Notes on Self-Experimentation with Sex Steroid Ablation for Regrowth of the Thymus

I periodically publish thoughts on self-experiments that seem interesting and relevant to aging. Despite the influence of the quantified self movement, the broader self-experimentation community is largely terrible on matters of research, rigor, reporting, and safety. My motivation is to something to raise the bar on all of these items.

For every discussion I've published on a particular self-experiment, there are half a dozen others sitting at some stage of research and interest. Over the past year or so, I've been on and off looking into sex steroid ablation as a mechanism for thymus regrowth. Since my company, Repair Biotechnologies, works on thymus regrowth as a way to reverse immune system aging, I have read up on most of the ways in which that has been attempted or achieved in animals and humans. The thymus is responsible for the maturation of T cells, a complicated process in which the cells are educated to attack pathogens and dangerous cells, but not normal tissue. The thymus atrophies with age, and by age 50 there is usually little active tissue left. Lacking a robust supply of new T cells, the immune system slowly declines into immunosenescence.

In humans, only two approaches to thymus regeneration have produced positive results. The small Intervene Immune trial used lengthy treatment with growth hormone alongside a few other substances to try to offset the negative effects of growth hormone. More impressive results, at least in the metrics measured, have been obtained from the use of sex steroid ablation in prostate cancer patients. For this approach, thymic growth has only been assessed in animal models. In human patients, the metrics have been naive T cell and recent thymic emigrant populations.

One has to ask the question: is it plausible for a 50-plus-year-old male to undergo a short period of sex steroid ablation in order to regrow the atrophied thymus, and rebuild the naive T cell population? Well, yes and no. Can it be done, yes. The evidence is pretty good. A fresh naive T cell population will last a decade or more without further reinforcement before it starts to become problematic - the normal course of aging, and people who have had the thymus surgically removed, tells us that. Many animal studies of castration and sex steroid ablation show thymic regrowth and revitalized production of T cells, for at least a short period of time. Based on trials of hematopoietic stem cell transplantation carried out in conjunction with sex steroid ablation, it is reasonable to think that three to six months would be required for a human naive T cell population to be reconstituted. Lesser duration of upregulated production of T cells would produce lesser benefits.

There is an interesting sidebar on what happens to the thymus in castrated mice. It doesn't grow in the sense of producing new cells. The cortical thymic epithelial cells grow in size. Then a few weeks later they shrink again. But this does produce upregulation of T cell production, and to a certain degree we don't really care that much about how the upregulation occurs so long as the T cells are of the right types and behavior. In human prostate cancer patients, increased T cell production is observed after months following sex steroid ablation, so the mechanisms are clearly distinct.

So why not conduct this self-experiment? Because the challenges and unknowns are not insignificant. Firstly, all human data and near all mouse data is in males - obviously necessarily the case in the prostate cancer studies. We have no good idea as to how to apply this sort of approach to females of either species. Secondly, while sex steroid ablation can be carried out very effectively using more modern drugs such as degarelix, a majority of the older prostate cancer patients it is used with do not recover the testosterone levels they exhibited prior to treatment. This is true even for the shortest treatment duration of 3-6 months, and appears to become worse the longer the treatment is continuously applied. There is really no established way of producing sex steroid ablation and very low testosterone levels with both rapid onset and rapid recovery, to get the overall duration to be any shorter than this.

Degarelix is an interesting drug. It causes very rapid sex steroid ablation, within a day or two, unlike other approaches that require a month or more to reach the desired point. When delivered intravenously, it leaves the system within a couple of days. But delivered subcutaneously, it forms a bolus that releases slowly over months. This is great for its intended use, but not so helpful if one wants a rapid recovery from sex steroid ablation. A single injection of degarelix is a 4-6 month commitment from start to (hopefully) final recovery - and the data indicates that the largely older prostate cancer patients do not tend to fully recover prior testosterone levels. Whether this is also true of younger individuals in the 40-50 age range is an interesting question with an unknown answer. Is the incomplete recovery inherent to age, or inherent to the usual secondary effects of sex steroid ablation on the systems of sex steroid regulation?

In principle one could inject degarelix intravenously every few days for a month to obtain a month of sharp onset and sharp recovery of testosterone levels. Then repeat every six months or so for a few years. One might expect that to greatly diminish side-effects, while still causing some degree of thymus regrowth. While the data exists for a single intravenous injection in humans, no-one knows what effects this might have if repeated extensively, or nor the proper calibration of dose and timing. Intravenous injection will produce spikes of much higher levels of the drug than occur for subcutaneous injection, and there is no data on what this does over time. That would all have to be discovered.

So while this might be an interesting project for someone with a high tolerance for personal risk, and an interest in conducting many trial runs with many blood tests along the way, it doesn't seem all that practical. Meanwhile, the practical approach of using degarelix in the well-established and well-characterized way it is used with prostate cancer patients, for the shortest period possible, produces lasting side-effects that most men might be somewhat unhappy to experience.

Setting aside that conclusion for a moment, if one was going to do this, how to measure outcomes? That should always be the question, regardless of whether or not a potential project is ever undertaken. Inability to come up with good metrics is a very compelling reason, in and of itself, not to carry out a self-experiment. In this case, there are good, well-established measures, however, and they will be applicable to any approach to restoration of thymic activity. Firstly, one can look at the Intervene Immune paper for some simpler approaches. In bloodwork to assess immune populations, the lymphocyte to monocyte ratio, CD4+ and CD8+ naive T cell counts, and recent thymic emigrants are all tests that exist and are to varying degrees easily accessed. One of course has to measure testosterone levels in order to prove that sex steroid ablation is taking place. For the thymus itself, careful CT scans have to be performed. The trick is to ensure that exactly the same location in cross-section is imaged at each assessment. There are papers covering how to do that, such as by taking increments in distance from the sternal notch as an anchor point. Assessing the cellularity of the thymus (it is unlikely to change in size) emerge from an examination of before and after images.

So on the whole, sex steroid ablation as an approach to regrowing the thymus and repopulating the naive T cell pool in older adults has good human evidence for efficacy in males, but is also risky and comparatively costly, and the existing tools are adapted to a use case that doesn't match well with this goal. Better approaches are needed, but developing them is likely beyond the means and interest of most self-experimenters.

Photobiomodulation to Enhance Mitochondrial Function as a Potential Therapy for Parkinson's Disease

There is evidence for near-infrared light to improve mitochondrial function, although exactly how it works, and how reliably it works, is far from settled. This wavelength of light penetrates tissue deeply enough to be considered as a treatment for neurodegenerative conditions, at least those in which mitochondrial dysfunction is strongly implicated, such as Parkinson's disease.

As the main driver of energy production in eukaryotes, mitochondria are invariably implicated in disorders of cellular bioenergetics. Given that dopaminergic neurons affected in Parkinson's disease (PD) are particularly susceptible to energy fluctuations by their high basal energy demand, it is not surprising to note that mitochondrial dysfunction has emerged as a compelling candidate underlying PD.

A recent approach towards forestalling dopaminergic neurodegeneration in PD involves near-infrared (NIR) photobiomodulation (PBM), which is thought to enhance mitochondrial function of stimulated cells through augmenting the activity of cytochrome C oxidase. Notwithstanding this, our understanding of the neuroprotective mechanism of PBM remains far from complete. For example, studies focusing on the effects of PBM on gene transcription are limited, and the mechanism through which PBM exerts its effects on distant sites remains unclear.

Also, the clinical application of NIR in PD proves to be challenging. Efficacious delivery of NIR light to the substantia nigra pars compacta , the primary site of disease pathology in PD, is fraught with technical challenges. Concerted efforts focused on understanding the biological effects of PBM and improving the efficiency of intracranial NIR delivery are therefore essential for its successful clinical translation. Nonetheless, PBM represents a potential novel therapy for PD.


Adjusting Macrophage Polarization for Therapeutic Effects is not Straightforward

The innate immune cells known as macrophages can adopt different packages of behaviors, known as polarizations, under different circumstances. The underlying reality is more a continuum than two clear categories, but researchers classify macrophages as being either M1, aggressive and inflammatory, or M2, pro-regenerative and anti-inflammatory. In theory, a number of issues that manifest with age could be slowed or reversed by forcing M1 macrophages to adopt M2 behaviors instead. The open access paper here is chiefly interesting for the discussion on why this is far from simple: a blunt approach to reprogramming macrophages is certainly feasible, but will probably do as much harm as good.

Compared to the classical phagocytotic "M1" macrophages, the alternatively polarized macrophages, called "M2" macrophages, function as modulators of cellular and humoral immunity and as mediators of tissue repair and remodeling. Transforming growth factor beta 1 (TGFβ1) is the most important growth factor enhancing tissue repair and fibrosis, and is believed to be produced and released by a subpopulation of M2 macrophages (M2c) in response to IL-10, in contrast to M2a macrophages which are primarily anti-inflammatory.

Prior work has shown that macrophages are the only inflammatory cells that infiltrate into the closed nucleus pulposus, and the number of macrophages is positively correlated with the severity of intervertebral disc degeneration. Moreover, there is evidence to suggest that macrophages may either directly play a role in phagocytosis, or synergistically regulate lumbar disc metabolism through a neuro-immune mechanism. Likewise, macrophage dysfunction can cause the aggregation, chemotaxis, and diffusion of inflammatory factors, leading to degradation of the extracellular matrix in the intervertebral disc, which in turn leads to lumbar disc degeneration. However, whether macrophage polarization is critical for the development of lumbar disc degeneration (LDD) and by what mechanism it may affect LDD, remains to be experimentally tested. This question was addressed in the current study.

The delicate phenotypic control of tissue macrophages is critical for proper tissue repair after injury and is very time-sensitive. Too much M1 polarization results in severe inflammatory responses, severe tissue damage and poor recovery. However, too much M2 polarization may result in insufficient inflammatory responses and incomplete pathogen- and cell debris removal. Importantly, M2-like polarization induces fibrosis, mainly mediated by TGFβ signaling.

In the current study, we co-blocked DNMT1 and TGFβ1 in macrophages. While DNMT1 suppression induced a general M2-like polarization in macrophages, specific inhibition of TGFβ1 may affect IL-10 production and secretion, which in turn reduces the generation of fibrotic M2c macrophages. Our results showed that co-blocking TGFβ1 did not attenuate the effects of DNMT1 inhibition on induction of M2-like macrophage polarization, but did reduce cell apoptosis and pain-associated MMP1, thereby promoting a favorable therapeutic outcome.


The Existence of Senolytics May Trigger the Shift Towards Targeting Root Causes of Disease

It is sadly true that little medical research and development involves targeting the root causes of the treated condition. This is exactly why therapies largely fail in trials, and are largely only marginally effective when approved. In the case of age-related disease, efforts to target root causes are a tiny faction of the field. This is slowly changing for the better, but the state of affairs today versus a decade ago is still only a matter of making that tiny fraction a little bit larger.

Senolytic therapies that selectively destroy senescent cells are the first rejuvenation therapies, treatments that target something close to a root cause of aging and age-related disease. The accumulation of senescent cells with age is highly detrimental to tissue function, even though they are only a tiny fraction of all cells even in late life. Removing senescent cells produces impressive benefits for dozens of age-related conditions in animal models. Going by the present data, senolytics are far and away more effective and useful than any other approach so far undertaken to intervene in the aging process.

Effective outcomes have a way of dragging the field along with them. It is hard to argue against large effect sizes and robust, replicated evidence of efficacy. For the period in which the majority of this data remains associated with animal models, it is only the research community engaged in revising its ideas on how to approach aging. As human data accumulates, we can hope that the broader medical and funding communities will follow, and then the public at large.

Insights from In Vivo Studies of Cellular Senescence

Recent observations using genetically-modified animal models indicate that the elimination of senescent cells attenuates aging and age-related diseases. These findings have opened new avenues to explore pharmacological approaches to induce apoptosis in senescent cells termed senolytics. With the absence of a unique marker for senescence, interventions have been developed to take advantage of some vulnerabilities that senescent cells have.

Senescent cells, like cancer cells, are resistant to apoptosis through the upregulation of BCL-2 anti-apoptotic proteins. Efforts in cancer research have found ABT263 (navitoclax), a potent inhibitor of BCL-2 and BCL-xL anti-apoptotic proteins, can be used to treat lymphomas and other types of cancer. Interestingly, due to the overexpression of BCL-2 in senescent cells, ABT263 exhibits senolytic activity and prolongs healthy lifespan in normally-aged mice. Importantly, using mouse models of age-related chronic diseases, including atherosclerosis and neurodegeneration, in which the accumulation of senescent cells is detrimental, treatment with ABT263 attenuated disease pathology.

Additional pharmacological interventions have been shown to target senescent cells in mice. These include a peptide that disrupts the interaction of FOXO4 and p53 leading to apoptosis, as well as nanoparticles that target senescence-associated β-gal positive cells. Interestingly, using a mouse model for osteoarthritis (OA), it was shown that senescent cells accumulate in articular cartilage and synovium promoting the development of OA. Consistent with the concept that senescent cells drive pathology, local administration of a new molecule named UBX0101, was used to disrupt the interaction of MDM2 and p53 to trigger apoptosis in senescent cells, yielding positive results in attenuating OA, plus validating UBX0101 as a senolytic. Consequently, UBX0101 was initiated in a clinical trial with adult patients diagnosed with femorotibial osteoarthritis to evaluate the safety, tolerability, and pharmacokinetics of the drug. It is currently being evaluated in phase 2 clinical trials for the effectiveness in treating musculoskeletal diseases with an emphasis on patients with OA.

Natural compounds, such as quercetin and fisetin, have also been used in combination with anti-cancer drugs, particularly the pan-tyrosine kinase inhibitor dasatinib to treat naturally aged mice and senescence-related diseases. The combination of dasatinib and quercetin (D + Q) target particular sensitivities of pro-survival pathways found in senescent cells, known as senescent cell anti-apoptotic pathways (SCAPs). These drugs could theoretically influence a broad spectrum of pathways in all cells, which may make it difficult to assess if senescence ablation occurs or there has been some amelioration of key features of senescent cells, such as the SASP. Nevertheless, these findings set a foundation to start clinical trials in adult patients with idiopathic pulmonary fibrosis and diabetic kidney disease. These studies have provided evidence that intermittent doses of senolytics can be systematically used in humans and tolerated.

The current philosophy of the healthcare system is to systematically treat chronic diseases with medications that, for the most part, address the consequence rather than the cause of the malady. Our desire to improve human care has motivated us to investigate how and why senescent cells accumulate with age and whether they may play therapeutically-relevant casual roles in age-related diseases. The objective is to start treating chronic diseases from the root and not the symptoms of the disease. As we are starting to enroll patients in "senolytics-clinical trials," it will be imperative to assess if senolysis efficiently targets the primary cause of disease or if it works best in combination with other drugs. Additional basic science research is required to address the fundamental role of senescent cells, especially in the established contexts of disease.

Complement C5 Protein is a Biomarker of Preclinical Atherosclerosis

At some point in the years ahead, the research community will develop effective means of reversing atherosclerotic lesions, the fatty, inflammatory deposits that build up in blood vessel walls to ultimately cause stroke or heart attack. Those therapies will be best applied in a preventative manner, not used in the late stage of the condition when lesions are large, complex, and greatly distort and weaken blood vessels. That in turn means that a reliable, low cost test to assess progression of preclinical atherosclerosis is required. Researchers here propose complement C5 protein levels in a blood sample as such a test, based on recent human data.

The purpose of this study was to analyze the temporal and topologically resolved protein changes taking place in human aortas with early atherosclerosis to find new potential diagnostic and/or therapeutic targets. The protein composition of healthy aortas (media layer) or with early atheroma (fatty streak and fibrolipidic, media, and intima layers) was analyzed by deep quantitative multiplexed proteomics. Plasma levels of complement C5 were analyzed in relation to the presence of generalized (more than 2 plaques) or incipient (0 to 2 plaques) subclinical atherosclerosis in 2 independent clinical cohorts, PESA (Progression of Early Subclinical Atherosclerosis) and NEFRONA (National Observatory of Atherosclerosis in Nephrology).

Proteins involved in lipid transport, complement system, immunoglobulin superfamily, and hemostasis are increased in early plaques. Components from the complement activation pathway were predominantly increased in the intima of fibrolipidic plaques. Among them, increased C5 protein levels were further confirmed by Western blot, enzyme-linked immunosorbent assay, and immunohistochemistry, and associated with in situ complement activation. Plasma C5 was significantly increased in individuals with generalized subclinical atherosclerosis in both PESA and NEFRONA cohorts, independently of risk factors. Moreover, in the PESA study, C5 plasma levels positively correlated with global plaque volume and coronary calcification.

In conclusion, activation of the complement system is a major alteration in early atherosclerotic plaques and is reflected by increased C5 plasma levels, which have promising value as a novel circulating biomarker of subclinical atherosclerosis.


Direct Reprogramming of Skin Cells into Photoreceptors to Restore Light Sensitivity in Mice

Researchers here demonstrate a direct form of cellular reprogramming, converting skin cells directly into another cell type without going through intermediary stages of induced pluripotency and differentiation. In this case the goal is to produce patient-matched photoreceptor cells to treat retinal degeneration. Proof of concept is demonstrated in blind mice that exhibit restored light sensitivity following treatment. The degree to which vision can be restored via this approach is an open question - that is much harder to assess in mice. In principle this type of reprogramming should be much more efficient, but it remains to be seen as to which approaches to building sources of patient-matched cells will emerge first into widespread clinical practice.

Up until now, researchers have replaced dying photoreceptors in animal models by creating stem cells from skin or blood cells, programming those stem cells to become photoreceptors, which are then transplanted into the back of the eye. In the new study, scientists show that it is possible to skip the stem-cell intermediary step and directly reprogram skins cells into photoreceptors for transplantation into the retina.

Scientists have studied induced pluripotent stem (iPS) cells with intense interest over the past decade. IPSCs are developed in a lab from adult cells and can be used to make nearly any type of replacement cell or tissue. But iPS cell reprogramming protocols can take six months before cells or tissues are ready for transplantation. By contrast, the direct reprogramming described in the current study coaxed skin cells into functional photoreceptors ready for transplantation in only 10 days. The researchers demonstrated their technique in mouse eyes, using both mouse- and human-derived skin cells.

Direct reprogramming involves bathing the skin cells in a cocktail of five small molecule compounds that together chemically mediate the molecular pathways relevant for rod photoreceptor cell fate. The result are rod photoreceptors that mimic native rods in appearance and function. The researchers performed gene expression profiling, which showed that the genes expressed by the new cells were similar to those expressed by real rod photoreceptors. At the same time, genes relevant to skin cell function had been downregulated.

The researchers transplanted the cells into mice with retinal degeneration and then tested their pupillary reflexes. Within a month of transplantation, six of 14 (43%) animals showed robust pupil constriction under low light compared to none of the untreated controls. Moreover, treated mice with pupil constriction were significantly more likely to seek out and spend time in dark spaces compared with treated mice with no pupil response and untreated controls. Preference for dark spaces is a behavior that requires vision and reflects the mouse's natural tendency to seek out safe, dark locations as opposed to light ones.


COVID-19 in the Context of Aging

It is widely appreciated that old people have a poor time of it when it comes to infectious disease. Seasonal influenza kills tens of thousands of older people every year in the US alone. The aged immune system functions poorly, and vaccinations for many conditions have low success rates in older people. Thus the vast majority of COVID-19 deaths are old people exhibiting immunosenescence. Given that the world at large seems to be entirely accepting of the yearly toll of influenza, while COVID-19 is classed as an apocalypse of some sort, one has to wonder how much of the hysteria surrounding COVID-19 stems from the rare - but highly publicized - deaths of younger individuals. Or perhaps if the rising toll of every influenza season was reported in the same way as deaths from COVID-19, more might be done? Human psychology is a strange thing.

An enormous amount of government and other funding will be directed towards fundamental infectious disease research in the years ahead, once things have settled down somewhat and COVID-19 has faded into the backdrop. That is one consequence of a pandemic that captures the attention of the world to the degree that this one has, deservedly or otherwise. This was perhaps the perfect storm, as ominous rumblings and awareness initiatives have been ongoing for some time regarding the threat of SARS-like viruses making the leap from animals to humans. A critical mass was finally reached. That COVID-19 has so far turned out to be less terrible than suspected at the outset is beside the point. The organizations of the world were primed to react in the way they are now to the first SARS-like virus that appeared remotely threatening.

The prospect of a large increase in funding for infectious disease and immunology research means that scientists in every relevant field of study are racing to position themselves to try to capture a portion of those funds. We outsiders don't see the ferocious pace of grant writing, but published papers are a visible sign of this energetic process. A few recent examples are noted below. Researchers involved in immunology and aging are taking this moment in history to remind the world that, yes, the immune system decays with age, old people bear the brunt of infectious disease as a result, and perhaps we should do something about this, now that we can target the mechanisms of aging - the cause of immunosenescence.

Covid-19 and Immunity in Aging Populations - A New Research Agenda

As we age, health conditions associated with aging, particularly noncommunicable diseases such as heart disease, cancers, and metabolic and autoimmune diseases, combined with treatments for these diseases and with immune senescence, substantially affect responses to vaccines and infectious diseases. Angiotensin-converting enzyme 2 (ACE2) has been identified as the receptor for SARS-CoV-2, the virus that causes Covid-19, and it has been suggested that differential levels of ACE2 in the cardiac and pulmonary tissues of younger versus older adults may be at least partially responsible for the spectrum of disease virulence observed among patients with Covid-19.

Even as the brunt of severe illness from Covid-19 is being borne by aging adults, we are navigating partially blind in efforts to develop vaccines and therapies to stop this and future pandemics, since we lack knowledge of the mechanisms of immunity to protect this population. If we can delineate principles of effective immunity in the elderly, we might also be able to develop new strategies for broader disease prevention and control in older populations.

COVID-19 is an emergent disease of aging

Here, we found that the case fatality rate for COVID-19 grows exponentially with age in Italy, Spain, South Korea, and China, with the doubling time approaching that of all-cause human mortality. In addition, men and those with multiple age-related diseases are characterized by increased mortality. Moreover, similar mortality patterns were found for all-cause pneumonia. We further report that the gene expression of ACE2, the SARS-CoV-2 receptor, grows in the lung with age, except for subjects on a ventilator. Together, our findings establish COVID-19 as an emergent disease of aging, and age and age-related diseases as its major risk factors. In turn, this suggests that COVID-19, and deadly respiratory diseases in general, may be targeted, in addition to therapeutic approaches that affect specific pathways, by approaches that target the aging process.

