advocacy, well-funded discovery, optimization, and highly-inclusive implementation of known and pending pre-adult interventions that enhance post-pubescent bio-physical potential for health, happiness, unity, productivity and longevity. Due to the decades required for so-upgraded bairns to significantly enter the adult population, and the mounting existential threats to society, implementation of the Uberkinder Program is profoundly urgent, and merits the highest level of advocacy and financial support by the Foresight Institute.

A monograph that includes deeper consideration, and numerous supportive references is available upon request. "
"SENthetic Lethality

Synthetic lethality is the biological phenomenon whereby inactivation of either gene A or B is tolerable, yet the loss of both is lethal. First described in 1922 (for Bar and Glass genes in drosophila), it wasn't until over 70 years later that Hartwell, Friend et al postulated that synthetic lethality could be used to target cancer, whereby one gene is a cancer-specific mutation and the second gene product acts as the therapeutic target.
This approach has seen great success, for example the development of PARP inhibitors for treating BRCA-deficient cancers. Whilst early attempts relied on RNA interference methods, the advent of CRISPR-based gene editing has made full genome screening more tangible/scalable to the point where numerous big pharma companies have synthetic lethality cancer drug discovery programmes.

I now propose that this method should be applied to longevity research to target senescent cells.

The approach would be to make KO cell lines of genes downregulated in senescence (e.g Lamin B1, SIRT1 etc) and then perform full genome CRISPR KO dropout screens to identify new ""SENthetic lethal"" drug targets.
Furthermore, a variation on the theme is Synthetic Dosage Lethality (SDL) whereby the downregulation of gene A coupled with the overexpression of gene B results in cell death. For this, overexpression cell lines of genes upregulated during senescence (e.g p16, p21 etc) would be used. An additional approach would be to utilise full genome CRISPR activation screens to allow the discovery of SDL targets whose overexpression in senescent cells leads to death - which could be exploited using RNA therapeutics."
"Hormone replacement therapy (HRT) involves supplementing certain hormones that decline in production with age. The main focus of HRT is on growth hormone (GH) and testosterone, as they are both associated with age-related diseases and have been shown to decline with age. By replacing these hormones, it is thought that they can help to slow down or even reverse the effects of aging.

Although this area of research has become increasingly popular in recent years, it is still not widely accepted. This is mainly due to the fact that there are still some major risks associated with hormone replacement therapy. These include side effects such as increased risk of cancer, stroke and heart attack; decreased fertility; and increased risk of blood clots. Additionally, there are concerns about long-term safety as well as potential for drug interactions when combined with other medications.

Despite the potential risks, many researchers believe that hormone replacement therapy could be a viable treatment for aging-related diseases and conditions. For instance, animal studies have shown promising results when using growth hormone in the treatment of osteoporosis, type 2 diabetes and cardiovascular disease. Similarly, testosterone has been studied for its potential to improve strength, muscle mass, libido and overall quality of life.

In conclusion, although hormone replacement therapy holds great promise as a way to extend lifespan or improve healthspan, it is still an underappreciated area of longevity research due to its associated risks. However, if further studies can provide evidence of safety and efficacy, then this approach could become an important tool in delaying aging-related diseases and ultimately extending the human lifespan."

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