Inflamm-Aging: Why Older Men Are the Most Susceptible to SARS-Cov-2 Complicated Outcomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by a high mortality of elderly men with age-related comorbidities. In most of these patients, uncontrolled local and systemic hyperinflammation induces severe and often lethal outcomes. The aging process is characterized by the gradual development of a chronic subclinical systemic inflammation (inflamm-aging) and by acquired immune system impairment (immune senescence).

Here, we advance the hypothesis that some key features of aging contribute to the disproportionate SARS-CoV-2 mortality suffered by elderly men. At least four well-recognized aging-related characteristics that are strongly expressed in older men go some way towards explaining why these patients account for the vast majority of fatalities: (i) the presence of subclinical systemic inflammation without overt disease, (ii) a blunted acquired immune system and type I interferon response due to the chronic inflammation; (iii) the downregulation of ACE2 (SARS-CoV-2 receptor), which triggers inflammation, particularly in patients with age-related comorbid diseases such as type II diabetes; and (iv) accelerated biological aging, as measured by epigenetic and senescence markers (e.g. telomere shortening) associated to the chronic inflammatory state.

The Aging of Muscle Stem Cells

Impaired function of muscle stem cells is presently thought to be the dominant contributing factor in the development of sarcopenia, the loss of muscle mass and strength that affects everyone with advancing age. The mechanisms that cause stem cell decline are less well mapped, both in terms of their relative effect sizes, as well as their positions in the complex web of cause and consequence that links fundamental forms of cell and tissue damage, the root of aging, to downstream manifestations of aging.

Adult skeletal muscle has its own stem cell population, namely muscle satellite (stem) cells (MuSCs). Under sedentary conditions in the adult stage, MuSCs are mitotically quiescent and reside beneath the basal lamina of the myofiber; this position between the myofiber and the surrounding extracellular matrix is crucial for maintaining the stem cell state. After muscle injury, quiescent MuSCs promptly get activated, resulting in proliferation and their differentiation into myoblasts. Hence, myoblast fusion is critical not only for skeletal muscle development but also for regeneration. However, the function of MuSCs gradually declines during physiological and pathological aging. Although loss of the muscular regenerative capacity in aging is partly due to this impairment of MuSC function, the precise mechanism of how stem cell function is maintained and impaired remains unclear.

Aged MuSCs are also more prone to undergo senescence or apoptosis than young MuSCs. In terms of the ability of MuSCs to differentiate, the adipogenic differentiation program is enhanced in cultured, aged MuSCs. In the context of acute injury, symmetric and asymmetric cell division promote the expansion of MuSCs and maintain homeostasis of the stem cell compartment. Impairment of this process in aged muscle leads to an impaired propensity to proliferate and produce myoblasts necessary for muscle regeneration. While there are reports demonstrating decreases in the number of MuSCs during aging, conflicting reports show no significant differences in the number of MuSCs between young and aged mice. Additionally, since MuSCs are very rare and the number of MuSCs differs in the type and location of skeletal muscles, it is difficult to reach conclusions on the frequencies of MuSCs within young and aged mice.

The decline of MuSC regenerative capacity is due to age-associated extrinsic/environmental changes as well as cell-intrinsic/autonomous changes. As extrinsic factors, inflammatory responses, extracellular components, and changes in interacting cell types definitely affect the function in MuSCs. MuSC function is also impaired by cell-intrinsic damages including oxidative stress, DNA damage, modified signaling pathways, damage to proteins, and altered metabolism. An accumulation of cell intrinsic damages leads to a "point of no return" in aged MuSCs as they go into a pre-senescent state or they undergo apoptosis. Alterations in several intracellular signaling pathways in aged MuSCs affect their self-renewability. The functional decline of MuSCs is partly due to the activation of FGF2, TGF-β, WNT pathways, JAK/STAT3, p16INK4a, and p38. Those pathway modulations could be a therapeutic target for muscle regenerative therapy in elderly.


The Trials of Running a Public Longevity Industry Company

Taking a company public is a deal with the devil. One does it to gain access to capital, and at the behest of early investors who want an exit. Thereafter, however, one has to deal with all sorts of complications and perverse incentives that make the process of developing therapies that much more challenging. A little of this topic is discussed in this article, in the context of longevity industry biotech companies. Only a few of these companies are public at the moment, but that will change as the field advances and broadens.

When we spoke to Aubrey de Grey recently, he highlighted the potential challenges facing public companies in the longevity field. Juvenescence portfolio company AgeX Therapeutics is publicly traded and Greg Bailey of Juvenesence points out that many of the macro challenges are the same as any other biotech business. "You can go months between positive news, and during that period of time, the shorts can play havoc with your stock. And the other thing is, you fundamentally always have to have two years of working capital. If you don't, the short sellers circle - knowing you're going to have to do a financing and that they'll be able to buy in when you do that financing at a 10 to 15% discount to market."

But the work conducted by companies working on anti-aging and regenerative therapies also adds another level of variability to the challenge. "Now you also need to be concerned about the fact that one of your competitor's trials fails and everybody says 'Ah, anti-aging is rubbish - all hype, no reality' and you get caught in the downdraft. And, unfortunately, there have been a number of downdrafts with public companies where their trials have not lived up to expectations."

With investors beginning to realise the scale of the Longevity market, Bailey feels that this sort of reaction will become less common, but that hasn't stopped Juvenescence from taking action to mitigate the risk by moving AgeX Therapeutics to a licencing model. "So now you can't short because we did a deal with a major Japanese company in January. We did a deal with UC Irvine that'll see us in clinical trials in 18 months from January. So now as a short investor you've got to worry that I sign a license tomorrow, and the stock jumps and you get caught offside."

Like almost everyone in the world at the moment, dealing with the impact of COVID-19 is a significant concern. However: "If Bank of America is right, and the fact that every government investor is going to be looking at boosting the immune system, which is definitely affected by aging, there's going to be an enormous amount of money available in this. So it's just about getting in front of the right people and them understanding there's going to be a company that is modifying aging that's going to be worth 100 billion dollars, sooner as opposed to later. You've just got to pick which model you think is the right business model to invest in to get your institution or entity correctly positioned."


How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Flagellin Immunization

This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess whether a ten week course of flagellin immunization will significantly and beneficially affect gut microbe populations. Flagellin is the protein making up a flagellum, the appendage that bacteria use to move themselves around. As it happens, the presence of flagellae correlates decently well with harmful gut microbes, and the absence of flagellae correlates decently well with helpful gut microbes. In principle, provoking the immune system into greater efforts to chase down and destroy anything with a flagellum will better manage the microbial populations of the gut. These populations change with age in ways that promote chronic inflammation and reduce the generation of beneficial metabolites.

Flagellin has been used in human clinical trials as an adjuvant to improve vaccination efficacy: it is recognized by the immune system, and helps produce a greater immune response. It was shown to have only minimal side effects in those trials. There is no human data for effects on the gut microbiome, but a very interesting paper describes the effects in mice. The outcome is a shifting of the relative population sizes of gut microbes in a beneficial direction that will reduce chronic inflammation. It is unknown as to how long such an effect might last - whether rousing the immune system is a short-term process, or whether it will continue to better guard against unwanted microbes.

The purpose in publishing this outline is not to encourage people to immediately set forth to follow it. There are, for example, important caveats in the above mentioned mouse study regarding links between flagellin immunity and human gut diseases. If you come away thinking that you should just jump in, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.


Why Self-Experiment with Flagellin Immunization?

Gut microbes are an important influence on long-term health and aging. If forced to guess, they might be in the same ballpark as exercise. The first changes occur around age 35, reducing the generation of beneficial metabolites. Later changes are more detrimental. There is at present little that people can do to reliably influence gut microbe populations, beyond improving diet, there is considerable variability between individuals, and only a few services such as Viome that offer assays to assess progress. Any sort of therapy that works for a majority of people without aggressive customization would be a step forward, and responsible self-experimentation can help to determine whether this is a viable path forward. These reasons must be balanced against a sober assessment of the risk involved in vaccinating oneself with a bacterial protein that has been used in only a few human trials, and an acceptance of personal responsibility for consequences should one choose to run those risks.

Caveats in More Detail

There are two areas of personal responsibility to consider here. Firstly, this involves injecting a bacterial protein that has little published data on human use (even when that data shows good safety in the short term). As the mouse study paper points out, human patients with inflammatory bowel disease exhibit greater immune reactivity to flagellin, and it is unclear as to whether this drives disease pathology or is a beneficial adaptation. That is a leap into the unknown. So is crossing the road, or indeed getting out of bed in the morning, but there are definitely different degrees of risk and comfort.

Secondly, obtaining flagellin in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is legal to proteins that are not defined as a therapy for research use, but illegal to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.

Choosing to purchase and use flagellin would therefore likely be a matter of civil disobedience, as is the case for anyone obtaining medicines or potential medicines outside the established national system of prescription and regulation. People are rarely prosected for doing so for personal use in the US - consider the legions of those who obtain medicines overseas for reasons of cost, despite the fact that doing so is illegal - but "rarely" is not "never." If you believe that the law is unjust, then by all means stand up against it, but accept that doing so carries the obvious risks of arrest, conviction, loss of livelihood, and all the other ways in which the cogs of modern society crush those who disagree with the powers that be.

Summarizing Flagellin Immunization

Flagellin is classified as a pathogen-associated molecular pattern, something that both innate and adaptive immune systems recognize and react to. The use of flagellin as an adjuvant to spur greater immune response to another, attached protein or protein fragment is an established field of development. This is largely because of the favorable characteristics of flagellin: it doesn't appear to be associated with mechanisms that might cause severe reactions. As noted earlier, human trials have taken place, and the safety data is good - at least over the short time frame in which safety is assessed.

Immunization with flagellin versus its use as an adjuvant is a matter of injecting the protein on its own at an appropriate dose, to rouse the immune system to greater activity against anything connected to flagellin. Given that we mammals, mice and humans, are already sensitized to flagellin, it is interesting that boosting that reaction has a noticable affect on gut microbes. That was only recently demonstrated in mice. A larger literature on this topic has yet to be established.

Establishing Dosage

For small molecule drugs, it is possible to produce a starting point for human dosage given a mouse dose via standard equations, as shown in "A simple practice guide for dose conversion between animals and human". Unfortunately this doesn't apply to vaccines. In fact, it is fair to say that there is no rigorous way to determine how vaccines scale from mice to people, as the systems involved are enormously complex. Outcomes depend on immune system behavior, not on animal size, and modeling the immune system is a speculative activity at best. Many approved vaccines are probably using wildly suboptimal doses.

Insofar as there is any common wisdom, it is that similar doses in mice and humans appear to be a good starting point, based on those cases in which rigorous dose-finding has been carried out. In the case of flagellin, the mouse study employed a 10 μg dose injected weekly for 10 weeks. A 2017 human clinical trial used two monthly doses of 1 to 10 μg via intramuscular injection. Studies in non-human primates used used doses of 1 to 10 μg.

Much larger doses have been given to animals without apparent toxicity, but the failure mode of immunization at too high a dose is intense inflammation or, worse, a cytokine storm that has the potential to be very harmful or even fatal. Whether such adverse consequences are triggered, and how likely they are, is species-dependent. There is no human data for higher doses than those mentioned above, so at the end of the day, the best starting point appears to be to stick with the 10 μg dose repeated weekly over 10 weeks via intramuscular injection, on the basis that this has been trialed in humans.

An Introduction to Injections

The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.

The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1 ml, and intramuscular is limited to 2-3 ml depending on location. These are all very fuzzy numbers, but these upper limits don't really matter for the purposes of injecting 10 μg of a protein: it can be dissolved in a very small amount of liquid, 0.5 ml or less.

Different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you'll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.

It seems sensible to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections. In the case of flagellin, it makes sense to stick with the human trial approach of intramuscular injections.

Considering Autoinjectors

Sticking a needle into one's own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately largely not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.

Option 1: Subcutaneous Autoinjection with Needle and Syringe

If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.

Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection

Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I'll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.

Obtaining a Needle-Free Injection System

There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. So far as I can tell, PharmaJet is the only available needle-free system that is capable of intramuscular rather than subcutaneous injection.

PharmaJet is the better engineered and more expensive of these two systems, and its specialized 0.5 ml syringes are built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.

The instructions for both of these systems are extensive, and include videos. They are fairly easy to use. One caveat is that needle-free systems produce a puncture that more readily leaks injected material back out again than is the case for needles. It is a good idea to have a less absorbent plaster ready to apply immediately after injection, such as one of the hydrocolloid dressings now widely available in stores.

Obtaining Vials of the Correct Size

If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.

There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place - this requires a crimping tool, and removing it requires the use of another tool.

There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.

It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.

Preparing Flagellin for Injection

If using a needle-free injection system, you will likely be limited to injecting 0.5ml amounts. Thus the objective here is to obtain 10 μg of flagellin dissolved in 0.5 ml of phosphate buffered saline in each of ten sealed vials, ready to be used with the injection system, with as little contamination as possible from the environment, and stored a freezer until it is ready to use. Depending on the size of the vial, it might contain doses for multiple injections, but stick to one dose per vial. It is not a good idea to carry out repeated freeze-thaw cycles on protein solutions, which is what you'll have to do if all the doses are in 5ml of solution in one vial. Multiple freeze-thaw cycles degrade the protein.

When ordering flagellin, it will typically arrive in 10 μg to 100 μg vials. Empty 100 μg of flagellin into a single vial holding 5 ml of phosphate buffered saline. Mix well. Place that vial and 10 empty vials into a vial rack. Then use a pipette to transfer 0.5 ml into each empty vial. Seal and crimp each vial as you go. Then put the whole set into the freezer.

Keeping Things Sterile is Very Important

Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. Laboratories use autoclaves, which sterilize with steam. These are largely expensive devices, but a range of smaller, cheaper options exists. There are many best practices guides and summaries available online. This extends to the injection itself. Even with needle-free systems, an injection site should still be wiped down with alcohol first. It is all too easy to infect an injection site if skipping the precautions, and this can have severe consequences.

Obtaining Flagellin

Not all flagellin is exactly the same molecule, nor is it created in the same way. The mouse study used a standard option of flagellin protein from Salmonella typhimurium (there are numerous strains, but strain differences are probably irrelevant). This is typically produced using recombinant protein manufacture techniques. There are many established companies that manufacture and sell proteins, including flagellin, to high standards of quality and safety, but as a general rule they will not ship to any customer other than an established and validated lab business. The usual way for everyone else to obtain cost-effective supplies of this sort is to search Alibaba for suppliers who offer that compound in their catalog, but unfortunately this isn't an option for flagellin. Instead one must search Alibaba for protein synthesis companies, pick a smaller one, and negotiate a price for flagellin synthesis.

As noted at the outset of this post, all of these efforts to obtain, ship, and use any random protein for self-experimentation are to some degree illegal - it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary protein isn't the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.

Open a Business Mailbox

A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.

Use Alibaba to Find Manufacturers

Alibaba is the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers. The company has done a lot of work to incorporate automatic translation, to reduce risk, to garden a competitive bazaar, and to make the reputation of companies visible, but it is by now quite a complicated site to use. It is a culture in and of itself, with its own terms and shorthand. There are a lot of guides to Alibaba out there that certainly help, even if primarily aimed at retailers in search of a manufacturer, but many of the specific details become obsolete quickly. The Alibaba international payment systems in particular are a moving target at all times: this year's names, user interfaces, and restrictions will not be the same as next year's names, user interfaces, and restrictions.

Start by searching Alibaba for protein synthesis companies. There are scores of biotech companies in China for any given specialty. Filter the list for small companies, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a protein, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices - they may vary widely, and it isn't necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.

Many manufacturers will state that they require a large (often ridiculously large) minimum order; that can be ignored. Only communicate with gold badge, trade assurance suppliers with several years or more of reputation and a decent response rate. Make sure the companies exist outside Alibaba, though for many entirely reliable Chinese businesses there are often sizable differences between storefronts on Alibaba, real world presence, and the names of owners and bank accounts. Use your best judgement; it will become easier with practice.

Arrange Purchase and Shipping via Alibaba

Given the names of a few suppliers, reach out via the Alibaba messaging system and ask for a quote for a given amount of flagellin; you will have to provide the sequence and a reference to the paper in which it is described. Buy more than you'll think you need, and make sure it is packaged into multiple vials, as one vial will be used to validate the identity and quality of the batch. Payment will most likely have to be carried out via a wire transfer, which in Alibaba is called telegraphic transfer (TT). Alibaba offers a series of quite slick internal payment options that can be hooked up to a credit card or bank account, but it is hit and miss whether or not those methods will be permitted for any given transaction. Asking the seller for a pro-forma invoice (PI), then heading to the bank to send a wire, and trusting to their honesty should work just fine when dealing with companies that have a long-standing gold badge.

To enable shipping with tracking via carriers such as DHL, the preferred method of delivery for Chinese suppliers shipping to the US or Europe, you will need to provide a shipping address, email address, and phone number. Those details will find their way into spam databases if you are dealing with more than a few companies, and will be, of course, sold on by Alibaba itself as well. Expect to see an uptick of spam after dealing with suppliers via Alibaba, so consider using throwaway credentials where possible.

Chinese manufacturers active on Alibaba are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized proteins. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier. Note that different carriers tend to have different processes and rates at which shipments are checked for validity.

Storing Flagellin

Proteins are shipped in a solid freeze-dried (lyophilised) form. While in this form they are easily stored in a refrigerator for the short-term or in a freezer for the long term. It has a much shorter life span once it has been mixed with liquid for injection, however, and should be kept frozen, and used within a matter of a few months at the most.

Validating the Purchased Flagellin

A protein may have been ordered, but that doesn't mean that what turns up at the door is either the right one or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a protein is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.

Obtain an Extra Vial from the Same Batch as the Others

Since it is extra work to attempt to split out microgram amounts of protein or mail protein in solution rather than lyophilised protein, just order an extra vial from the same batch and send it off to be tested.

Use Science Exchange to Find Lab Companies

Science Exchange is a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Here again, pick a small lab company to work with after searching for LC-MS (liquid chromatography and mass spectrometry) services. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Companies registered with Science Exchange largely don't provide their rates without some discussion, but a little over $100 per sample is a fair price for LC-MS to check the identity and purity of the compound.

Work with the Company to Arrange the Service

The process of request, bid, acceptance, and payment is managed through the Science Exchange website, with questions and answers posted to a discussion board for the task. Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by NovoPro or other sources.

Ship the Samples

Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.

Examine the Results

Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor or other sources.

Establishing Tests and Measures

There are a few options for testing before and after, the most compelling of which is the gut microbiome. Changes there, given a stable diet, should be indicative that something happened. But to assess outcomes beyond that, inflammatory markers should be tested at the very least. It might also be interesting to look at DNA methylation assessments of biological age, though it is an open question as to what exactly these tests are measuring, and whether they are all that useful to an individual.

Gut Microbiome

The most direct measure of effectiveness is to assess the gut microbe populations directly. There are lab services that do this, but few commercial direct to consumer services. One US based service is Viome, though it doesn't present results in a useful tabular form, and does not provide the raw data. Nonetheless, it is a measurable endpoint. Gut microbe populations are fairly slow to change, given a consistent diet, so large differences over a ten week course of flagellin injections would be interesting to observe.


There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Since inflammatory markers are the topic of interest, a service that offers more individual tests rather than packages might be a better choice. The Life Extension Foundation offers a wide range of tests, for example, including a selection of inflammatory markers.

DNA Methylation

DNA methylation tests can be ordered from either vendors such as Epimorphy / Zymo Research - note that it takes a fair few weeks for delivery. From talking to people at the two companis, the normal level of variability for repeat tests from the same sample is something like 1.7 years for the Zymo Research test. The level of day to day or intraday variation between different samples from the same individual remains more of a question mark at this point in time, though I am told they are very consistent over measures separated by months. Nonetheless, it is wise to try to make everything as similar as possible when taking the test before and after a treatment: time of day, recency of eating or exercise, recent diet, and so forth.

Guesstimated Costs

The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.

  • Business mailbox, such as from UPS: $250 / year
  • Cytokine blood panel test from LEF: $300 / test
  • MyDNAage kits: $310 / kit
  • Viome kits: $150 / kit
  • Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: $60
  • Small pack of 13mm sterile serum vials: $35
  • PharmaJet Needle-free Injection Kit: $1020
  • Comfort-in Needle-free Injection Kit: $470
  • Bulk 13mm serum vial parts and capping tools: $750
  • 100 μg of flagellin via Alibaba: $1000
  • Shipping and LC-MS analysis of a sample: $200

Practice Before Working with Flagellin

Do you think you can reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with saline solution rather than finding out that your manual dexterity and methods are lacking while handling an expensive protein. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case.

Schedule for the Self-Experiment

One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of flagellin to take a couple of months. Once all of the decisions are made and the materials are in hand, pick a start date. The schedule for the self-experiment is as follows:

  • Day 0: Perform the various tests: bloodwork, gut microbiome assessment, etc.
  • Day 1: Intramuscular injection of 10 μg of flagellin.
  • Day 8: Intramuscular injection of 10 μg of flagellin.
  • Day 15: Intramuscular injection of 10 μg of flagellin.
  • Day 22: Intramuscular injection of 10 μg of flagellin.
  • Day 29: Intramuscular injection of 10 μg of flagellin.
  • Day 36: Intramuscular injection of 10 μg of flagellin.
  • Day 43: Intramuscular injection of 10 μg of flagellin.
  • Day 50: Intramuscular injection of 10 μg of flagellin.
  • Day 57: Intramuscular injection of 10 μg of flagellin.
  • Day 64: Intramuscular injection of 10 μg of flagellin.
  • Day 65: Repeat the tests.

Where to Publish?

If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial. Equally if you run your own website or blog, why not there?

When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results! Publish whatever the outcome.

Implicating Glymphatic System Dysfunction in Glaucoma

There is growing interest in the systems of drainage that carry away cerebrospinal fluid and molecular waste from the brain. The failure of these systems due to the damage and dysfunction of aging may be an important cause of neurodegenerative conditions, allowing protein aggregates such as amyloid-β to build up to pathological levels in brain tissue. One branch of this work is focused on drainage through the cribriform plate, while another is focused on the comparatively recently discovered glymphatic system. Here, researchers note that a portion of the glymphatic system is implicated in glaucoma, a retinal degeneration that is proximately caused by rising pressure in the eyeball. Underlying causes are thought to include autoimmunity, senescent cells, and chronic inflammation in general.

Instead of a traditional lymphatic system, the brain harbors a so-called glymphatic system, a network of tunnels surrounding arteries and veins through which fluid enters and waste products drain from the brain. In a new study researchers show that the rodent eye also has a glymphatic system that takes out the trash through spaces surrounding the veins within the optic nerve. They also found that this system may be compromised in glaucoma and is capable of clearing amyloid-β, the build up of which has been implicated in the development of Alzheimer's disease, glaucoma, and age-related macular degeneration.

The majority of the aqueous humor - the fluid that fills the chamber between the cornea and the lens - drains from the eye to the surrounding vasculature through a circular lymph-like vessel called Schlemm's canal. This helps regulate intraocular pressure. Researchers decided to connect the knowledge about the front of the eye with their questions about the back of the eye. Because a 2012 study that the brain's glymphatic system was capable of clearing amyloid-β, they used that molecule to investigate the existence of an ocular glymphatic system.

The researchers found that the constriction of the pupil in response to light in both mice and rats increased glymphatic clearance from the eye. They also showed that the pressure in the eye, which is higher than that in the cranium, is necessary to drive the drainage through the ocular glymphatic system. Because of this, they hypothesized that glymphatic clearance might also be disrupted in glaucoma, a disease that typically involves increased intraocular pressure.

The research team determined that there was more amyloid-β clearance from the eyes of two different mouse models of glaucoma than from age-matched control mice. But the increased drainage did not seem to be caused by changes in intraocular pressure. Instead, excessive outflow was actually leakage into the spaces between axons in the optic nerve, not into the glymphatic system, that was related to a breakdown in the barrier between the eye and the optic nerve. The researchers hypothesized that this barrier normally diverts fluid, facilitating solute transport along the optic nerve's veins, a process that may fall apart in a diseased eye.


Autoimmunity in Parkinson's Disease

Is Parkinson's disease in part an autoimmune condition? Parkinson's is an age-related neurodegenerative condition in which the primary motor control symptoms result from the death of a specialized population of neurons that generate dopamine. There are also other harms done to neurological function, however. Under the hood, processes such as chronic inflammation, mitochondrial dysfunction, and aggregation of α-synuclein contibute to that cell death. Researchers here speculate on an autoimmune component to the pathology of Parkinson's disease, in that these mechanisms also drive the immune system into greater inflammatory activity that harms healthy tissue.

A new study adds increasing evidence that Parkinson's disease is partly an autoimmune disease. In fact, the researchers report that signs of autoimmunity can appear in Parkinson's disease patients years before their official diagnosis. Scientists have long known that clumps of a damaged protein called alpha-synuclein build up in the dopamine-producing brain cells of patients with Parkinson's disease. These clumps eventually lead to cell death, causing motor symptoms and cognitive decline.

An earlier study showed that alpha-synuclein can act as a beacon for certain T cells, causing them to mistakenly attack brain cells and potentially contribute to the progression of Parkinson's. This was the first direct evidence that autoimmunity could play a role in Parkinson's disease. The new findings shed light on the timeline of T cell reactivity and disease progression. The researchers looked at blood samples from a large group of Parkinson's disease patients and compared their T cells to a healthy, age-matched control group. They found that the T cells that react to alpha-synuclein are most abundant when patients are first diagnosed with the disease. These T cells tend to disappear as the disease progresses, and few patients still have them ten years after diagnosis.

The researchers also performed an in-depth analysis of one Parkinson's disease patient who happened to have blood samples preserved going back long before his diagnosis. This case study showed that the patient had a strong T cell response to alpha-synuclein ten years before he was diagnosed with Parkinson's disease. Again, these T cells faded away in the years following diagnosis. "One of the most important findings is that the flavor of the T cells changes during the course of the disease, starting with more aggressive cells, moving to less aggressive cells that may inhibit the immune response, and after about 10 years, disappearing altogether. It is almost as if immune responses in Parkinson's disease are like those that occur during seasonal flu, except that the changes take place over ten years instead of a week."

Therapies exist to treat inflammation from autoreactive T cells, and these TNF therapies are associated with lower incidence of Parkinson's disease. Going forward, the researchers are especially interested in using a tool called a T cell-based assay to monitor patients already at risk for Parkinson's to see if they could benefit from TNF therapies.


Speculating on Circumstances in Which Group Selection of Aging Can Occur

The consensus view on the evolution of aging is that it is a consequence of a race to the bottom in terms of competition for early life reproductive success. The result is mechanisms and systems that aid early fitness at the cost of later dysfunction - and consequent aging and death. This is known as the antagonistic pleiotropy hypothesis. So we exist, do pretty well at the outset of life, but are equipped with a biochemistry that is incapable of repairing itself well enough for the long term. Some metabolic byproducts cannot be broken down, and accumulate to cause issues. The adaptive immune system must store information, and eventually runs out of capacity. And so on.

There are other minority viewpoints on the evolution of aging, numerous varieties of the programmed aging hypothesis. In this view, degenerative aging is directly selected rather than a side-effect. It is in some way advantageous to fitness. Looking at today's research materials, the variety of programmed aging hypothesis that springs to mind is the one invoking group selection: aging exists to control the population so that ecosystem collapse is avoided. Speaking generally, group selection is not well regarded, and not thought a valid mechanism of evolution. But are there circumstances in which researchers believe that group selection could be involved in the evolution of aging? The example here is a collection of organisms that are all clones of one another - such as microbes, or some lower animals such as nematodes.

Some worms programmed to die early for sake of colony

Evolutionary theorists originally believed that ageing evolved to reduce the population in order to increase food availability for the young, but scientists have since shown this cannot be true for most animal species as longer-lived non-altruists would usually be favoured by natural selection. However, certain organisms possess what appear to be self-destruct programmes, preventing them from living beyond a certain age. For example, in the tiny roundworm C. elegans, mutations to particular genes can massively increase their lifespan (from two to three weeks under laboratory conditions, to close to 20 weeks), presumably by switching off the life-shortening programme.

Researchers investigated the specifics of the C. elegans life cycle to understand why programmed death may work for them, by devising computer models of a C. elegans colony growing on a limited food supply. They tested whether a shorter lifespan would increase the reproductive capacity of colonies, by generating the equivalent of colony seeds (a dispersal form of worm called a dauer). They found that shorter lifespan, as well as shorter reproductive span and reduced adult feeding rate, increased the reproductive success of the colony.

"Our findings are consistent with the old theory that ageing is beneficial in one way, as they show how increasing food availability for your relatives by dying early can be a winning strategy, which we call consumer sacrifice. But adaptive death can only evolve under certain special conditions where populations of closely related individuals don't mix with non-relatives. So this is not predicted to apply to humans, but it seems to happen a lot in colonial microorganisms."

Shorter life and reduced fecundity can increase colony fitness in virtual Caenorhabditis elegans

In the nematode Caenorhabditis elegans, loss of function of many genes leads to increases in lifespan, sometimes of a very large magnitude. Could this reflect the occurrence of programmed death that, like apoptosis of cells, promotes fitness? The notion that programmed death evolves as a mechanism to remove worn out, old individuals in order to increase food availability for kin is not supported by classic evolutionary theory for most species. However, it may apply in organisms with colonies of closely related individuals such as C. elegans in which largely clonal populations subsist on spatially limited food patches.

Here, we ask whether food competition between nonreproductive adults and their clonal progeny could favor programmed death by using an in silico model of C. elegans. Colony fitness was estimated as yield of dauer larva propagules from a limited food patch. Simulations showed that not only shorter lifespan but also shorter reproductive span and reduced adult feeding rate can increase colony fitness, potentially by reducing futile food consumption. Early adult death was particularly beneficial when adult food consumption rate was high. These results imply that programmed, adaptive death could promote colony fitness in C. elegans through a consumer sacrifice mechanism. Thus, C. elegans lifespan may be limited not by aging in the usual sense but rather by apoptosis-like programmed death.

A Cardiac Patch Without Cells Improves Regeneration Following Heart Attack

It is a sad truth that near all transplanted cells in near all cell therapies die quickly, and do not integrate with tissues to improve function. The benefits that do occur result from the signals secreted by the transplanted cells before they die. The research community has been undertaking a range of strategies to address this issue. One is to produce a scaffold material that mimics tissue sufficiently well to give cells the support they need to survive, populate it with suitable cells, and then transplant the resulting structure. This can produce 10% survival of transplanted cells, an enormous improvement over delivery of cells alone.

One manifestation of this approach is a heart patch, a thin structure that is placed onto the surface of an injured heart. Heart patches have performed fairly well to date in the lab, but like all tissue engineering work, that they use cells makes them logistically challenging and expensive to deploy. Researchers here report on how well a patch works if the cells are left out of the equation: just transplant the scaffold, a structure that can be mass manufactured and stored comparatively easily, and see whether it encourages native cells to greater regeneration.

Cardiac patches are being studied as a promising future option for delivering cell therapy directly to the site of heart attack injury. However, current cardiac patches are fragile, costly, time-consuming to prepare and, since they use live cellular material, increase risks of tumor formation and arrhythmia.

"We have developed an artificial cardiac patch that can potentially solve the problems associated with using live cells, yet still deliver effective cell therapy to the site of injury." Researchers built the patch by first creating a scaffolding matrix from decellularized pig cardiac tissue. Synthetic cardiac stromal cells - made of a biodegradable polymer containing cardiac stromal cell-derived repair factors - were embedded in the matrix. The resulting patch contained all of the therapeutics secreted by the cells, without live cells that could trigger a patient's immune response.

In a rat model of heart attack, treatment with the artificial cardiac patch resulted in ~50% improvement of cardiac function over a three-week period compared to non-treatment, as well as a ~30% reduction in scarring at the injury site. The researchers also conducted a seven-day pilot study of heart attack in a pig model, and saw ~30% reduction in scarring in some regions of the pig hearts, as well as stabilized heart function, compared to non-treatment. Additional experiments demonstrated that artificial patches that had been frozen were equally potent to freshly created patches.


GDF11 in the Regeneration and Aging of Skin

GDF11 was identified in parabiosis studies as beneficially influencing stem cell and tissue function. Levels of GDF11 decline with age. There was some debate over whether or not the early research was correct, but GDF11 is presently in clinical development as a basis for regenerative therapies for the old. Researchers here outline a role of GDF11 in the regeneration and tissue maintenance of skin, focusing on its anti-inflammatory role. The chronic inflammation of aging is present in all tissues, skin included, and is detrimental to health and tissue function. Anti-inflammatory effects are likely important in the observed benefits from upregulation of GDF11 in old animals.

GDF11 regulates essential cell differentiation and proliferation responses and is expressed in numerous tissues, including the skin, heart, skeletal muscle, and developing nervous system. Its expression is at the highest level in young adult organs and seems to decline during aging. Some studies have shown that GDF11 can reverse age-related dysfunction in muscle, nervous and cardiovascular systems. Although serum GDF11 levels were found to be decreased in old mice, supplementation with GDF11 "rejuvenated" them, thereby suggesting that GDF11 is a key player in mammalian life span. It has also been suggested that GDF11 is involved in the age-related global physiological decline in function, and that restoring circulating blood levels of GDF11 could reverse some of the cellular and physiological dysfunctions observed in aged mice.

Tumor necrosis factor-α (TNF-α) plays a key role in inflammatory diseases, including skin inflammation, while GDF11 inhibits inflammatory reactions. GDF11 treatment antagonizes TNF-α-induced inflammation in macrophages, and the administration of GDF11 appears to attenuate skin inflammation. Studies show that TNF-α-induced activation of the nuclear factor kappa B (NF-κB) signaling pathway, which is known to participate in various inflammatory conditions, is limited by GDF11 treatment.

Heat shock proteins (HSPs) are molecular chaperones essential for the maintenance of cellular functions, but they can be released extracellularly upon cellular injury or necrosis. GDF11 induces protective effects in various tissues through the suppression of oxidative stress and the expression of HSPs. As the key member of the TGF-β superfamily, GDF11 represents a promising therapeutic agent for the treatment of a number of inflammatory skin diseases, including psoriasis.


To What Degree is Chondrocyte Hypertrophy in Osteoarthritis Due to Cellular Senescence?

Senescent cells are large. They do not replicate, that function is disabled, but it is as if they go to the effort of producing all the material needed for replication, and thus swell up in size. A lot of the distinctive behavior of senescent cells seems quite connected to the fact that they are large. Insofar as aging is concerned, the important aspects of senescent cells are (a) whether or not they are being cleared rapidly and efficiently enough to keep their numbers down, and (b) the inflammatory, damaging signals they secrete. As senescent cell numbers grow, they cause ever more dysfunction in the surrounding tissue and the body at large.

In today's open access paper, researchers tie together observations of enlargement in the chondrocyte cells that make up cartilage tissue, that growth in size involved in the transformation of cartilage to bone, with the incidence of cellular senescence in those cells. Osteoarthritis, involving chronic inflammation and degeneration of cartilage, may be largely driven by cellular senescence. If examining large chondrocytes, there is no doubt some degree of overlap between dysfunction in which too much transformation to bone is taking place, versus rising levels of cellular senescence. But how much overlap?

Research into cellular senescence is at a peculiar stage at the moment. Senescent cells are clearly involved in near every age-related disease, but the research community at large has only become earnestly engaged in this topic over the last five years or so. There are a great many diseases of aging, and only so many scientists. So cellular senescence, despite being of great importance to the treatment of aging, is still poorly explored in the specific context of most conditions. Researchers are likely to learn more by deploying one of the proven senolytic drugs to destroy senescent cells than they are through analysis of the disease state without intervention - but again, many diseases and only so many research teams.

The Role of Chondrocyte Hypertrophy and Senescence in Osteoarthritis Initiation and Progression

Osteoarthritis (OA) is the most common joint disorder throughout the whole human population. OA accompanies progressive degradation of the articular cartilage, which leads to a loss of joint mobility and function and eventually to a low quality of life in patients due to both pain and restricted lifestyles. Healthy chondrocytes usually display moderate metabolic activity and proliferation under normal conditions; however, some articular chondrocytes lose their differentiated phenotype under diseased conditions and enter an endochondral ossification (EO)-like state of proliferation along with abnormal hypertrophic differentiation. Cellular senescence can also occur alongside hypertrophy due to similar stimuli. Cellular senescence and hypertrophy share various markers and processes, and both events are reported to play a role in the development of OA.

Chondrocyte hypertrophy and cell death are natural phenomena that usually occur during a developmental process called EO. Hypertrophic chondrocytes appear and play a crucial role in EO. Hyaline cartilage can be divided into two groups, (1) temporary and (2) permanent cartilage. Healthy cartilage is usually called permanent cartilage or resting chondrocytes, which are present in the articulating joint. Usually, permanent cartilage has a low proliferation rate and does not undergo terminal differentiation and EO. Temporary cartilage is initially formed as cartilage, but the final product is bone. Chondrocytes undergo active proliferation and generate a cascade of cells; whereas some of them undergo enlargement, others undergo hypertrophical changes and become hypertrophic chondrocytes. These cells increase their volume dramatically and the surroundings become mineralized to develop bone tissue.

Although various cell types are involved in OA pathology, chondrocytes are primarily thought to play a major role in OA induction by cellular senescence. When senescent cells were transplanted into the knee joint of wild type mice, an OA-like state was induced, which included pain, impaired mobility, and morphological and histological changes. The senescence-associated secretory phenotype of senescent cells can alter the tissue microenvironment and impair tissue regeneration induced by stem cells or progenitor cells, which can eventually lead to the senescence of the neighboring cells. Beside chondrocytes, synovial fibroblasts are also thought to initiate or progress OA through senescence. Nuclear expression of p16 was detected in higher amounts in OA synovial tissue samples when compared to that of normal synovial tissues, which indicates senescence in OA synovial fibroblasts.

The molecular mechanisms of OA initiation and progression require considerable further study, despite significant progress in recent years. OA is mainly caused by trauma induced by an external force or cartilage damage accumulated during aging. During these processes, chondrocyte hypertrophy and senescence are thought to play a critical role in OA initiation or progression. However, the remaining question is: which came first, the chicken or the egg? There is still little understanding of whether these two independent processes (i.e., chondrocyte hypertrophy and senescence) are dependent on penetration in the other. Further study on which event is the cause or the effect should be conducted to better understand these processes.

The Importance of Mitophagy in Aging

Mitochondria are bacterial-like cell components, hundreds of them in each cell working to create the adenosine triphosphate (ATP) energy store molecule used to power cellular processes. Mitochondria are dynamic structures, and constantly fuse together, split apart, and replicate like bacteria. Worn mitochondria are removed on a regular basis by the cellular quality control mechanism of mitophagy, a specialized form of autophagy. The survivors replicate to make up the losses.

With advancing age, mitochondrial dynamics shift to favor fusion over fission, producing larger structures that are resilient to mitophagy. The processes of mitophagy (and autophagy in general) are also thought to decline in efficiency for other reasons. This leads to the accumulation of malfunctioning, worn, broken mitochondria in cells throughout the body, and a consequent loss of cell function and tissue function. This is most likely an important component of degenerative aging, and thus restoration of mitophagy and mitochondrial function are important goals in the field of rejuvenation research.

Mitochondria are important for cellular life and death, implying that mitochondrial homeostasis must be tightly controlled and fine-tuned when cells respond to stress. Mitophagy is the primordial mechanisms for mitochondrial quality and quantity control and multiple mechanisms control this process. Some studies indicate an ample crosstalk between different mitophagy pathways that may coordinate and complement to deal with environmental challenges.

Exercise has long been known to promote healthy aging and decrease the susceptibility to age-related diseases probably, depending on the induction of autophagy. Mitophagy may also be involved in the beneficial effects of exercise. A recent study has shown that exercise activates the AMPK-ULK1 cascade to provoke the removal of damaged mitochondria via mitophagy. Caloric restriction is yet another way to extend healthy lifespan. Similar to exercise, nutrient deprivation activates the AMPK-ULK1 cascade that is required for mitophagy to remove damaged mitochondria and promote cellular survival.

Some compounds exert their lifespan extending effect via mitophagy. Thus, urolithin A extends lifespan and improves fitness during C. elegans aging and improves muscle function and exercise capacity in rodents. In-depth analysis demonstrates that mitophagy is required for the beneficial effect of urolithin A. Nicotinamide adenine dinucleotide (NAD) levels decrease with age, while the upregulation or replenishment of NAD metabolism has been shown to exhibit beneficial effects against aging and age-associated diseases. Treatments that increase intracellular NAD+ improve mitochondrial quality via mitophagy. Rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), prolongs life in yeast, worms, flies, and mice. Recent studies indicate that eliminating damaged mitochondria via mitophagy may be one of the mechanisms responsible for the beneficial effects of rapamycin.

In conclusion, dysfunction of mitochondria is one of the major characteristics of aging and age-related disease. Increasing evidence shows that mitophagy (by removing damaged mitochondria) is significantly involved in counterbalancing age-related pathological conditions. Thus, chronic stimulation of mitochondrial turnover by enhancing mitophagy is a promising approach to delay age-related diseases and to extend healthspan and lifespan.


An Update on Oisin Biotechnologies and OncoSenX

This short interview with Gary Hudson of Oisin Biotechnologies (and more recently covers some of the history and the present status of the company and its spinoff OncoSenX. Oisin Biotechnologies is one of the more ambitious senolytics companies working on means of destroying senescent cells in old tissues in order to produce rejuvenation. The company is using a programmable gene therapy approach rather than the small molecule development that the majority of other programs are undertaking.

"As we all know, the dilemma faced by any company that wishes to attack aging as a disease is that the FDA doesn't yet accept that premise, and requires biotechs to identify an indication - a specific disease - to be treated by any new investigational drug. While we can accommodate the FDA rules by picking a specific indication, the normal pressures of business will inevitably require that whatever indication is first chosen will dictate the path for the company into the far future. An aging-focused start-up will thus become a cancer or heart or kidney disease company, and lose its ability to attack the basis for most age-associated maladies. I wanted to avoid that trap at all costs."

This led to a model where Oisín is the platform company, providing the technology to use its unique DNA-plasmid and nanoparticle approach to kill cells based on their internal state. "Once the platform is proven scientifically and pre-clinically demonstrated, our plan was that Oisín would spin out specific indications or classes of indications, either via a conventional out-license to other biotechs or big pharmas, or as partially or wholly-owned subsidiary ventures. The oncology spinout, OncoSenX, is an example of this. In this way, Oisín controls the platform technology and manufactures the therapeutics to maintain quality control, while following the FDA-mandated path to clinical for new drugs. Additionally, since some investors will be more comfortable with conventional indication-focused ventures, this model opens up new avenues for funding, partnerships and collaborations with other companies, broadening the market appeal of our technology."

""We've raised over $8m to date for both Oisín and OncoSenX, and are currently in the process of raising new funding rounds. Oisín has a Series Seed round underway for up to $5m while OncoSenX is raising a Series A of up to $30m. Oisín's funding will be used for additional preclinical studies prior to an IND for an aging-related disease indication while OncoSenX's will be used for the Phase 1 and 2 oncology trials in humans." The company's next goal is to conduct a pre-clinical trial application meeting for OncoSenX to clear the path to a Phase 1 safety and efficacy trial against human solid tumors to be conducted in Canada within a year.


Nrf2, Excessive Autophagy in Skeletal Muscle, and Age-Related Sarcopenia

Autophagy is the name given to a collection of processes responsible for recycling damaged, harmful, or unwanted proteins and structures in cells. In general autophagy declines with age and this is a problem, allowing long-lived cells to accumulate damage and dsyfunction. There is considerable focus in the research community on ways to enhance autophagy, based on evidence that upregulation of autophagy occurs as a beneficial response to stress, improving health and lengthening life. Calorie restriction is the most studied example of this response.

Nothing is simple and universal in biochemistry, however. In muscle tissue, autophagy instead increases with age, to the point at which it becomes harmful to cell and tissue function. This may be one of the contributions to sarcopenia in older individuals, the progressive loss of muscle mass and strength - though the size of the effect in comparison to the many other contributing factors can be debated. In today's open access paper, the authors report on their investigation of the proximate causes of this excessive autophagy. They point to a loss of Nrf2 expression, which may or may not be a useful target for potential interventions.

Nrf2 deficiency promotes the increasing trend of autophagy during aging in skeletal muscle: a potential mechanism for the development of sarcopenia

Autophagy is an evolutionary conserved housekeeping cellular degradation and recycling process, whereby misfolded proteins and exhausted organelles are degraded to maintain cellular homeostasis. Skeletal muscle is the most abundant tissue in human body, accounting for about 40-55% of the body weight. Autophagy plays a key role in the regulation of muscle mass, either excessive or impaired autophagy leads to muscle mass wasting. Deficiency in the basic autophagy function causes accumulation of misfolded proteins and exhausted organelles and results in skeletal muscle cell dysfunction and death. On the contrary, excessive autophagy can also be deleterious by causing cellular stress and muscle protein degradation.

Muscle mass declines with increasing age, which is termed sarcopenia. Several animal models have showed that ablation of autophagy function results in precocious aging and muscle wasting in mice. However, it is still controversial about the changes of autophagy function in skeletal muscle with increasing age. Based on the evidence from lower organisms and non-muscle tissue, most literature held the concept that skeletal muslcle autophagy declines with aging. On the contrary, a recent study showed that autophagy and mitophagy in mice muscle were enhanced during aging, which may contribute to the decline in organelle contents and muscle mass, but serve to maintain a healthy organelle pool and muscle cells function.

Inconsistent results in the literature suggested that the measurement of autophagy-related proteins at the static level can often lead to discrepancies in interpretation, because autophagy is a dynamic process. Therefore, measurement of autophagy flux is necessary to reflect the real condition of autophagy within muscle cells during aging. Until a recent study, no previous studies have investigated the alterations of autophagy during aging in skeletal muscle using autophagy flux measurement. In contrast with most previous studies, our study showed that increasing age lead to a trend of increased autophagy in skeletal muscle, using autophagy flux measurements.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a cytoprotective gene which mainly functions to protect cells against oxidative stress and toxicants. In recent years, increasing evidence has revealed the role of Nrf2 in the regulation of autophagy. Our study showed that Nrf2 knockout decreased the levels of autophagy-related proteins, which was in consistent with previous studies. However, the explanation of our results was different from previous studies. Using autophagy flux measurements, we proved that Nrf2 knockout significantly increased autophagy in the muscle tissue. Therefore, we attributed the decreased levels of autophagy-related proteins in the Nrf2 knockout mice to the faster clearance of these proteins by autophagy.

Several previous studies have investigated the effect of aging on the expression of Nrf2 and its downstream cytoprotective genes in the skeletal muscle but the results were inconsistent. In recent years, increasing studies have recognized that expression of Nrf2 and its downstream genes in the skeletal muscle can be activated by physical exercise. Elderly humans who have a physically active lifestyle have an even higher expression level of Nrf2 and its downstream cytoprotective proteins compared with young subjects. This can partially explain the inconsistent previous studies regarding the effect of aging on the expression of Nrf2 and its downstream cytoprotective genes.

In conclusion, our study demonstrated that Nrf2 deficiency promoted the increasing trend of autophagy during aging in skeletal muscle. Nrf2 deficiency and increasing age may cause excessive autophagy in skeletal muscle, which can be a potential mechanism for the development of sarcopenia.

Indicators of Frailty Start to Appear Early, Probably Due to Lifestyle Choices

Clinicians classify frailty in a symptomatic way, looking at factors such as weight loss, weakness, walking speed, and so forth. This is a method of assessment designed for use with elderly people, but researchers here apply it to a study population that includes people in the 40-60 age range. They find that in this range, a fair number of individuals exhibit signs of what in the elderly would be called prefrailty - meaning just a few symptoms are present, rather than a majority. One logical possibility is that this is a manifestation of a sedentary, increasingly overweight population. Physical activity and good dietary choices (largely eating fewer calories) are required to minimize declines in capacity as time marches on. It requires greater neglect to be truly out of shape at age 40 as compared to age 60, but it is certainly possible to achieve.

This study reports on pre-frailty in 656 presumed healthy, independently living community-dwellers aged 40 to 75 years. We used an established frailty phenotype with two objective components (grip strength, walking speed) and three self-report measures (unintentional weight loss, physical activity, exhaustion). This phenotype was developed on people aged 65+ years and has been reported to sensitively identify pre-frailty and frailty states in this population. Our research indicates that using this frailty phenotype, pre-frailty is detectable in younger community dwellers aged 40-75 years. Moreover, neither age nor gender was significantly associated with any frailty state.

Our frailty rates (1.8% frail, 39% pre-frail, and 59.2% not frail) are comparable with those published recently from analysis of data from a large UK biobank, reporting on 493,737 people aged 37-73 years (3% frail, 38% pre-frail, and 59% not frail).

Setting unintentional weight loss aside (which requires medical investigation), our findings suggest that there are many people aged 40 years or older whose frailty status could potentially be addressed by increasing physical activity, building muscle, improving exercise tolerance, nutrition and mental health. It is reasonable to propose that chronic disease self-management and population health interventions to improve physical activity, such as workplace or community wellbeing programs, could significantly attenuate reverse or slow the onset of pre-frailty in community dwellers aged 40 years or more, and their subsequent risk of progression to frailty.


Cyclin D1 as a Potential Basis for an Exercise Mimetic

Researchers continue to delve into the mechanisms by which exercise produces benefits in older individuals, with an eye to producing exercise mimetic drugs. The study here is an example of the type, comparing some of the biochemistry of exercise in young, old, exercising, and sedentary mice. This part of the field is likely to evolve in much the same way as the development of calorie restriction mimetics over the past twenty years - slowly, in other words. Cellular metabolism is very complex, and picking out target mechanisms has a fair rate of failure.

Researchers gave mice that were about 20 months old, the equivalent of being 60-70 years old in humans, and mice that were 3 to 4 months old, the equivalent of 20- to 30-year-old humans, access to an exercise wheel and allowed them to run at will. Young mice averaged about 10 kilometers each night, and the older mice covered about 5 kilometers. Two other groups of young and old mice were given wheels that didn't rotate to serve as controls.

Subsequent analysis showed that the muscle stem cells of the exercising animals remained quiescent, and that the animals did not develop significant numbers of new muscle fibers in response to the exercise. After three weeks of nightly aerobics for the active groups, the researchers compared the ability of the animals to repair muscle damage. They found that, as expected, the aged, sedentary mice were significantly less able to repair muscle damage than younger sedentary mice. However, the older animals that had exercised regularly were significantly better at repairing muscle damage than were their counterparts that did not exercise. This exercise benefit was not observed in the younger animals.

The researchers also showed that injecting blood from an old mouse that had exercised into an old mouse that hadn't conferred a similar benefit in stem cell function, suggesting that exercise simulates the production of some factors that then circulate in the blood and enhance the function of older stem cells. Further studies indicated that the exercise-induced rejuvenation observed by the researchers could be mimicked by increasing the expression of a signaling molecule called cyclin D1, which is involved in rousing resting muscle stem cells in response to damage. The discovery suggests that it may one day be possible to artificially activate this pathway to keep aging muscle stem cells functioning at their youthful best.


Alzheimer's Therapies are Achieving Goals in Patient Biochemistry, But Not in Outcomes

If a therapy targets an important cause of a condition, and does so effectively, there should be little ambiguity in the results that emerge from clinical trials. The size of effect will be large, there will be no need to frown and interpret and try to find subgroups in the which the results are enough to declare some sort of success. Sadly, this latter position is much the state of the Alzheimer's clinical development community, still largely focused on immunotherapies targeting amyloid-β. Such an enormous amount of funding is devoted to these efforts that there is considerable incentive for the sponsoring entities to find some way to declare success, any success, at the end of the day. But are patients doing any better? Not really.

The trend in the field is towards an ability to measurably reduce aggregated amyloid-β and tau in the brain, as well as other biomarkers associated with neurodegeneration, but to see no benefit to cognitive function as an outcome. Why this is the case remains an open question, given the very robust evidence for amyloid-β and tau to be driving the progression of the condition. Or are they? Perhaps Alzheimer's disease is a self-sustaining inflammatory condition in which the immune cells of the brain have been driven into sufficient dysfunction that removing the trigger of aggregated amyloid-β and tau will do little. Or perhaps vascular dysfunction is a lot more important than has been thought, and success will be elusive until it is also addressed. It is with this in mind that one has to parse remarks on trial data.

Confused About the DIAN-TU Trial Data? Experts Discuss Nuances

At the virtual AAT-AD/PD Focus meeting, clinicians and funders involved in the Dominantly Inherited Alzheimer's Network Trials Unit (DIAN-TU) discussed results from the first DIAN-TU treatment and prevention trial of the monoclonal antibodies solanezumab and gantenerumab. DIAN-TU's principal investigator had presented topline data analyses of the primary outcome, which was head-scratchingly negative. He also presented the first analyses of several of the trial's biomarker measures, which were robustly positive. What does it all mean?

"It was gratifying to see all biomarkers that were presented moving toward normal. Brain amyloid down, cerebrospinal fluid (CSF) Aβ42 up, tau and p-tau down, and the neurofilament light protein (NfL) increase prevented. This shows the biology of the antibody is working. The whole field is witnessing that antibodies designed to remove amyloid do their jobs and are followed by other biomarkers going in the right direction. Why did this not translate into clinical benefit? Is there a threshold we need to hit? Do we need to go down to zero? Or is it dose exposure over time?"

In DIAN-TU, Gantenerumab Brings Down Tau. By a Lot. Open Extension Planned

Topline data suggest that the first two drug arms of the DIAN-TU trial platform - solanezumab and gantenerumab - were not a complete bust. Instead, the analyses finished to date point to nuanced effects of dose, time, disease stage, and biology. To be sure, the data did substantiate the earlier announcement that both therapeutic antibodies had fallen short on the trial's primary endpoint, the DIAN-TU multivariate cognitive endpoint. What happened? In short, symptomatic participants had descended into moderate dementia even before they could be titrated up to a high dose, whereas asymptomatic participants stayed stable throughout the trial regardless of whether they were on drug or placebo. This left the trial's main question unanswered.

For solanezumab, a monoclonal antibody targeting soluble amyloid-β, this indeed marks the end of its exploration within DIAN, the global research network for families with autosomal-dominant Alzheimer's disease. But all is not lost for gantenerumab, a monoclonal targeting aggregated forms of amyloid-β. Besides removing amyloid plaques from the brain and normalizing CSF Aβ42, this antibody reversed toward normal the elevated levels of CSF total tau and p-tau181, an AD-specific, pathological form of this neuronal protein. Gantenerumab further stemmed the rise of the general neurodegeneration marker CSF neurofilament light.

The effect sizes of this biomarker response were so large that they prompted the DIAN investigators to invite DIAN participants - who have devoted four to seven years of their lives to this trial, depending on when they enrolled - to join an open-label extension. It will explore high-dose gantenerumab therapy for several additional years. Its goal? To see if sustained gantenerumab therapy near the highest tolerated dose removes both plaques and tangles all the way down to a hypothesized, yet-to-be-defined threshold at which cognition and function might start to benefit.

Chronic Kidney Disease Accelerates Many Aspects of Aging, Such as Cardiovascular Disease Risk

Chronic kidney disease is an unpleasant condition. There is little that can be done for patients at the present time, though there is hope that senolytic drugs might be able to turn back the fibrosis characteristic of the condition. The kidneys are important to the correct function of tissues throughout the body, and consequently chronic kidney disease accelerates the degenerative aging of many other organs, including the cardiovascular system and brain. Finding ways to restore kidney function in older patients would be a big deal.

Chronic kidney disease (CKD) is a systemic pathology that affects approximately 10% of the population. The prevalence of CKD has increased markedly over the past decades due to aging of the population worldwide and increase in incidence of diabetes mellitus, which has become the primary cause of CKD. Nowadays, CKD is considered a public health problem that causes high rates of mortality in the population due to the association with cardiovascular diseases (CVDs). Multiple studies support the notion that patients with renal disease suffer accelerated aging, which precipitates the appearance of pathologies, including CVDs, usually associated with advanced age.

Considerable efforts have been made to slow the progression of the disease and improve the quality of life in patients with CKD. New pharmacological strategies do slow the progression of CVDs, and reduce the morbidity and mortality of CKD patients. Likewise, methods of renal replacement therapy currently offer increased purification capacity and reduced adverse effects. However, the development of CVDs in patients with CKD has not yet been halted. This may be because when CKD is diagnosed, vascular pathology is already advanced and irreversible.

The causes of vascular damage in CKD are exceptionally complex. Among the theories proposed in recent years to explain the high frequency of CVDs in renal patients, one states that senescence of peripheral blood cells (known as immunosenescence) and vascular cells (known as vascular senescence) may be involved in the initiation and perpetuation of vascular pathology that appears early in patients with CKD.

The aging process that occurs due to uremia is associated with numerous changes at the cellular and molecular level, which coincide with changes observed during the physiological aging process. These changes may explain some of the complications that typically occur in patients with CKD and CKD-associated CVDs. Expanding our understanding of the factors and molecules involved in accelerated senescence will serve to identify possible targets associated with this process. This will lead to improved methods of diagnosis and monitoring of these patients. Understanding the similarities between accelerated senescence and normal physiological aging will help establish new treatments.


Heart Attack Risk is Age-Related, but Aging Also Makes Treatments Less Effective

Aging makes everything worse. Its mechanisms of damage and consequence degrade tissue function to the point of catastrophic failure, as in a heart attack. That same damage also makes the immediate consequence of a heart attack worse, and reduces regenerative capacity and the ability to respond to therapies. All in all degenerative aging is an unpleasant business, lacking an upside. The right way forward is to periodically repair the damage before it reaches a pathological level, rather than working on ways to mitigate the consequences of a sizable burden of damage.

Aging elevates the susceptibility of the heart to ischemia and myocardial infarction (MI): over 50% of ischemic heart diseases occur in people older than 70s. Cardiac contractility declines suddenly in post-ischemia infarction, which can induce heart failure and even cause death. The EF and FS drop significantly in response to cardiac stress in advanced age, which is implicated by the progressive degeneration and reduction in cardiac myocytes. Beside changes in cellular level, a number of molecular alternation contribute to age related stress intolerance, including Ca2+ handling impairment, mitochondrial dysfunction, free radical accumulation, and alteration of myosin protein expression. Recently, the crucial role of epigenetic alteration in the cardiac aging process has attracted much attention.

The therapeutic effect of ischemic cardiac dysfunction varies in old patients. Patients older than 80 have worse survival rate than 70-year-old patients after cardiac ischemic therapy, but the survival rate for patients who undergo coronary artery bypass grafting is not affected by age. Early intervention for aging patients with ischemic heart disease will decrease the mortality rate. The difference in therapeutic effects between the aged and young groups also existed following cell therapies. Aged mice with cardiac injuries that underwent cardiosphere-derived cell (CDCs) transplantation showed no improvement in cardiac function, while cardiac function was improved in the young.

Furthermore, the results of cardiac regeneration therapy for aging people is still in a matter of debate. The effectiveness of stem cell therapies is always influenced by aging-related environmental changes. For example, ageing-induced low grade systematic inflammation cause poor survival rate of injected stem cells. Similarly, stem cell-derived exosome therapies are also largely limited by recipient cell senescence which causes a deteriorated cell proliferation ability and a weaken cardiac myocyte performance.


MItochondria in Age-Related Hearing Loss

In today's open access paper, researchers present evidence to suggest that the mitochondrial dysfunction that accompanies aging may be a meaningful cause of the loss of neurons that contributes age-related hearing loss, in the sense that it increases the incidence of necroptosis, a form of programmed cell death. Present thinking on the progressive deafness of old age is that the sensory hair cells of the inner ear largely remain intact, but their connection to the brain atrophies - the nerve cells in question dying in excessive numbers for reasons that continue to be explored.

Mitochondria are the power plants of the cell, producing chemical energy store molecules necessary for cellular processes to run, but they are also deeply involved in the various ways in which cells can undergo programmed cell death. With age, mitochondria become less efficient, they become larger the balance between fission and fusion changes, and the quality control mechanism of mitophagy, responsible for removing damaged mitochondria, falters. This contributes to most of the manifestations of aging in some way, and thus it is important for the research community to push ahead in the development of potential means of restoring mitochondrial function in older people.

Mitochondrial Damage and Necroptosis in Aging Cochlea

Approximately one in five people over the age of 50 has imperfect hearing, and almost half of those aged over 65 years have hearing difficulties. Presbycusis, also termed age-related hearing loss (ARHL), is an irreversible hearing impairment associated with aging due to limited repair capacity of sensorineural tissues in the cochlea. Unfortunately, there is no effective cure for the patients, and future treatment development is still questionable due to lack of mechanistic insight. In order to identify pathologic changes in the aged cochleae, we utilized C57BL/6J mice to investigate the pathophysiology of ARHL, as this strain displays accelerated, high-frequency hearing loss by 3-6 months of age and profound hearing impairment by 15 months of age.

Mitochondria are involved in the metabolic dysregulation associated with ARHL pathology. Morphologically, damage is apparent in the outer hair cells (OHCs) of animal ARHL models. Mitochondria are the principal source of reactive oxygen species (ROS), the production of which is closely associated with ARHL progression. Antioxidants alleviate the deleterious effects of ROS and effectively treat oxidative stress-related diseases in an animal model of ARHL. As mitochondria play key roles in both the respiratory chain and cell death, animal models of ARHL often exhibit defects in mitochondrial enzyme activities and mitochondrial-mediated apoptosis.

Neuronal cell death occurs through various pathways in sensorineural tissue, leading to hearing impairment. Necroptosis is a programmed cell death that exhibits necrosis-like morphological characteristics. The most defined molecular pathway of necroptosis is mediated by TNF-α receptor through receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) and the pseudokinase mixed-lineage kinase domain-like (MLKL).

We identified increased RIPK3 level in the aging cochlea, especially in the inner and outer hair cells and stria vascularis. Pronounced reduction in cytochrome c oxidase subunits 1 (COX1) and 4 (COX4), indicating that the mitochondria of the aging cochleae were dysfunctional, correlated with the degree of mitochondrial morphological damage. Hearing impairment found in aging animals was associated with a loss of sensory hair cells and neuronal filaments. Our data suggest that mitochondrial degeneration and necroptosis may play a critical role in the pathophysiology of ARHL and provide mechanistic insights for future therapeutic development.

Age-Related Changes in the Gut Microbiome and Alzheimer's Disease

Researchers here discuss potential links between (a) the detrimental changes that take place with age in the microbial populations of the gut and (b) the development of Alzheimer's disease. Given the present areas of interest in these fields of study, it seems likely that chronic inflammation is the primary point of overlap: changes in the gut microbiome promote inflammation, and inflammation drives the development of Alzheimer's.

One of the important factors that is influencing human health and attracting increasing attention of scientists during the last two decades is the gut microbiome. There are ~1,000 species and ~7,000 strains of bacteria that inhabit the human intestine, among which the most common are bacteria attributed to Firmicutes (51%) and Bacteroidetes (48%). However, over the last 15 years, the functions of the intestinal microbiome have been revised owing to the establishment of a direct link between density and species composition of the intestinal microbiome and a number of pathological conditions including diabetes, obesity, and cardiovascular diseases. These diseases, in turn, are the established risk factors for the development of Alzheimer's disease (AD), and there is data indicating that gut microbiome influences brain functions. Moreover, recent studies have revealed the significant differences in quantity and quality of gut microbiome in AD patients compared to mentally healthy individuals of the same age.

On the other hand, negative lifestyle aspects, among people living in our modern societies, are also considered important risk factors for the development of AD. The most striking result is that radical increases in Alzheimer's disease in Japan and substantial increase in developing countries are associated with changes in national diets. Furthermore, there are many undesirable lifestyle factors in the modern society that may contribute to AD development. These factors include unhealthy diet, lack of sleep, circadian rhythm disturbance, chronic noise, sedentary behavior etc., and, in turn, gut microbiome is highly sensitive to these factors. From this point of view, studying the links between modern lifestyle, gut microbiome and Alzheimer's disease is an important task that requires special attention.

Reducing the number and species diversity of many beneficial anaerobes such as Bifidobacterium and Lactobacillus, as well as a shift in the diversity of the intestinal microbiota toward pathogenic microorganisms, results in changes in local intestinal chemical and immunological parameters and induces the translocation of the gut bacteria into lymphoid tissue. These factors contribute to an increase in permeability of the intestinal barrier and blood-brain barrier and the penetration of pathological microflora and their metabolites into the brain.

On the other hand, intestinal bacteria are able to excrete functional amyloid peptides and lipopolysaccharides (LPS) in large quantities. Amyloid peptide in bacteria contributes to various physiological processes on the surface of bacterial cells, such as biofilm formation, adhesion, interaction with other bacterial and eukaryotic cells, etc. Its structure and biophysical properties are similar to human pathological amyloid. In addition to the amyloid peptide, many intestinal bacteria secrete LPS. LPS are the main components of the outer cell wall of gram-negative bacteria and, in the case of penetration from the intestinal cavity into the bloodstream, can cause neuroinflammatory reactions. Published data indicates that the LPS level in the blood plasma of patients suffering from AD is three times higher than the physiological age norm.


Potential Mechanisms to Restore Lost Function in Aged Hematopoietic Stem Cells

Hematopoietic stem cells are responsible for generating blood and immune cells. They are vital to the function of the immune system. With age their function alters in unfavorable ways, leading to the production of too great a proportion of myeloid cells versus lymphoid cells, but also declines in total. This is one of the contributing factors in the aging of the immune system, which is itself very influential of the progression of aging and age-related frailty. Thus potential ways to restore the hematopoietic stem cell population to a more youthful capacity to generate immune cells is an important part of the toolkit for human rejuvenation that lies somewhere ahead of us.

A key step in hematopoietic stem cell (HSC) aging research was achieved in 1996, revealing that HSCs from old mice were only one-quarter as efficient as those from young mice at homing to and engrafting the bone marrow (BM) of irradiated recipients. Aged HSCs are inferior to young HSCs and show incomplete reconstitution potential. This discovery established that the HSC aging process is accompanied by functional decline. Since then, differences between young and aged HSCs have been elucidated from multiple aspects, and the mechanisms of HSC aging have been gradually illustrated.

Different studies have explored the mechanisms by which aged HSC dysfunction occurs. Altered expression levels of multiple genes and mutation of some specific genes were shown to lead to HSC aging. In addition, inhibition of some signaling pathways, such as the mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways, was closely related to HSC aging. Furthermore, epigenetic perturbations also drove both cellular functional attenuation and other aging manifestations. Finally, some factors in the HSC niche, such as cytokines and enzymes, are also crucial during the aging process.

Currently, there is no doubt that HSCs show declining function during aging, but whether this dysfunction is reversible remains unclear. Notably, researchers showed that prolonged fasting can rejuvenate HSCs. Prolonged fasting reduces circulating IGF-1 levels and protein kinase A (PKA) activity in various cell populations and promotes stress resistance, self-renewal, and lineage-balanced regeneration. Further, HSC aging is accompanied by alterations in gene expression. Therefore, overexpressing knocking down the expression specific genes might be strategies to prevent HSC dysfunction. Reduced Satb1 expression was found in aged HSCs and associated with compromised lymphopoietic potential, and forced Satb1 overexpression partially restored that potential.

Aged mice exhibit increased mTOR signaling in HSCs, and mTOR inhibitor rapamycin can enhance the regenerative capacity of HSCs from aged mice, improve their immune response, and extend their life span. Cdc42 regulates diverse cellular functions, including cellular transformation, cell division, migration, enzyme activity, and cell polarity. Aged HSCs show elevated Cdc42 activity, and Cdc42 inhibition has been demonstrated to rejuvenate HSC functions. Further, inhibition of p38 MAPK reduces reactive oxygen species (ROS) levels and contributes to HSC rejuvenation. TN13, a cell-penetrating peptide-conjugated peptide, inhibited p38 activity and rejuvenated aged HSCs by reducing ROS.

One strategy to delay aging is to restore cell functions, while another is to clear senescent cells. Senescent cells accumulate with age and contribute to the development of aging-related diseases. Depletion of senescent cells mitigated irradiation-induced premature aging of the hematopoietic system and rejuvenated aged HSCs in normally aged mice.


DNA Damage During Cell Replication is Probably Not Important in Mammalian Aging

The size of the contribution of stochastic nuclear DNA damage to aging is debated. It causes cancer, when rare combinations of cancerous mutations occur and suppression of those early cancerous cells fails, but can it give rise to a meaningful degree of tissue dysfunction otherwise? The present consensus is that most such damage is irrelevant, occurring in cells that will not replicate further all that many times, and in genes that are not active. However, mutations in stem cells and progenitor cells can spread widely throughout tissue. Indeed, evidence shows that mice and humans exhibit a patterning of such distributed mutations. No robust evidence yet exists to pin down a size of effect of this spread of mutations on the progression of aging, however.

There are many ways in which DNA can become damaged, and cells possess highly efficient DNA repair mechanisms that quickly fix almost all issues. In today's open access paper, researchers show that the damage that occurs during replication of DNA does not have a significant influence on aging in mammals, despite the fact that it does appear to affect aging in short-lived lower species. The researchers engineered mice to improve repair of replicative DNA damage, but these mice did not live longer as a result. This is an interesting addition to the debate over the relevance of stochastic DNA damage to aging.

Supraphysiological protection from replication stress does not extend mammalian lifespan

In recent years, replication stress (RS) has been acknowledged as an important source of endogenous DNA damage. RS is a type of DNA damage that occurs when obstacles to replication lead to an accumulation of single stranded DNA (ssDNA) at stalled replication forks, which is recognized by ssDNA binding protein RPA. This initiates a signaling cascade involving Ataxia Telangiectasia and Rad3-related (ATR) kinase and CHK1 which promotes DNA repair, cell cycle arrest, and apoptosis.

Similar to other types of DNA damage, RS has been linked to aging. For instance, aged hematopoietic stem cells (HSCs) exhibit increased levels of RS compared to young HSCs. In addition, mutations in the ATR gene cause Seckel syndrome in humans, which is characterized by progeria, growth retardation, microcephaly, mental retardation, and dwarfism. The involvement of RS in premature aging has also been shown experimentally with a mouse model for Seckel syndrome. ATR-Seckel mice exhibit a phenotype similar to that of human patients, which is further aggravated in combination with several cancer-driving mutations such as the Myc oncogene or the absence of the tumor suppressor p53. ATR-Seckel mice show high levels of RS during embryonic development, accelerated aging in adult life and early lethality.

Interestingly, mice harbouring extra alleles of Chk1 (Chk1Tg) or of the ribonucleotide reductase (RNR) regulatory subunit Rrm2 (Rrm2Tg), which is a limiting factor for dNTP production, improved the lifespan and alleviated the progeroid phenotype of ATR mutant mice. These Chk1 and Rrm2 transgenic mice carry bacterial artificial chromosome (BAC) alleles of the respective genes, including exons and introns, under their own endogenous promoters. This strategy provides supraphysiological levels of CHK1 and RRM2 while preventing overexpression in tissues where these genes are normally not expressed, and was proven successful with the Trp53 BAC-transgenic mouse mode.

Collectively, these studies suggested that RS might have important implications in mammalian aging. However, the effect of Chk1 and Rrm2 expression levels on normal aging, in mice with physiological levels of ATR, remains to be elucidated. In the current study, we investigated the effect of supraphysiological levels of CHK1 and RRM2, which confer extra protection against RS, on normal aging. We utilized cohorts of wild type, Chk1Tg, Rrm2Tg and Chk1Tg;Rrm2Tg mice to assess tumor-free survival of these mice. We found no differences in survival between the genotypes and all mice exhibited similar signs of aging. Thus, supraphysiological levels of CHK1 and RRM2 do not affect normal aging in mice.

Degenerative Protein Modifications in the Aging of the Brain

Researchers here discuss the connection between a declining flow of oxygen to tissues and the level of modified proteins in tissue, particularly in the brain. Modifications tend to render proteins either harmful or at the very least useless for their intended task. This affects cell function and thus also tissue function. It is an open question as to the degree to which impaired clearance versus a faster pace of creation is the important issue, but there is evidence for both to be an issue when the supply of oxygen diminishes, either abruptly due to blood vessel rupture, or more gradually due to vascular aging (loss of capillary density, heart failure, and so forth).

Proteins are the building blocks of life as they are not only the structural constituents of the living organisms but also a final functional molecule governing most of the biological functions. The proteins undergo alterations by spontaneous non-enzymatic Degenerative Protein Modifications (DPMs) including oxidation, deamidation, carbamylation, carbonylation, glycation, etc. The DPMs change protein charge state, hydrophobicity, and three-dimensional structure that influence functional activities and induce aggregation.

These protein modifications and accumulation of modified proteins are allied to aging and the development of age-associated pathologies like neurodegenerative diseases. DPMs like spontaneous protein deamidation characterized by the modification of glutaminyl and asparaginyl residues were hypothesized as a molecular timer of biological events including protein turn over, development and aging. Protein deamidation progressively disrupts structural integrity of the protein and alter their biological activity. Other DPMs including glycation, advanced glycation end products, oxidation, carbonylation, carbamylation, etc., impart deleterious structural and functional changes in proteins and impair their normal function.

Hypoxia, a condition where oxygen supply to tissue is inadequate, induces free radical generation leading to oxidative protein modifications and tissue damage. Oxygen supply also acts as a modulator of aging processes. The cerebrovascular disorders and hypoxia-ischemia injuries in the brain are projected as a primary cause of protein pathologies that leads to cognitive impairment and dementia. In short, hypoxia-ischemia injury in the brain persuades DPMs that can lead to aging, age-associated diseases and neurodegeneration.


Using the CellAge Database to Find Genes Associated with Inhibition of Cellular Senescence

The CellAge database was announced last year, a repository of information on genes linked to cellular senescence. Cells become senescent in response to a variety of stresses, or upon reaching the Hayflick limit. A senescent cell ceases replication and secretes inflammatory and pro-growth signals. The process serves a useful function when such cells are present for a short time and then destroyed, aiding in suppression of cancer and in wound healing. When senescent cells linger, they cause chronic inflammation and significant disruption to tissue function, however. This is one of the contributing causes of aging, and selective removal of these cells via senolytic therapies will likely be the first form of rejuvenation therapy to see widespread use. Meanwhile, some research groups are instead looking for ways to inhibit entry into the senescent state, a task that starts by identifying relevant mechanisms that might be points of intervention.

Research has sought to ascertain the genetic program and prodrome underlying the development and phenotype of senescent cells. Expedited by recent advances in genomic and transcriptomic sequencing, alongside high-throughput genetic screens, a wealth of publicly available data now exists which has furthered the understanding of senescence regulation. Unfortunately, despite our increasing knowledge of cellular senescence (CS), determining whether a cell has senesced is not clear-cut.

Common senescence markers used to identify CS in vitro and in vivo include senescence-associated β-galactosidase (SA-β-gal) and p16INK4A (p16). However, β-galactosidase activity has been detected in other cell types such as macrophages, osteoclasts, and cells undergoing autophagy. Furthermore, some forms of senescence are not associated with p16 expression, while p16 has been detected in non-senescent cells. As such, there are now over 200 genes implicated in CS in humans alone.

Biological networks can be built using protein interaction and gene co-expression data. Here, we present the network of proteins and genes co-expressed with the CellAge senescence genes. Assaying the networks, we find links between senescence and immune system functions and find genes highly connected to CellAge genes under the assumption that a guilt-by-association approach will reveal genes with similar functions. In this study, we look at the broad context of CS genes - their association with aging and aging-related diseases, functional enrichment, evolutionary conservation, and topological parameters within biological networks - to further our understanding of the impact of CS in aging and diseases. Using our networks, we generate a list of potential novel CS regulators and experimentally validate 26 genes using siRNAs, identifying 13 new senescence inhibitors.


A Phenoptosis Perspective on the Evolution of Exceptional Human Longevity

The conclusion to today's open access paper opens with the following declaration: "There is still no agreement among gerontologists as to the main aging-related issue: whether it is an accidental accumulation of damage in the organism or a result of the operation of a specially evolved program." This is true in the sense that a minority of scientists (one in ten, perhaps - it is hard to count heads on this topic) consider aging to be programmed, a phenomenon that is under evolutionary selection, rather than an unselected side-effect of other selected traits.

The consensus views on the evolution of aging is that it is an outcome of antagonistic pleiotropy. Selection operates most strongly on factors leading to early life reproductive success, regardless of later consequences. Evolution thus produces outcomes such as (a) an adaptive immune system that cannot operate indefinitely because it must store information about every pathogen encountered, or (b) mammalian biochemistries that cannot effectively break down certain rare metabolic byproducts, and so this metabolic waste accumulates over a lifetime to cause late-life pathologies. In other words, systems and organs that function well at the outset, but accumulate damage and dysfunction and fall apart over time.

Programmed aging theories, on the other hand, are somewhat more varied. There are some, like the hyperfunction theory, focused on processes of development that do not stop and run wild with age, are hard to distinguish from antagonistic pleiotropy. Others, such as the concept that aging is a group selection outcome that exists because other options lead to ecosystem collapse due to excessive reproduction, are quite alien in comparison to the consensus. But the core idea is that aging is a selected process, not just an unfortunate side-effect of selection and the fact that early reproduction is always favored.

The authors of this paper are on the programmed aging side of the house, seeing aging as experienced by humans as simply a slower form of phenoptosis, the abrupt decline and death following mating that is observed in species such as salmon. They are also interested in oxidative stress in aging, being one of the groups that worked on mitochondrially targeted antioxidants capable of improving mitochondrial function and modestly slowing aging in short-lived laboratory species. Armed with that understanding, it is worth reading the paper for their take on on exceptional human longevity and why it evolved. Humans have much longer lives than other primates, and in some ways this appears to be an extension of childhood features into later life, a process called neoteny - though by no means clearly so. This increased life span may have been driven by our intelligence, and then our technology (in the broadest sense), as described by the Grandmother hypothesis. It is a selection effect that promotes longer survival of grandparents once they can assist in increasing the fitness of their descendants. But that isn't the only possible explanation.

Perspectives of Homo sapiens lifespan extension: focus on external or internal resources?

The nature of the selection factors underlying the evolution of aging remains controversial. Many specialists in evolutionary gerontology support a set of ideas called the "evolutionary theory of aging". This theory is based on the idea that the selection efficiency decreases with age. It is also assumed that vitality and fertility are high in youth at the cost of reduced fitness at later ages. An alternative view is that programmed aging and death may be favored by some kind of selection.

A theoretical experiment called the "Fable about Fox and Hares" has been suggested. Two young hares differing "intellectually" have equal chances to escape from a fox since both hares are running faster than a fox. However, with age, the clever hare acquires some advantage, which becomes of crucial importance when the running speed of hares lowers to that of a fox. Now, the clever hare has a better chance to escape and, hence, to produce clever leverets than the stupid hare. Such an effect becomes possible due to age-dependent lowering of the running speed as a result of the operation of an aging program. This will facilitate the selection for cleverness.

The evolutionary changes in humans compared to other primates have the following distinguishing characteristics: large brain, exceptionally large life span, high paternal investment in offspring, and the role of older individuals as helpers in upbringing the children. The large brain is associated with a change in psychological characteristics: enhanced learning and cognition. Even human sleep is shorter, deeper, and has more rapid eye movement phases than that in other primates. Supposedly, the selection pressure in the direction of the reduction in sleep duration and its "quality" improvement were activated in the early stages of human evolution due to the change in the ecological niche and the development of overnight stays on the ground and not in the tree branches.

The evolution of these life history characteristics and extremely high intelligence was probably related to some degree to the dietary transition to high-quality, solid and hard-to-get food resources. In humans, technical progress leads to a sharp decrease in infant mortality and an increase in life expectancy, especially in comparison to wild chimpanzees. Despite the huge variation in the life span of various human populations, starting with preagricultural tribes and ending with the urban population in the developed countries, the differences between their survival curves are still smaller than those between the preagricultural human populations and the chimpanzees living in the wild. This relationship can be explained by the fact that neoteny prolongs life span and health span.

The change in survival curves of humans compared to chimpanzees occurs for two reasons: neoteny and very rapid technical progress. An analysis of time scales and survival curves allows us to separate these two causes. Thus, the evolution of neoteny requiring much more time may be responsible for the difference in the mortality curves of chimpanzees and hunter-gatherers, while technical progress is responsible for the great differences in the mortality curves of hunter-gatherers and Swedish individuals in the 20th century.

Physical Activity as a Treatment for Age-Related Frailty

How much of the very prevalent manifestation of age-related frailty is due to the widespread lack of exercise in this era of comfort and sloth? Research suggests a sizable fraction, an answer that we might suspect to be the case simply because interventions such as strength training produce significant improvements in older patient populations. This is a personal choice for all of us: "use it or lose it" is a very real decision. Unpleasant consequences for health and well-being accompanying the worse of the two options.

Frailty, a consequence of the interaction of the aging process and certain chronic diseases, compromises functional outcomes in the elderly and substantially increases their risk for developing disabilities and other adverse outcomes. Frailty follows from the combination of several impaired physiological mechanisms affecting multiple organs and systems. And, though frailty and the age-related loss of muscle mass and strength known as sarcopenia are related, they are two different conditions. Thus, strategies to preserve or improve functional status should consider systemic function in addition to muscle conditioning.

Physical activity/exercise is considered one of the main strategies to counteract frailty-related physical impairment in the elderly. Exercise reduces age-related oxidative damage and chronic inflammation, increases autophagy, and improves mitochondrial function, myokine profile, insulin-like growth factor-1 (IGF-1) signaling pathway, and insulin sensitivity. Exercise interventions target resistance (strength and power), aerobic, balance, and flexibility work. Each type improves different aspects of physical functioning, though they could be combined according to need and prescribed as a multicomponent intervention. Therefore, exercise intervention programs should be prescribed based on an individual's physical functioning and adapted to the ensuing response.


Reporting on a Phase 1 Trial of a Drug to Suppress Inflammation in the Brain

Inflammation following injury in the brain causes much of the subsequent lasting damage. Further, chronic inflammation in the brain is an important aspect of the development of neurodegenerative disease. Judging from the direction of present research in the Alzheimer's community, it might be the most important mechanism driving these conditions. This may or may not be largely a matter of senescent cells in the brain; senolytic drugs have shown considerable promise in reversing pathology in animal models by destroying senescent supporting cells such as microglia and astrocytes. There are other ways in which the immune system can fall into a state of chronic inflammation beyond cellular senescence, however. Thus drugs that sabotage specific mechanisms of inflammation in the brain are under development at various stages; the ideal situation is not a blanket suppression, as inflammation is a necessary activity, but rather prevention of excessive or chronic inflammation.

Despite advances in our understanding of cellular and molecular neuroinflammatory mechanisms underlying adverse outcomes following injury, approved therapeutics that target this pathological process are lacking. Although there have been significant advances in the medical management of patients with acute brain injuries, there is a clear and urgent need for interventions that improve neurologic recovery and outcomes. To address this medical need, we developed a small-molecule drug candidate, MW01-6-189WH, hereafter called MW189. MW189 was developed as a selective suppressor of injury- and disease-induced glial proinflammatory cytokine overproduction associated with destructive glial inflammation/synaptic dysfunction cycles, and their long-term neurotoxic effects.

MW189 is efficacious in vivo in animal models of acute brain injury, in which upregulation of proinflammatory molecules is implicated in disease progression. By attenuating the inflammatory responses of overstimulated glia, MW189 may limit the pathological progression and neurocognitive dysfunction that complicate a variety of central nervous system disturbances.

We report first-in-human, randomized, double-blind, placebo-controlled phase 1 studies to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending intravenous doses of MW189 in healthy adult volunteers. MW189 was safe and well tolerated in single and multiple doses up to 0.25 mg/kg, with no clinically significant concerns. The most common drug-related treatment-emergent adverse event was infusion-site reactions, likely related to drug solution acidity. No clinically concerning changes were seen in vital signs, electrocardiograms, physical or neurological examinations, or safety laboratory results

A pilot pharmacodynamic study administering low-dose endotoxin to induce a systemic inflammatory response was done to evaluate the effects of a single intravenous dose of MW189 on plasma cytokine levels. MW189 treatment resulted in lower levels of the proinflammatory cytokine TNF-α and higher levels of the anti-inflammatory cytokine IL-10 compared with placebo treatment. The outcomes are consistent with the pharmacological mechanism of MW189. Overall, the safety profile, PK properties, and pharmacodynamic effect support further development of MW189 for patients with acute brain injury.


Transplanted Neurons Derived From Induced Pluripotent Stem Cells Restore Function Following Stroke in Rats

Like much of the nervous system, the brain doesn't regenerate well at all. Lost cells remain lost, and lost function is often permanent. One of the most important goals in the field of regenerative medicine is repair of the brain, which might be achieved in the decades ahead via delivery of new neurons that can integrate with existing neural circuits. Far from being a class of therapy only deployed following evident injury such as the aftermath of a stroke, this could take the form of periodic treatments that maintain the brain by repairing the lesser damage and loss of neurons that accumulates in an ongoing fashion over a lifetime.

As illustrated by the painful and usually partial recovery that can be achieved by some people following injury to the brain, the brain is capable of adaptation. Uninjured areas can take on new functions. This is why it is reasonable to expect therapies based on delivery of new neurons to allow restored function following injury. Indeed, it is demonstrated by researchers in this paper, in which human neurons derived from induced pluripotent stem cells integrate with the existing neural networks of a damaged rat brain to restore motor control and other capabilities. The researchers engineered a series of tests to prove that the human neurons were active and responsible for the restored functions in treated rats - this isn't just a matter of transplanted cells secreting signals that assist regeneration undertaken by native cells.

Researchers successfully repair stroke-damaged rat brains

Researchers have succeeded in restoring mobility and sensation of touch in stroke-afflicted rats by reprogramming human skin cells to become nerve cells, which were then transplanted into the rats' brains. Several previous studies have shown that it is possible to transplant nerve cells derived from human stem cells or from reprogrammed cells into brains of rats afflicted by stroke. However, it was not known whether the transplanted cells can form connections correctly in the rat brain in a way that restores normal movement and feeling.

"We have used tracking techniques, electron microscopy, and other methods, such as light to switch off activity in the transplanted cells, as a way to show that they really have connected correctly in the damaged nerve circuits. We have been able to see that the fibres from the transplanted cells have grown to the other side of the brain, the side where we did not transplant any cells, and created connections. No previous study has shown this. It is remarkable to find that it is actually possible to repair a stroke-damaged brain and recreate nerve connections that have been lost. The study kindles hope that in the future it could be possible to replace dead nerve cells with new healthy nerve cells also in stroke patients, even though there is a long way to go before achieving that."

Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior

Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell-derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell-derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. We find that at 6 months after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons.

We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side.

Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain's neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.

The Need for a Robust Measure of Biological Age

The research community is engaged in a the search for a reliable, agreed upon way to measure biological age, the burden of cell and tissue damage that causes dysfunction, disease, and death. Given such a reliable, cost-effective measure, potential rejuvenation therapies could be much more rapidly discovered, validated, and optimized than is presently the case. Researchers here review the present state of the art in this part of the field, and the challenges faced in trying to measure - or even rigorously define - biological age.

Putting together a conclusive theory of aging has been difficult due to the inability to properly quantify and define aging. Consequently, the efficacy of various geroprotective interventions remains subject to controversy. Without general agreement as to what constitutes aging and biological age (BA), and how to measure their progression, conclusions on the benefits of particular therapies are likely to be biased. Meanwhile, the very existence of a reliable way to measure BA remains under question.

It is possible that aging has no distinct genetic signature and is in essence a multitude of simultaneous damage accumulation processes. If that is true, BA as a concept is unlikely to be a property of objective reality but should be treated as an artificial construct. If there is indeed no singular process behind all the manifestations of aging, measuring BA is infinitely harder than in the case of single-source aging. It would require either (a) finding a common denominator for the majority of aging-related processes or (b) arbitrarily weighing all such processes according to their perceived importance to form the final "age score".

The problem of multiple aging processes is further confounded by individual variability. There are indications that people age according to different trends that can be grouped into several "ageotypes" defined by the hierarchical clustering of their biomarkers. Moreover, the fluidity of individual ageotypes can cause unstable performance of an aging score within any singular individual. Nonetheless, a reliable and universally agreed upon way to quantify BA is a necessity for modern biogerontology. Most importantly, it would enable flexible experimental designs and in many cases would remove the need to follow up human subjects for decades to evaluate the benefits of a geroprotective intervention. Moreover, it could be used as a criterion to test the relevance of specific diseases, pathways or processes in the context of aging research.


Reviewing the Influence of β-hydroxybutyrate on Metabolism and Age-Related Disease

β-hydroxybutyrate is a ketone body that is produced in greater amounts during exercise and calorie restriction. It has a range of beneficial effects that can influence the pace of aging, such as suppressing the pace at which cells become senescent. Senescent cells accumulate with age, and cause increasingly harmful tissue dysfunction and chronic inflammation. There is good supporting evidence for this and other mechanisms of interest related to β-hydroxybutyrate to exist, the question (as always) is the effect size. How much of the known and well calibrated benefits of exercise and calorie restriction are due to greater levels of β-hydroxybutyrate? Without an answer to that question, it doesn't do to get too excited about this sort of thing.

Accumulating data demonstrate that a ketogenic diet elevates the levels of β-hydroxybutyrate (β-HB), improving many age-related diseases. Indeed, β-HB appears to act as a regulator of cellular signaling via numerous pathways in various cellular organelles in a manner that is independent of nicotinamide adenine dinucleotide (NAD) levels. Reports have verified that β-HB controls many cellular signals via its function as a ligand, regulates gene expression, inhibits or activates protein functions, and plays a role in neuronal functions. Thus, identification of the molecular targets of β-HB will provide a better understanding of how calorie restriction or a ketogenic diet improves age-related disease symptoms. Stemming from the current evidence, investigation of the detailed molecular capacity of β-HB will provide new opportunities for its application as a therapeutic target for the treatment or prevention of human diseases.

Approaches to increase circulating β-HB by dietary manipulation or ingestion of supplements have been examined via four different routes: a ketogenic diet, calorie restriction, ketone ester (KE) administration, and sodium-glucose transport protein 2 (SGLT2) inhibition. The ketogenic diet, calorie restriction, and SGLT2 inhibition induce ketogenesis in the liver through lipolysis. In particular, SGLT2 inhibition decreases insulin secretion from β cells, resulting in lipolysis in adipose tissues, regulation of ketone body reabsorption in the kidney, and increased β-HB. KEs are hydrolyzed by nonspecific gut esterases in the small intestine and liberate β-HB and (R)-1,3-butanediol, thus increasing the level of β-HB in the circulation.

β-HB supplementation extends the lifespan of C. elegans by 20% through the DAF-16/FOXO and SKN-1/Nrf pathways and the regulation of aging and longevity. In mammals, β-HB decreases the senescence-associated secretory phenotype (SASP) and the senescence of vascular cells. Moreover, the ketogenic diet significantly extended the median lifespan of mice and resulted in the preservation of the physical function of aged mice. Thus, β-HB and ketogenic diets can be considered important mediators with regenerative potential that also have the capacity to retard aging-associated phenotypes.

Since individual differences make it difficult to control the optimal circulating β-HB levels by calorie restriction or a ketogenic diet, it is necessary to develop adjustable treatment options, such as KE administration. As abrupt changes in circulating β-HB may disrupt energy homeostasis, the chiral enantiomer s-β-HB may offer a potential option for therapeutics, as this molecule cannot be used as an alternative energy metabolite. Furthermore, s-β-HB is not consumed by the physiological system, and the half-life of s-β-HB in circulation is longer than that of β-HB. As β-HB alleviates various age-associated disease symptoms and aging phenotypes via diverse and yet unknown molecular mechanisms, evaluation of β-HB and/or s-β-HB as a therapeutic agent is an important approach for the treatment of the aging population.


Persistent CMV Infection Provokes Greater Senescent Cell Accumulation

With the newfound acceptance of senescent cell accumulation as an important contributing cause of aging, a viewpoint that has really only flourished in the research community over the last five years or so, many fields of research relevant to aging are retrofitting senescent cells into their theories and understanding of the aging process. Today's open access paper is an example of this process. Researchers interested in the role of persistent cytomegalovirus (CMV) infection in the aging, a field that has itself seen a surge of interest over the past decade, link it to cellular senescence and the growth of chronic inflammation that occurs in old age.

CMV is a type of herpesvirus that is very prevalent in the population; near everyone is exposed to it at some point in time. There is good epidemiological evidence associating CMV exposure to worse health in later life. CMV provokes the adaptive immune system into specializing ever more resources to tackling it, a futile effort as it cannot be cleared from the body. It hides, latent, to emerge again. The thymus, where T cells of the adaptive immune system mature, atrophies with age, and the supply of new T cells diminishes. Without reinforcements, this continual specialization to CMV depletes the immune system of cells capable of handling other tasks.

One of those tasks is the destruction of senescent cells, rapidly enough to prevent their inflammatory secretions from disrupting tissue maintenance and organ function. The pace of clearance of senescent cells declines with age, and this is one of the contributing factors leading to an increased number of such errant cells in old tissues. Further, the pressure that CMV puts on the immune system produces other more direct issues, such as forcing a greater replication of immune cells that drives them into senescence faster than would otherwise be the case. Senescent immune cells are just as problematic as senescent cells in tissues.

The Immune Response Against Human Cytomegalovirus Links Cellular to Systemic Senescence

Clearance of senescent cells is an important role of the adaptive immune system in relation to healthy aging. This regulatory role can be compromised by aging itself, HIV infection, or many other immune stressors that weaken immune function and/or promote immunosenescence. The deficits in adaptive immunity observed in HIV/CMV co-infection, accompanied by an increased prevalence of age-associated pathologies termed accelerated aging, together comprise a unique setting within which to study senescent cell accumulation and its physiological impact.

Immunological stress, as imposed by chronic infection, chronic inflammation, or requirements for homeostatic proliferation, can promote progression of lymphocytes to cellular senescence and impose immune deficits that impair clearance of senescent cells from tissues, thus, compounding the accumulation of senescent cells and the negative implications of their senescence-associated secretory phenotype (SASP). The homing of abundant circulating CMV-specific CD8+ T cells to such inflamed tissues could fuel a further feedback loop of activation, proliferation, and telomere attrition. Therefore, not only are dysfunctional immune cells indirectly contributing to senescent cell accumulation, but are themselves progressing towards senescence, reinforcing a vicious cycle that may be a key factor in "premature aging" of persons living with HIV and unhealthy aging in others.

If CMV infection is a major factor in shifting the adaptive immune system towards chronic senescence, the most dramatic effects should be revealed in the context of HIV infection, where increased chronic inflammation and exaggerated anti-CMV immune responses may optimally promote progression of age-independent chronic immunosenescence. Within this setting, CMV infection is associated with CD8+ T-cell progression towards cellular senescence, increased inflammation, and greater risk of age-related morbidities. This may represent acceleration of the same effects that CMV infection produces in old elderly individuals who develop an immune risk profile with its connotations for immunosenescence, unhealthy aging and mortality. Extensive proliferation and oligoclonal dominance of CMV-specific CD8+ T cells are the hallmarks and potentially drivers of these associations.

Mitochondrial DNA Damage in the Context of Atherosclerosis

Mitochondria swarm by the hundreds in every cell, acting much like power plants by generating chemical energy store molecules (adenosine triphosphate, ATP) to power cellular operations. The progressive loss of mitochondrial ATP production that occurs with age is harmful to cell and tissue function. One way in which mitochondrial dysfunction leads to tissue damage is through stochastic damage to mitochondrial DNA. Some forms of damage, such as large deletions, can change mitochondrial function in ways that allow them to both malfunction and outcompete their functional peers. A cell becomes overtaken by broken mitochondria, and pollutes the surrounding tissue with damaging, reactive molecules. These can cause cholesterol and other lipids to become oxidized, and this contributes to the development of atherosclerosis, as oxidized lipids cause macrophages to become dysfunctional and falter in their task of keeping blood vessel walls free from fatty lesions. This may not be the only relevant mechanism, however.

The link between mitochondrial dysfunction and atherosclerosis has been the subject of extensive research. A model that could link the mitochondrial DNA (mtDNA) mutation-induced mitochondrial dysfunction and deficient autophagy may help understanding of the importance of these events in the age-related nature of inflammation and identifying potential points of therapeutic intervention. Based on the available data, we propose a plausible mechanism, according to which, phagocytosis stimulation by circulating large associates of modified LDL activate the pro-inflammatory response of the innate immune system.

According to this hypothesis, atherogenic modified LDL circulating in the blood of atherosclerotic patients induces lipid accumulation in the arterial wall cells. Modified LDL particles form self-associates that penetrate the cell by nonspecific phagocytosis, stimulation of which by LDL associates activates the pro-inflammatory response of macrophages in the form of secretion of inflammatory cytokines. Secretion of cytokines leads to increased accumulation of intracellular lipids. If the innate immunity functions normally, the pro-inflammatory reaction resolves rather quickly and further lipid accumulation does not occur. However, when macrophages contain mtDNA mutations, the pro-inflammatory response does not arrest, but rather intensifies with each repeated pro-inflammatory stimulation. Local inflammation in the vascular wall becomes chronic and accompanied by uncontrolled lipid accumulation giving rise to an atherosclerotic lesion.

Another intriguing possibility is that cells may recognize the dysfunctional mitochondrion as a pathogen that presents foreign epitopes, therefore triggering the immune response. This may be a consequence of the bacterial origin of mitochondria, due to which defective mitochondria could be recognized by immune cells as pathogens triggering the innate immunity response.

The proposed concept allows speculation that atherogenesis is due to two errors made by the cell of the arterial wall. The first one is that the cell perceives the associates of modified LDL as a pathogen that is taken up by phagocytosis, which causes an inflammatory response and the accumulation of intracellular lipids, which in turn is a trigger of atherogenesis at the cellular level. The second is that due to mutations, the mitochondria becomes dysfunctional and due to a defect in mitophagy, the cell cannot free itself from this mitochondrion and perceives it as a bacterium-like pathogen. This triggers an ongoing inflammatory signaling that may lead to inflammasome activation.


Blood Metabolites as a Marker of Frailty

Frailty in older people is usually diagnosed in a symptomatic way, by assessment of physical weakness. The condition has other components, however, such as chronic inflammation, cognitive decline, greater immune dysfunction, and so forth. Researchers here produce a biomarker for frailty based on a selection of metabolites in blood. This is a step towards a more rigorous class of test that might be able to pick out those in the earlier stages of frailty who are more likely to progress absent some form of intervention, such as strength training or therapies like senolytics that will reduce the burden of inflammation.

Researchers looked at 19 elderly patients, all above the age of 75, and measured whether they suffered from frailty through three clinical analysis tests - the Edmonton frail scale (EFS), the Montreal cognition assessment (MoCA-J), and the Timed Up and Go Test (TUG). "Both the EFS and the MoCA-J gave us an indication of the individuals cognitive function, whereas the TUG allowed us to assess their motor ability. Between them, they also showed health status, mood, short-term memory, and other indications, so they gave us a clear idea of who suffered from the disorder." By using these three tests, the researchers found that nine out of the 19 individuals fit into the category of being frail whereas the other ten did not, however some still did suffer from cognitive impairment or hypomobility, a syndrome which hinders movement.

Next, the researchers took blood samples from the 19 patients and had a close look at the metabolites - small molecules of amino acids, sugars, nucleotides, and more that make up our blood. They tested 131 metabolites and found that 22 of them correlated with frailty, cognitive impairment and hypomobility. Patients who suffered from these disorders tended to have lower levels of most of these metabolites. The 22 metabolites identified included antioxidant metabolites, amino acids and muscle or nitrogen related metabolites. Fifteen of them correlated with frailty, six indicated cognitive impairment and twelve indicated hypomobility. The metabolites that correlated with frailty overlapped with five of those that indicated cognitive impairment and six that indicated hypomobility.

These metabolites include some of the aging markers in healthy people reported by the same group in 2016. This suggests that the severity of biological aging, which varies between individuals, could be monitored from an early stage of old age by measuring blood biomarkers. The research indicates that frailty has a distinct metabolomic profile when compared to other age-related disorders. By demonstrating a link between these metabolites and the symptoms of the disorder, these findings could lead to a different approach to diagnosing and treating frailty.


Cellular Senescence in Diseases of the Eye

Cellular senescence contributes to many age-related diseases. Senescent cells arise naturally as a result of the Hayflick limit on cellular replication, as well as injury, or due to molecular damage or a toxic environment that might give rise to cancer. A senescent cell ceases replication and secretes a potent mix of signals that produce inflammation and disrupt nearby tissue structure and function. In youth, senescent cells are near all quickly removed, via programmed cell death or the actions of the immune system, but these removal mechanisms falter with age. Senescent cells accumulate as a result, and the more of them there are, the worse the outcome. These errant cells are thought to be responsible for a sizable fraction of the chronic inflammation of aging, for example, and produce many other ill effects besides.

While good evidence has existed for decades to point to senescent cells as an important cause of aging, the research community at large has only gradually accepted this hypothesis over the last decade. Thus the contribution of senescence to age-related disease is only well studied in a handful of the hundreds of varied age-related diseases. This is very much the case for the eye. There is some recent evidence for senescence to be involved in cataracts and glaucoma, but for any number of other conditions the role of senescence remains to be investigated in depth.

This situation is repeated throughout the body. Since the first senolytic therapies capable of selectively destroying a meaningful fraction of senescent cells already exist, it seems likely that advances in knowledge will be driven by trying the treatments and watching the results, rather than by more passive investigation. This is probably for the best, and certainly much faster if the goal is rapid progress towards effective treatments that can turn back age-related conditions by addressing deeper causes.

The Emerging Role of Senescence in Ocular Disease

Cellular senescence is a state of irreversible cell cycle arrest in response to an array of cellular stresses. An important role for senescence has been shown for a number of pathophysiological conditions that include cardiovascular disease, pulmonary fibrosis, and diseases of the skin. As a central mechanism, senescent cells can impact the surrounding tissue microenvironment via the secretion of a pool of bioactive molecules, termed the senescence-associated secretory phenotype (SASP). However, whether senescence contributes to the progression of age-related macular degeneration (AMD) has not been studied in detail so far.

Acute senescence is mostly beneficial and presumably does not contribute to aging; it relies on the coordinated action of senescent cell production and subsequent elimination - the processes involved in wound healing, tissue remodeling, and embryogenesis. Paradoxically, while chronic senescence can initially have beneficial effects, its long-term existence could potentially aggravate age-related diseases. "Chronic" senescence develops gradually because of progressive damage over time as seen in aging and age-related diseases. During chronic senescence, the switch from temporal to persistent cell cycle arrest appears to be random, induced by the multiple inducing factors acting simultaneously on a cell. These results in arrest of proliferation and ultimately cells become dysfunctional and most importantly negatively affect local environment.

Aging is considered one of the most obvious predisposing factors for the development of AMD because prevalence of this disease rises in those over 60. With aging, the human retina undergoes various structural and physiologic changes. Several independent studies suggest senescence contributes to the development of many ocular diseases. Aging has been associated with fewer retinal neurons along with numerous age-related quantitative alterations such as decreased areas of dendritic and axonal arbors and decreased density of cells and synapses. One study found that retinal pigment epithelium (RPE) cells were lost in large numbers in the periphery of the human retina while a second study reported overall RPE to photoreceptor ratio dropped with age throughout the retina. Furthermore, protein levels of canonical senescence markers such as p16, p21, and p53 were shown to increase in the RPE isolated from aged human donors.

Retinal microaneurysms overexpress canonical senescence markers, suggesting that cellular senescence is associated with the pathogenesis. Apoptosis also cooccurs with cellular senescence in old-age retinal microaneurysms. The age-related decrease in the anterior segment outflow is largely responsible for the elevated intraocular pressure, one of the factors attributing to the development of glaucoma. Markers of cellular senescence are found in the trabecular meshwork of patients with primary open-angle glaucoma and aging of these cells leads to their decreased function and a consequent decreased outflow facility.

Beyond loss of retinal cells, aging is also associated with the accumulation of both intracellular and extracellular deposits. The finding that amyloid-β (Aβ) is also elevated in aging retina and is a component of drusen suggests that Aβ may be a key factor in AMD pathology. Aβ has been recently shown to induce RPE cells to enter senescence. A recent study shows the role of RPE senescence in the retinal degeneration induced by Aβ peptide as characterized by upregulation of senescence markers. Hence, cellular senescence of RPE or neuronal cells induce different age-related retinal diseases and targeting them could be a viable therapeutic strategy.

Elevated Brain Amyloid-β Levels Correlate with Worse Cognitive Performance in Clinically Normal Old People

It seems reasonable to believe, based on the evidence, that amyloid-β aggregation is associated with the onset of Alzheimer's disease, but the question has always been whether it was a suitable target to reverse the condition. The failure of reductions in brain amyloid-β via immunotherapy to produce meaningful clinical success has brought other views of the condition to the forefront. For example, raised amyloid-β may be a side-effect of persistent infections that produce chronic inflammation, and it is the inflammation that is important. Or amyloid-β may provoke sufficient inflammation and cellular senescence in supporting cells of the brain for it to become self-sustaining as the core of the condition, even once the amyloid is removed.

A logical next step at the present time would be to test senolytic therapies that can pass the blood-brain barrier, as this should both clear senescent cells and reduce the chronic inflammation in the brain that results from senescent cell signaling. If this produces results in humans that are as promising as those in mice, that might be a good indication that the primary driving mechanism of Alzheimer's disease (and perhaps many other neurodegenerative conditions) is chronic inflammation.

The Anti-Amyloid Treatment in Asymptomatic Alzheimer disease (A4) Study is an ongoing prevention trial in clinically normal older individuals with evidence of elevated brain amyloid. The large number of participants screened with amyloid positron emission tomography (PET) and standardized assessments provides an unprecedented opportunity to evaluate factors associated with elevated brain amyloid.

This cross-sectional study included screening data in the A4 Study collected from April 2014 to December 2017 and classified by amyloid status. Data were was analyzed from 2018 to 2019 across 67 sites in the US, Canada, Australia, and Japan and included 4486 older individuals (age 65-85 years) who were eligible for amyloid PET, clinically normal, and cognitively unimpaired.

Amyloid PET results were acquired for 4486 participants (71.29 ± 4.67 years; 2647 women), with 1323 (29.5%) classified as amyloid-β (Aβ)+. Aβ+ participants were slightly older than Aβ-, with no observed differences in sex, education, marital or retirement status, or any self-reported lifestyle factors. Aβ+ participants were more likely to have a family history of dementia (3320 Aβ+ [74%] vs 3050 Aβ- [68%]) and at least 1 APOE ε4 allele (2602 Aβ+ [58%] vs 1122 Aβ- [25%]). Aβ+ participants demonstrated worse performance on screening Preclinical Alzheimer Cognitive Composite results and reported higher change scores on the Cognitive Function Index.

In conclusion, elevated brain amyloid was associated with family history and APOE ε4 allele but not with multiple other previously reported risk factors for AD. Elevated amyloid was associated with lower test performance results and increased reports of subtle recent declines in daily cognitive function. These results support the hypothesis that elevated amyloid represents an early stage in the Alzheimer's continuum.


Many People Aged 40 to 50 Exhibit Rapid Progression of Preclinical Atherosclerosis

Researchers here show that many people in their 40s have measurable signs of preclinical atherosclerosis, the early stages of the development of fatty lesions that narrow and weaken blood vessels. The data shows that these early lesions also progress more rapidly than was expected at this time of life. In its later stages, atherosclerosis results in stroke or heart attack as important vessels rupture or are blocked by debris from a fragmented lesion. At present there is little that can be done to meaningfully reverse existing lesions: lowering blood cholesterol levels only slows progression somewhat. Despite considerable interest in the research community in achieving reversal of established lesions, there has been little practical progress towards viable therapies in recent decades.

The PESA ('Progression of early subclinical atherosclerosis') study has been monitoring 4200 healthy middle-aged men and women with noninvasive imaging technology and omics biomarkers for more than 10 years. The use of noninvasive imaging technologies "allows us to identify the progression of the disease earlier than is possible with classical markers, such as the presence of coronary calcium detected by computed tomography (CT), thus allowing us to identify individuals at higher risk who could benefit from early intervention. The results show that ultrasound of the peripheral arteries is a more efficient method for detecting atherosclerosis progression than the study of coronary calcium by CT."

Atherosclerosis is characterized by the accumulation of fatty deposits in the artery walls. The disease is normally detected at an advanced stage, when it has already caused clinical events such as a heart attack or stroke. Treatment of the disease at this symptomatic stage is of limited effectiveness, and most patients experience a decline in quality of life. The treatment of these patients, moreover, places a significant burden on health care resources.

"This study is the first to analyze the progression of atherosclerosis at frequent intervals. The previous view was that the disease progressed very slowly throughout life. However, the new results show that the disease progressed very rapidly in 40% of the individuals analyzed. Future data from the PESA study will show whether this progression is associated with subsequent cardiovascular events. Until now, the speed of atherosclerosis progression has not been a factor in assessing individual risk."

"The key finding of the study is that over a short follow-up of just 3 years, 40% of individuals aged between 40 and 50 years showed major progression of atherosclerosis in distinct locations, including the carotid, femoral, and coronary arteries. This rapid disease progression could make these individuals more vulnerable to developing symptoms or having clinical events such as a heart attack or stroke." The researchers conclude that the findings, while they await validation from the occurrence of events in the PESA cohort in the future, will be of great value for the identification of strategies to stall the epidemic of cardiovascular disease.


A TAT Peptide Based Approach to Upregulation of Proteasomal Activity

The proteasome is a construct in cells that shreds damaged, misfolded, or unwanted proteins, reducing them to component parts that can be reused. It is a part of the ubuiquitin-proteasome system: molecules to be destroyed are tagged with ubiquitin, and drawn into a proteasome for recycling. Greater proteasome activity is thought to be a good thing, improving cell function. This is of particular relevance to aging, as proteasomal function declines with age, contributing to faltering cell and tissue function, particularly in the long-lived cells of the nervous system.

While established drugs exist to inhibit activity of the proteasome, useful in cancer therapies in which cell death is a goal, improving proteasomal activity is less well explored. The one approach shown to work well to date is to increase expression of some of the individual rate-limiting proteins that make up proteasomal structure. This has been shown to extend life in short-lived laboratory species, an enhancement therapy that partially compensates for the progressive loss of proteasomal function with age. Researchers here outline their discovery of a different methodology, one that may be more amenable to the production of a drug capable of upregulating proteasome function.

New Peptide-Based Pharmacophore Activates 20S Proteasome

As the central protease of the ubiquitin-proteasome pathway, the proteasome has long been considered an attractive target for drugs potentially affecting multiple aspects of cell physiology. Indeed, small molecules targeting the proteasome have entered the clinic with great success. However, their scope at present is very limited: all proteasome-modifying compounds currently approved or clinically tested as drugs are competitive inhibitors and all are used to treat advanced blood cancers. Here we turn to the opposite side of pharmacological intervention into the proteasome: augmentation of catalytic activity. Since dysfunction of proteasome-mediated controlled protein degradation is a hallmark of both cellular aging and neurodegenerative diseases, enhancement of the enzyme's activity should be considered an attractive therapeutic option.

The complex structure of the catalytic core 20S proteasome (the "core particle") presents fascinating options for allostery-based augmentation. The peptidase responsible for post-hydrophobic (chymotrypsin-like, ChT-L) cleavages is considered a rate-limiting "workhorse" and is the major target for inhibitors and activators alike. Indeed, overexpression of a catalytic subunit harboring the active site of the ChT-L peptidase has been shown not only to extend lifespan but also to reduce age-related cognitive decline in animal models. However, reports on pharmacological augmentation are limited to in vitro and cell culture studies.

Here, we describe a series of short, modified peptides based on the basic domain of the viral Human Immunodeficiency Virus-1 (HIV-1) Tat protein. Among many intracellular effects, the HIV-1 Tat protein inhibits the core proteasome. In our previous studies, we noted that short peptide fragments of HIV-1 Tat displayed peculiar in vitro properties: they inhibited detergent-treated core particle but mildly activated the latent core. However, treatment with detergent, although convenient for in vitro assays, yields mildly denatured proteasome and, under these far-from-physiological conditions, likely with destroyed natural allosteric routes.

Therefore, we turned our attention to the activating properties of HIV-1 Tat protein-derived "TAT peptides". After observing a strong in vitro proteasome augmentation by modified HIV-1 Tat-derived peptides, we tested selected compounds in cell culture. In a separate study, we found that proteasome stimulation by TAT peptides partially prevented cognitive deficits and mortality in animal models of Alzheimer's disease. The very encouraging results included increased proteasome-mediated turnover of amyloid precursor protein (APP) and β-secretase (which cleaves APP to generate β-amyloid peptide), concomitant with lowered levels of β-amyloid, lowered mortality and protection against cognitive decline. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

Soluable α-klotho Reduces Cardiac Fibrosis in Mice

Klotho is a longevity-related gene. In mice, greater expression extends life while reduced expression shortens life. Most study has focused on beneficial effects on cognition and kidney function, with some debate over where in the body it acts - it may be that benefits to brain function are entirely the consequence of improved kidney function. That isn't all that klotho does, however. The delivery of soluble α-klotho has emerged as a basis for building therapies based on klotho biochemistry, and Unity Biotechnologies is one of the groups working on clinical development of this line of work. Here, researchers show that the scarring of fibrosis in heart tissue, a feature of aging, can be reduced via delivery of α-klotho. The same questions apply here as for the brain; is this a local mechanism of action, or something mediated by effects in another organ and systemic signaling throughout the body?

Heart disease is the leading cause of death worldwide. The major cause of heart failure is the death of the myocardium caused by myocardial infarction, detrimental cardiac remodeling, and cardiac fibrosis occurring after the injury. This study aimed at discovering the role of the anti-aging protein α-klotho (KL), which is the co-receptor of fibroblast growth factor-23 (FGF23), in cardiac regeneration, fibrosis, and repair.

FGF23 is the most recently discovered fibroblast growth factor and functions as an endocrine hormone that regulates phosphate homeostasis through binding to FGFR and KL, its coreceptor in the kidney and parathyroid glands. Elevated levels of circulating FGF23 have been associated with left ventricular hypertrophy, and it has been suggested that FGF23 exerts a direct effect on the myocardium. Interestingly, the co-receptor of FGF23, KL, has been shown to exhibit renal protective functions independent of FGF23/FGFR signaling.

We found that the anti-apoptotic function of soluble KL in isoproterenol-treated cardiomyocytes was independent of FGF23 in vitro. In vivo, isoproterenol-induced cardiac fibrosis and cardiomyocyte and endothelial cell apoptosis were reduced by KL treatment. Moreover, the number of Ki67-positive endothelial cells and microvessel density within the isoproterenol-injured myocardium were increased upon KL treatment. However, by using genetic fate-mapping models, no evident cardiomyocyte proliferation within the injured myocardium was detected with or without KL treatment. Collectively, the cardioprotective functions of KL could be predominantly attributed to its anti-apoptotic and pro-survival activities on endothelial cells and cardiomyocytes. KL could be a potential cardioprotective therapeutic agent with anti-apoptotic and pro-survival activities on cardiomyocytes and endothelial cells.


Transcriptomic Analysis of Microglia in Mice Shows Greater Inflammatory Activity with Advancing Age

Microglia are innate immune cells of the brain, akin to macrophages elsewhere in the body, but equipped to undertake an additional set of tasks relating to neural function. A range of evidence strongly suggests that the progression of neurodegenerative conditions is strongly driven by greater inflammatory activity in the microglia of older individuals. This is perhaps largely due to cellular senescence, perhaps largely due to greater adoption of the aggressive M1 phenotype. Underlying causes include greater leakage of the blood-brain barrier due to the molecular damage of aging, allowing unwanted compounds and cells into the brain that will rouse an inflammatory response.

Accordingly, there is greater interest nowadays in strategies that might reduce inflammation in the brain, whether senolytic drugs targeting senescent cells, small molecules that might force microglia into the more helpful M2 phenotype, or other approaches to selectively sabotaging mechanisms of the immune response. Repair of underlying damage beyond cellular senescence that causes the chronic inflammation of aging is still a fairly low priority in the research community, alas.

Aging and Alzheimer's disease (AD) are both associated with diminished blood-brain barrier (BBB) integrity and an opening for T cell migration into the central nervous system (CNS). In the parenchyma, bidirectional crosstalk occurs between the infiltrating cells and the resident glial cells; activated microglia impair BBB function by releasing several inflammatory modulators and thus lead to hyperpermeability; and the resulting T cell infiltration, in turn, favors increased microglial activation by secreting proinflammatory cytokines or acting in a protective manner toward senescent microglia.

We performed RNA-seq analyses on microglia and astrocytes freshly isolated from wild-type and APP-PS1 (AD) mouse brains at five time points to elucidate their age-related gene-expression profiles. Our results showed that from 4 months onward, a set of age-related genes in microglia and astrocytes exhibited consistent upregulation or downregulation (termed "age-up"/"age-down" genes) relative to their expression at the young-adult stage (2 months). Most age-up genes were more highly expressed in AD mice at the same time points. Bioinformatic analyses revealed that the age-up genes in microglia were associated with the inflammatory response, whereas these genes in astrocytes included widely recognized AD risk genes, genes associated with synaptic transmission or elimination, and peptidase-inhibitor genes.

The results of this study indicate that microglia exhibit an increase in responsiveness to inflammation stimuli with age, which is reflected by the consistently elevated expression of inflammatory-response genes, whereas astrocytes appear to function as "preservers" of inflammation, which is reflected by the upregulation of peptidase-inhibitor genes upon aging.


Galactose Conjugation Makes Navitoclax a Safer Senolytic Drug

If given a way to more effectively target senolytic drugs towards senescent cells, reducing off-target effects, then navitoclax is a good drug to test with. Navitoclax is arguably the worst of the first set of drugs found to be meaningfully senolytic; it certainly compares unfavorably with dasatinib. While navitoclax can kill a usefully large fraction of senescent cells in aged tissues, the dose required for that outcome will also kill a lot of normal cells along the way. More unpleasant drugs have since been discovered to be usefully senolytic, such as cardiac glycosides, but there is much less data on their senolytic use at this stage. In comparison, a good deal of data exists for the ability of navitoclax to destroy senescent cells. Its unpleasant side effects are quantified as a result of its development as a potential chemotherapeutic.

Finding ways to target highly toxic drugs to specific cell populations is a well established line of work in the cancer research community. Even if it were not the fact that many senolytic drugs were initially characterized for use as cancer therapeutics, it would not be surprising to find analogous cell targeting research taking place in the field of senolytics. This is a well understood approach to making toxic drugs more useful.

You may recall that researchers have been exploring the possibility of conjugating toxic drugs with galactose, producing a far less harmful prodrug molecule. Senescent cells are characterized by the high expression of senescence-associated β-galactosidase. This enzyme acts to remove the galactose from the prodrug, restoring the original toxic molecules that then kill the cell. In today's open access paper, an variant on this approach is demonstrated to improve the characteristics of navitoclax as a senolytic drug.

Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity

Recent research has identified targetable vulnerabilities of senescent cells that can be exploited by a novel group of drugs called senolytics. These compounds preferentially kill senescent cells by different mechanisms. Senolytics include the BCL-2 family inhibitors Navitoclax (ABT-263) and ABT-737; the flavonoid fisetin; combinations of tyrosine kinase inhibitors and flavonoids (e.g. dasatinib and quercetin); FOXO4-p53 interfering peptides; HSP90 chaperone inhibitors; and other compounds such as piperlongumine and cardiac glycosides. Senolytics have emerged as promising agents for treatment of pulmonary fibrosis, atherosclerosis, osteoarthritis, type 1 diabetes mellitus, type 2 diabetes mellitus, and neurocognitive decline. They can also rejuvenate aged hematopoietic stem cells and muscle stem cells and extend the lifespan of naturally aged mice.

Despite successful preclinical proofs-of-concept for senolytics, their potential translatability is hampered by their associated toxicities, necessitating the development of more specific, and less toxic, second-generation senolytics. Navitoclax has been validated in a variety of preclinical models showing high potency in killing senescent cells - however, it also has significant on-target haematological toxicity, including thrombocytopenia.

One consistent feature of senescent cells is their enrichment in lysosomes and lysosomal proteins, including senescence-associated β-galactosidase (SA-β-gal) which is widely used as a marker of senescence. We previously showed that the encapsulation of nanoparticles with galacto-oligosaccharides (GalNPs) is an efficient method to preferentially deliver cytotoxic drugs and tracers to the lysosomes of senescent cells where SA-β-gal activity digests the galacto-oligosaccharides, thereby releasing the cargo. We have also shown that a fluorescent probe covalently linked to multi-acetylated galactose is preferentially digested by senescent cells, releasing the free fluorophore.

Here, we have modified Navitoclax with an acetylated galactose to exploit the enriched SA-β-gal activity of senescent cells. Using a variety of model systems, we show that galacto-conjugation of Navitoclax, which we name Nav-Gal, results in a prodrug with selective, pro-apoptotic senolytic activity released in senescent cells that is dependent on GLB1 activity. Galacto-conjugation of Navitoclax reduces thrombocytopenia in treated mice at therapeutically effective doses, as well as apoptosis of platelets in human blood samples treated ex vivo. Overall, we propose galacto-conjugation of cytotoxic drugs as a versatile methodology for developing second-generation prodrugs with high senolytic activity and reduced toxicity. We provide evidence of the efficacy of combining senescence-inducing chemotherapies with senotherapies in cancer, with potential for clinical application.

Intermittent Fasting Increases Neurogenesis in Mice

Neurogenesis is the process by which neurons are created and then integrated into neural circuits. It is essential to the processes of memory and learning, but also vital to the maintenance of brain tissue over a lifetime. It may or may not take place throughout the brain, versus only in areas connected to memory function, and mice may or may not be a good model for human neurogenesis. Most of the work to date has taken place in mice, as working with human brain tissue is beyond the reach of most research groups.

Neurogenesis declines with age, likely largely because the stem cell populations responsible for generating new neurons become less active, as is the case for stem cells throughout the body. Greater neurogenesis is generally considered to be a good thing, but the research community has yet to produce therapies reliably proven to achieve this outcome in humans. A number of approaches work in mice to varying degrees. Fasting, calorie restriction, and exercise all appear to improve neurogenesis, but a larger effect size than is produced via better lifestyle choices would be desirable.

Dietary restriction (DR) is defined as a decrease in energy consumption without reducing nutritional value. This simple dietary intervention has been shown in a wide range of experimental animals to extend lifespan and decrease the incidence of several age-related diseases. The definition of DR has been expanded from an alternative description of caloric restriction (CR) to also encompass a broader scope of interventions, including short-term starvation, periodic fasting, fasting-mimetic diets, and intermittent fasting (IF).

IF has been proven to be advantageous to various organ systems in the body and acts as a mild metabolic stressor. It has been postulated that IF is able to cause powerful changes in the metabolic pathways in the brain via an increase in stress resistance, and breakdown of ketogenic amino acids and fatty acids. Experimental studies have also shown that IF is neuroprotective against acute brain injuries such as stroke, and neurodegenerative diseases. In addition, recent studies have also shown that IF can lead to an increase in neurogenesis levels in the hippocampus.

We evaluated the impact of 3 months of IF (12, 16, and 24 hr of food deprivation on a daily basis) on hippocampal neurogenesis in mice using immunoblot analysis. We investigated the expression levels of molecular and cellular components of the hippocampal region, focusing specifically on Notch activation and associated proteins that are known to promote hippocampal neurogenesis such as brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB).

Three-month IF significantly increased activation of the Notch signaling pathway, neurotrophic factor BDNF, and downstream cellular transcription factor, cAMP response element-binding protein (p-CREB). The expression of postsynaptic marker, PSD95, and neuronal stem cell marker, Nestin, was also increased in the hippocampus in response to 3-month IF. These findings suggest that IF may increase hippocampal neurogenesis involving the Notch 1 pathway.


The Beneficial Metabolic Adaption Provoked by Consistent Exercise

Consistent exercise produces sweeping changes in metabolism. It is clearly beneficial at any age, and there is a mountain of data to support that assertion. With more modern tools of analysis, greater efforts are being made to catalog the beneficial changes in cellular metabolism that result from exercise, rather than just the improvements to health at a high level. The open access paper noted here is an example of this sort of work.

Exercise provides many health benefits, including weight loss, improved lipid profiles, and improved insulin sensitivity. It is particularly relevant in the era of high-prevalence childhood and adult obesity and cardiometabolic disease. Exercise is a core tenet of all cardiovascular prevention guidelines, and degree of physical fitness is a strong predictor of cardiovascular mortality.

Metabolites are a diverse array of biochemicals that together capture an individual's metabolic state. They are particularly useful in the investigation of cardiometabolic diseases. Furthermore, they can characterize response to both acute and chronic exercise. Several studies have revealed key changes in lipolysis, glycolysis, glycogenolysis, citric acid cycle, and amino acid metabolism after a single/acute aerobic exercise session and identified differences in metabolite substrate use between fit and unfit individuals. However, much less work has been done with respect to metabolic changes following chronic exercise training. These studies reported increases in microbiome-derived tryptophan metabolites and acylcarnitines, and decreases in adenine nucleotides.

We analysed changes in metabolomic profiles at the end of an 80-day exercise intervention compared to baseline, and the association of metabolite changes with changes in clinical parameters. Global metabolism was dramatically shifted after the exercise training programme. Fatty acids and ketone body substrates, key fuels used by exercising muscle, were dramatically decreased in plasma in response to increased aerobic fitness. There were highly significant changes across many classes of metabolic substrates including lipids, ketone bodies, arginine metabolites, endocannabinoids, nucleotides, markers of proteolysis, products of fatty acid oxidation, microbiome-derived metabolites, markers of redox stress, and substrates of coagulation.

For the first time, therefore, we were able to provide an accurate report of the degree of increased consumption of fatty acid and ketone body substrates by trained, energy-efficient muscle. We also captured heretofore unseen, in terms of scale and scope, shifts in metabolism across many different substrates. These findings have important implications in cardiovascular disease prevention and risk reduction regimes.


NOXO1 Knockout as an Example of Counterintuitive Results in Animal Longevity

The balance between oxidative damage and adaptive responses to oxidative damage appears important in the way in which the operation of metabolism determines natural variations in longevity. Not as important as the factors that determine order of magnitude differences in life spans between species, but important enough to cause 10-20% changes in life expectancy in mice when manipulated, for example. That is the case in this study, in which researchers assess what appeared to be a damaging genetic alteration, a knockout of the NOXO1 gene associated with production of reactive oxygen species that has detrimental effects on intestinal function, and found that mice lived 20% longer or so as a result.

There have been a lot of surprising results in the manipulation of oxidative metabolism. It is a very complex system. Oxidative molecules are generated by mitochondria or during inflammation, and they both damage cellular components and act as signals to provoke greater cellular maintenance activities, or other tissue functions such as the adaptive growth of muscle in response to exercise. Too much oxidative damage to the point of overloading antioxidant and repair systems is directly harmful. Too little oxidative damage can also be harmful in the long term because too little cellular maintenance takes place. It is hard to predict how a reactive biological system of countless interacting component parts will behave when one part is broken or enhanced.

This particularly study may also be an example of the point that the effects of calorie restriction are large in comparison to most other studied mechanisms. Because NOXO1 knockout degrades intestinal function, these mice weigh less, and may thus be benefiting more from a reduced calorie intake than they lose from a suboptimal oxidative biochemistry. Though the usual signs of the calorie restriction response appear absent - the researchers did do some digging on this topic. All in all, investigating the complexities of metabolism is a challenging business; given the modest size of the effect observed here, this particularly line of work is unlikely to be worth the effort from anything other than the purely knowledge-driven perspective.

NoxO1 Knockout Promotes Longevity in Mice

According to the free radical theory of aging, reactive oxygen species (ROS) have been proposed to be a major cause of aging for a long time. Meanwhile, it became clear that ROS have diverse functions in a healthy organism. They act as second messengers, and as transient inhibitors of phosphatases and others. In fact, their detrimental role is highly dependent on the context of their production. NADPH oxidases (Nox) have been discovered as a controllable source of ROS. NoxO1 enables constitutive ROS formation by Nox1 by acting as a constitutively active cytosolic subunit of the complex. We previously found that both Nox1 and NoxO1 were highly expressed in the colon, and that NoxO1-/- deficiency reduces colon health. We hypothesized that a healthy colon potentially contributes to longevity and NoxO1 deficiency would reduce lifetime, at least in mouse.

In contrast, here we provide evidence that the knockout of NoxO1 results in an elongated life expectancy of mice. No better endothelial function, nor an improved expression of genes related to longevity, such as Sirt1, were found, and therefore may not serve as an explanation for a longer life in NoxO1 deficiency. Rather minor systemic differences, such as lower body weight occur. One effect of low body weight might be the expression and activity of Sirt1 and PGC1α, which are upregulated in response to caloric restriction. However, neither Sirt1 nor PGC1α was upregulated in NoxO1 deficient organs. Accordingly, the Sirt1 downstream target eNOS was not differentially activated in NoxO1-/- mice. We conclude that Sirt1 is not the preliminary effector to prolong the lifespan of NoxO1-/- mice, when compared to wildtypes. Although not significantly different from wildtype mice, low body weight in NoxO1 mice may have other beneficial effects.

As a potential reason for longer life, we suggest better DNA repair capacity in NoxO1 deficient mice. Although final fatal DNA damage appears similar between wildtype and NoxO1 knockout animals, we identified less intermediate DNA damage in colon cells of NoxO1-/- mice, while the number of cells with intact DNA is elevated in NoxO1-/- colons. We conclude that NoxO1 deficiency prolongs lifetime of mice, which correlates with less intermediate and potentially fixable DNA damage at least in colon cells.

Correlating Autonomic Nervous System Aging and Cognitive Impairment

Heart rate variability is known to be a good way to assess the function of the autonomic nervous system. (That said, most of the commercially available tools for those who want to measure heart rate variability at home are quite unreliable; it is challenging for a self-experimenter to obtain results that are as useful as those provided by medical equipment used by medical staff). The autonomic nervous system, like all aspects of our biology, is negatively impacted by the progression of aging. The same mechanisms of molecular damage that drive autonomic nervous system aging will be involved in cognitive decline and neurodegenerative conditions, so it should be no great surprise to see that these forms of age-related degeneration correlate with one another.

Changes in cognitive performances and cardiovascular disorders represent a normal phenomenon of the aging process. Cardiovascular risk factors, such as smoking, obesity, diabetes mellitus, increased cholesterol, and systemic blood pressure (BP) levels, and an inadequate lifestyle may compromise also cerebral blood flow, which in turn can negatively affect cognitive performance. Moreover, the same age-related anatomical and functional cardiac changes, including also the autonomic nervous system (ANS), determine cardiac output alteration, causing cerebral blood flow modulation. This variation could interfere with microcirculation and cause cerebral ischemia, particularly in those brain sites that control the different cognitive domains.

Evidence supports the relevance of ANS study by heart rate variability (HRV) assessment as a tool for the noninvasive analysis of cardiovascular autonomic function. HRV, defined as a marker identifying the balance between sympathetic and parasympathetic tone, predicts total mortality, sudden death, cardiovascular disease risk, as well as other morbidities. It represents the measure of physiological variation in the interval between consecutive heart sinus beat or in the fluctuations between instantaneous heart rates and provides the importance that the ANS has regarding cardiovascular health and prognosis.

We assessed the relationship between long-term heart rate variability (HRV), as a measure of autonomic nervous system (ANS) functioning, and cognitive performance in elderly patients representative of outpatients in a real-life setting. 117 patients underwent anthropometric evaluation, cardiac assessment by 12-lead electrocardiogram, 24-hour electrocardiogram recording, and blood pressure (BP) measurement, as well as global cognitive evaluation. Our results show that an increased sympathetic activity, but not decreased vagal activity, is associated with better cognitive performances. These results support the sympathetic autonomic function, in that the relationship between better cognitive performances and a moderate prevalence of autonomic function appears dependent on long-term changes in heart rate, mediated by sympathetic activation.


The Autophagy-Lysosomal Pathway and Protein Aggregation in Neurodegenerative Disease

Researchers here review some of the evidence for a two-way relationship between the age-related failure of autophagy and the accumulation of harmful protein aggregates that characterizes many neurodegenerative conditions. Autophagy is a collection of cellular maintenance processes responsible for recycling unwanted or damaged proteins and structures, but isn't just the case that failure of autophagy can lead to the spread of protein aggregates. It is also possible that the presence of these aggregates can cause a loss of autophagy, implying a vicious cycle of progression of these age-related conditions.

A hallmark of many neurodegenerative diseases is the progressive formation of insoluble protein aggregates. There are two main factors that cause protein aggregation in neurodegenerative diseases: mutations in genes encoding aggregate-prone proteins and the decline of cellular degradation functions, in particular of the autophagy-lysosomal pathway (ALP).

ALP is a major process for degrading intracellular macromolecules and generating energy or building blocks to make other macromolecules. ALP relies on the engulfment of cargos to be degraded (macromolecules or damaged organelles) in double-membrane vesicles (autophagosomes), which, therefore, fuse with endosomes/lysosomes to form autolysosomes, where autophagosome contents are degraded by lysosomal enzymes. ALP plays a key role in protein homeostasis and in the clearance of protein aggregates (processes that are particularly important in non-dividing neurons).

Mounting evidence also shows that protein aggregation itself may affect ALP, thus generating a vicious cycle, which boost protein aggregation and toxicity. Different works have shown that the aggregated forms of α-synuclein can bind the lysosome, thus impairing the chaperone-mediated autophagy or inducing lysosomal rupture. The interplay between protein aggregation and ALP dysfunction is crucial in driving neurodegenerative processes in a number of neurological conditions.


Loss of Autophagy in Hematopoietic Cells Contributes to Osteoporosis

Today's open access paper is an example of one of the less well known connections between processes of aging. Loss of efficiency of the cellular maintenance processes of autophagy is a characteristic of cells in old tissues. Here, researchers note that this dysfunction in the hematopoietic cells responsible for creating blood and immune cells also results in structure changes in bone marrow that contribute to the development of osteoporosis, the loss of bone mass and strength that occurs with age. Just because such connections are obscure doesn't mean that they are unimportant.

Osteoporosis is the outcome of an imbalance between osteoblasts that create bone and osteoclasts that break down bone tissue. Both cell populations are constantly active throughout life; bone is a dynamically remodeled tissue. With age a number of processes lead to greater osteoclast activity - easy enough to point out, but difficult to pick apart the causes and decide on a point of intervention. Cellular senescence is a contributing factor, of course, as it contributes to near all age-related conditions. Here, reduced autophagy is proposed to interfere in maintenance of the osteoblast population.

There are a few types of autophagy, involving different ways of identifying and moving unwanted proteins and structures to be engulfed by a lysosome. A lysosome is a membrane-wrapped package of enzymes that can break down most of the molecules a cell will encounter. Exactly why autophagy declines with age is an interesting question. A great many papers cover what is known of proximate causes, changes in expression of protein machinery that regulates or is needed by different parts of the process, all of which seem to fail in their own way, as well as the accumulation of persistent metabolic waste in lysosomes, molecules that cannot be broken down by our biochemistry as it stands. The latter is fairly straightforward, but the changes in regulation and expression of proteins are ever a challenge to chase back to their underlying cause.

Deterioration of hematopoietic autophagy is linked to osteoporosis

Previous studies on osteoporosis overwhelmingly focused on the etiology within bone tissue that locally induces the disease. In this study, we showed that osteoporosis is highly associated with reduction in hematopoietic autophagy activity in humans. We showed that an autophagy defect in the hematological system leads to severe bone loss. The disturbed osteocyte homeostasis is apparently caused by impaired type H blood vessels and possibly an aberrant alteration in the extracellular matrix (ECM) pathways that govern osteocyte homeostasis in hematopoietic autophagy-defective mice. Our results thus suggest that autophagy in the adjacent hematopoietic cells is essential to maintain bone homeostasis, and chronic hematopoietic autophagy deficiency can result in the development of osteoporosis in both mice and humans

While the osteal impact on hematopoiesis, in particular on the formation of bone marrow hematopoietic stem cell niches, is well documented, studies of hematopoietic regulation of osteocyte function have been inadequate. Hematopoietic regulation of osteoblast proliferation and differentiation was previously discussed largely with skepticism speculation. However, a recent study showed that loss of the hematopoietic stem cell factor GATA2 in the osteogenic lineage impairs trabecularization and mechanical strength of bone. Our present study of RNA sequencing revealed that impairment of hematopoietic cells by autophagy defect also led to enhanced iron activity, which may eventually lead to iron overload, a major cause of osteoporosis.

Hematopoietic cells and osteocytes are adjacent in the bone marrow niche environment. Normal hematopoiesis and bone homeostasis are interdependent. Men with low bone mineral density (BMD) or greater BMD loss have decreased circulating erythrocytes and lymphocytes and increased myeloid cells, and anemia or low blood cell counts are associated with declining BMD or increased fracture risk in the aged population. On the other hand, chronic disorders affecting hematopoiesis, such as sickle cell anemia and thalassemia, demonstrate clear skeletal phenotypes, including bone loss and increased fracture risk.

Bone marrow HSCs have been found to be capable of differentiating to osteoblasts through a mesenchymal intermediate. These findings suggest that the developmental capacity of HSCs is not restricted to hematopoietic lineages, but extends to osteogenic differentiation, possibly via the HSC potential to transdifferentiate to osteocytes. HSCs do not rest passively in their niche, but instead directly participate in bone formation and niche activities. Therefore, HSC functions and bone turnover are coupled in osteoporosis.

Finally, screening for expression of selected genes and an immunohistological assay identifies severe impairments in H vessels in the bone tissue, which results in disconnection of osteocytes from hematopoietic cells in the autophagy-defective mice. We therefore propose that hematopoietic autophagy is required for the integrity of H vessels that bridge blood and bone cells and that its deterioration leads to osteoporosis.

Engineered Stem Cells Survive Longer and Improve Outcomes in a Heart Patch

In most cell therapies, the transplanted cells do not survive for long, or in large numbers. They produce beneficial effects, such as reduced inflammation or enhanced regeneration, via signaling that changes the behavior of native cell populations. Considerable effort is going into finding ways to make cells used in therapy survive for a longer period of time following transplantation. The approach taken here is to engineer a fraction of the transplanted cells to produce a growth factor that improves the survival of the others. The results are demonstrated in an animal model, showing a greater regeneration of heart muscle.

Human mesenchymal stem cells (hMSCs) have been considered as one of the most promising cell sources for cell-based cardiac regeneration therapy because of their proven safety and notable paracrine effects to secrete numerous antiapoptotic and angiogenic growth factors, which enabled them to be a more competitive agent for clinical applications. However, unlike promising results obtained from preclinical models of myocardial infarction (MI), recent multiple meta-analyses have debated whether the therapeutic potential of hMSC treatment is sufficient. While these clinical trials successfully demonstrated the feasibility and safety of hMSC treatment, the researchers were unable to show significant functional benefit.

In response, diverse approaches have been attempted to enhance the therapeutic efficacy of hMSCs in treating MI. For instance, genetically engineered hMSCs overexpressing a number of antiapoptotic proteins, growth factors, or prosurvival genes - such as vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and hepatocyte growth factor (HGF) - showed increased survival and retention in vivo resulting in improved cardiac function and myocardial angiogenesis in MI-induced hearts. However, these approaches require genetic modification and, therefore, are incompatible with clinical applications.

Another strategy to bolster the therapeutic potential of hMSCs is priming/preconditioning the hMSCs - which exposes them to physical treatments (e.g., hypoxia and heat shock), pharmacological agents, growth factors, distinct types of biomaterials, modified culture conditions, or other various molecules, including microRNAs - in vitro before transplantation into the hearts. However, it appears that the priming application only provides a short-term benefit.

Consequently, for hMSCs to be used more effectively for comprehensive cardiac repair, an innovative method that can maintain the priming effect of hMSCs more consistently and effectively must be developed. In the present study, we sought to develop a strategy, namely, in vivo priming, which could prime hMSCs in intact hearts in vivo. To induce and maintain the beneficial effects of priming persistently in situ, we loaded MSCs isolated from human bone marrow (BM-MSCs) together with genetically engineered HGF-MSCs (HGF-eMSCs) that continuously secrete HGF within a three-dimensional (3D) cardiac patch, which was implanted in the epicardium of MI-induced hearts. Subsequently, we demonstrated that the primed BM-MSCs had a higher survival rate compared with unprimed BM-MSCs in the patches while they were attached to the MI hearts, which led to a significant improvement in cardiac function and an enhancement of vessel formation after MI.


CCN2 Inhibition Reverses Fibrosis in Overuse Injury

Fibrosis is a characteristic feature of aging, degrading tissue and organ function in the lungs, kidneys, heart, and elsewhere. It is a failure of regeneration and tissue maintenance, involving the inappropriate formation of scar-like collagen structures. Fibrosis is also found in overuse injury in muscle. In the case of aging, recent research has shown that cellular senescence has a prominent role in fibrosis, most likely mediate by inflammatory signaling. Removal of senescent cells reverses fibrosis in animal models, making senolytic therapies interesting in this context. Here researchers do not discuss senescent cells, but do show that inhibition of CCN2 reverses muscle fibrosis due to overuse injury. CCN2 inhibition underwent a successful human trial for lung fibrosis, which in turn suggests that perhaps senolytic therapies would usefully treat overuse injuries.

Overuse-induced musculoskeletal disorders are widely understood to be injuries and disorders affecting the musculoskeletal system. Tissue fibrosis is a pathological hallmark of overuse-induced muscle injuries and is considered to play key roles in associated motor dysfunction. Such fibrosis is thought to distort dynamic properties of tissue and contribute to functional declines due to adherence of adjacent structures. We have shown that inflammation is a key driver of further fibrosis, and that early use of anti-inflammatory drugs, ergonomic task reduction and manual therapy treatments are able to prevent their development. However, treatments aimed at reducing established muscle and other tissue fibrosis have proved to be more difficult, because once deposited and cross-linked, the extracellular matrix becomes resistant to degradation.

Blocking CCN2 signaling has shown promise for many fibrotic disorders. Downregulation of CCN2 reduces liver fibrosis and limits hypertrophic scarring without affecting wound healing. We recently found that CCN2 is critical to the early progression of chronic overuse-induced muscle fibrosis and grip strength declines in rats that performed an operant reaching, grasping, and lever-pulling task at high repetition high force (HRHF) levels for three weeks. CCN2 inhibition reduced this early progression of fibrosis and improved motor declines. However, continued performance of the HRHF task for 18 weeks, untreated, induces even greater muscle fibrosis and motor declines than at earlier weeks. Therefore, we examined for the first time whether inhibition of CCN2 using this antibody is able to reduce established skeletal muscle fibrosis in our operant rat model of overuse injury.

We show here that 6 weeks of rest combined with systemic inhibition of CCN2 significantly reduced established skeletal muscle fibrosis and improved motor function, compared to control rat levels. We again show that increased muscle fibrosis was mirrored by increased serum levels of CCN2, adding further support to its use as a serum biomarker of underlying tissue fibrosis occurring with overuse injuries as well as other diseases associated with enhanced fibrogenic activity.


Rejuvenation of Immune Function is One of the More Important Outcomes to Engineer through the Treatment of Aging

One would hope that it does not require an ongoing pandemic and related hysteria to point out that old people have poorly functioning immune systems, and thus suffer disproportionately the burden of infectious disease. But perhaps it does. The 2017-2018 seasonal influenza, a modestly more severe occurrence of something that happens every year, killed something like 60,000 people in the US alone, with little notice or comment. There is nothing so terrible that it won't be accepted - ignored, even - if it is normal.

Floodgates of funding for infectious disease research and development have been opened in response to COVID-19, and while no doubt all too little of it will be spent wisely or usefully (public funding being the very definition of waste and corruption) it has certainly prompted many groups to try to position themselves to benefit. Those who have, all along, been working on ways to try to make older people more resilient via improvement in their immune function are perhaps more deserving than others, but it really isn't the case that much of this work is closer than five to ten years away from practical realization and completed human trials.

Of the ways to restore immune function in the old, the worst are the small molecule drugs that show signs of adjusting metabolism in the right direction. For example, the mTOR inhibitors that just failed a phase III trial for reducing influenza incidence in the old. Better drug or drug-like approaches are those that target regrowth of the atrophied thymus. The thymus is where T cells of the adaptive immune system mature, and the production rate is reduced to a trickle in the old - a major cause of immune aging. In humans, there is data for the growth hormone approach of Intervene Immune, and better data for sex steroid ablation, to restore the production of T cells.

Further, regeneration of lymph nodes, vital to coordination of an immune response, and regeneration of the hematopoietic stem cell population that creates all immune cells will be beneficial - but existing approaches to these challenges are by no means close to readiness for clinical trials. Selective destruction of malfunctioning, senescent, and exhausted immune cells is also likely to be beneficial - but only removal of senescent cells via senolytic therapies is a very near term prospect at the present.

At the end of the day, therapies capable of making a 70 year old exhibit the immune profile and response of a 40 year old would be transformative. The world has come to accept that sizable numbers of older people die from infectious disease every year, and that this is set in stone and little can be done about it. That is simply not the case - a great deal can be done about it. It just requires the will and funding to move ahead with the most plausible programs of immune rejuvenation.

It is worth noting that the pandemic statistics referenced in today's open access paper require some interpretation and none should be taken either at face value or as usefully applicable across the board. Context is everything. Testing for COVID-19 is presently very selective for symptomatic, more severe cases. No-one yet has a good grasp on how many mild cases there are, and that is everything for determining actual mortality risk. Further, circumstances such as an enclosed cruise liner are not representative of the way matters progress in the broader population. And so on.

Geroprotective and senoremediative strategies to reduce the comorbidity, infection rates, severity, and lethality in gerophilic and gerolavic infections

Aging is a complex, multifactorial process that leads to loss of function and is the primary risk factor for major human pathologies including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases. Although there is still much debate in the scientific community, proposals have been made to classify aging as a disease in order to develop therapeutic strategies to prevent or delay the onset of age-related illnesses. Increasing frailty with age leads to an increased risk of many diseases. These diseases are commonly referred to as age-related. Many pathogens are more infectious and prevalent in the elderly, and may be referred to as gerophilic (from Greek, géros "old man" and philia, "love"). Some infections, including COVID-19, are not exclusively gerophilic, as younger people may also become infected. However, these individuals have mild symptoms or remain asymptomatic, while the elderly experience substantially more severe symptoms and lethality. The term gerolavic (from Greek, géros "old man", and epilavís, "harmful") may more appropriately describe infections that cause the most harm in the elderly.

Statistics from the COVID-19 pandemic indicate that COVID-19 is a gerolavic infection, one that disproportionately affects the elderly. According to Worldometers, an online resource aggregating data on COVID-19, of the 139,580 people infected worldwide as of March 13, 2020, 70,733 patients had recovered and 5,120 had died. Based on these data, the mortality rates (number of deaths/number of cases), or the probability of dying if infected by the virus, were determined to be 3.6% for individuals aged 60-69, 8% for individuals aged 70-79, and 14.8% for patients aged 80 years or older. The majority of the infected population are 50 and older, while the majority of the deceased are 60 and older.

An open coronavirus analysis project provides further insight into the mortality rates of COVID-19, specifically using data from the Diamond Princess Cruise, where all passengers were exposed to SARS-CoV-2 for an extended period. Of the approximately 1,690 passengers over 65 years of age, 7 passengers died, suggesting a death rate of 0.41%. This death rate is approximately 4.3 times higher than that of influenza. As more countries start reporting statistics, these death rates are likely to be adjusted. These statistics indicate that the infectivity of SARS-CoV-2, and the severity and lethality of COVID-19, are age-related.

One of the possible causes of the age-associated increases in COVID-19 infection rate, severity, and lethality is immunosenescence. Immunosenescence is a well-known age-related process contributing to the global burden of disease. Among the factors contributing to immunosenescence is the chronic involution of the thymus with increased age. Indeed, the infection rates of COVID-19, separated by age, are correlated with involution of the thymus. The thymus gland is most active early in life, reaching maximum size within the first year. Its activity then declines with age until an individual reaches 40 to 50, after which there are negligible traces of the thymus remaining, replaced by fibrotic tissue. As a result of thymic involution, the number of naïve T cells exiting the thymus decreases significantly, with substantial declines in older age.

Age-associated immunosenescence leads to a reduced ability to resist infection, while infection produces biological damage and loss of homeostasis. This ultimately contributes to accelerated aging and the development of age-related diseases, and further accelerates immunosenescence. In support of this model, infections and other age-related diseases are among the main causes of death in the developed world and in developing countries.

Due to the gerolavic nature of COVID-19, the classical preventative measures and treatment strategies used for targeting infectious diseases may not be as effective, and there is a need for alternative geroprotective and senoremediative strategies. Here we compare the expected benefit of treatments for elderly populations (60 years and older) that are currently in development, including standard preventative strategies such as vaccines and antivirals targeting SARS-CoV-2, and the potential added benefit of speculative geroprotective strategies such as rapalogs, NAD+ boosters, senolytics, and stem cell treatment. These additional measures may be used in isolation or as adjuvant therapies to reduce infection risk, symptom severity, or improve vaccine efficacy.

Therefore, interventions that enable immunocompromised elderly to mount an immune response to newly developed vaccines are necessary to help eradicate the disease and reduce the associated mortality. To avoid substantial loss of life and quality of life, primarily among the elderly and vulnerable populations, governments and healthcare systems should investigate preventative and intervention strategies stemming from recent advances in aging research. As discussed in this paper, small clinical studies have shown that several geroprotective and senoremediative interventions, such as treatment with sirolimus and rapalogs, can induce immunopotentiation, increase resistance to infection, and reduce disease severity in the elderly, without severe side effects.

Many of these predicted geroprotectors are available as supplements; however, no meta-analysis or metaclinical trials have been performed at scale to evaluate their effectiveness. The COVID-19 pandemic highlights the paucity of clinical trials on the effects of dietary supplements and drugs on aging and immunosenescence. The existence of pseudoscience and anecdotal promotion in the supplement industry does not mean that protective compounds do not exist. Dietary supplement vendors and pharmaceutical companies need to actively engage in preclinical and clinical research to evaluate the effectiveness of the currently available products on immunosenescence and aging.

There Are Many Ways to Influence Known Longevity Pathways

The paper here is an example of the point that there are many ways to influence any given longevity-related mechanism. The vast majority of the diverse approaches shown to slow aging in short-lived laboratory species, such as nematode worms, in fact act through a small number of core mechanisms. These are related to cellular stress responses, such as an increased operation of maintenance processes that include, prominently, autophagy. Unfortunately they also have diminishing returns as species life span increases: it is possible to produce large gains in short-lived species via manipulation of these mechanisms, but not in humans. Other approaches are needed if the goal is rejuvenation of the old.

Physiological aging is a complex process, influenced by a plethora of genetic and environmental factors. While being far from fully understood, a number of common aging hallmarks have been elucidated in recent years. Among these, transcriptomic alterations are hypothesized to represent a crucial early manifestation of aging. Accordingly, several transcription factors (TFs) have previously been identified as important modulators of lifespan in evolutionarily distant model organisms.

Based on a set of TFs conserved between nematodes, zebrafish, mice, and humans, we here perform a RNA interference screen in C. elegans to discover evolutionarily conserved TFs impacting aging. We identify a basic helix-loop-helix TF, named HLH-2 in nematodes (Tcf3/E2A in mammals), to exert a pronounced lifespan-extending effect in C. elegans upon impairment. We further show that its impairment impacts cellular energy metabolism, increases parameters of healthy aging, and extends nematode lifespan in a reactive oxygen species dependent manner.

We then identify arginine kinases, orthologues of mammalian creatine kinases, as a target of HLH-2 transcriptional regulation, serving to mediate the healthspan-promoting effects observed upon impairment of hlh-2 expression. Consistently, HLH-2 is shown to epistatically interact with core components of known lifespan-regulating pathways, i.e. AAK-2/AMPK and LET-363/mTOR, as well as the aging-related TFs SKN-1/Nrf2 and HSF-1. Lastly, single-nucelotide polymorphisms (SNPs) in Tcf3/E2A are associated with exceptional longevity in humans. Together, these findings demonstrate that HLH-2 regulates energy metabolism via arginine kinases and thereby affects the aging phenotype dependent on ROS-signaling and established canonical effectors.


Olympic Athletes Have a Lower Mortality than the General Population

A number of studies have shown that elite athletes have a significantly lower mortality and longer life expectancy than the general population. It is unclear as to whether this is due to physical activity and training for fitness versus other mechanisms, as elite chess players appear to have similar advantages. This study is par for the course, looking at Japanese Olympic participants. Interestingly, it hints at the upper end of the dose-response curve for physical activity, in that a longer career as a professional athlete may be detrimental in comparison to lesser degrees of exercise and training.

From this large, retrospective cohort study targeting 3546 Japanese Olympic athletes, we observed significant lower mortality among Olympians compared with the Japanese general population. The overall standardised mortality ratio (SMR) was 0.29. The results were consistent with previous studies conducted in other non-Asian countries, but the SMR was lower than in previous studies. A retrospective cohort study targeting 203 French Olympic rowers reported that the SMR for all causes of death was 0.58. Another retrospective cohort study targeting 2403 French Olympic athletes reported a SMR of 0.49 among women and 0.51 among men. A third retrospective cohort study of 233 Croatian male Olympic medalists reported a SMR of 0.73.

In the analysis by total number of participation in the Olympic Games, significantly higher mortality was observed among those who participated in the Olympics more than twice compared with those who participated only once. The underlying reason for this may be that those who participated in Olympic Games many times may have had long careers as elite athletes. To be continuously successful, they could have been exposed to exercises with high intensity for long periods, which may have led to higher mortality compared with those who participated only once.

We conducted cohort classification by sport discipline according to the nine combinations of static and dynamic intensity of sports disciplines, and demonstrated the association between intensity of sports disciplines and mortality. We did not observe significantly higher mortality among athletes who participated in disciplines of highest static and highest dynamic intensity. This could be explained in part by their exercise habits after retiring from international competitions. Although a study reported that those capable of prolonged vigorous physical exercises lived longer compared with the general population, Olympic athletes involved in sports disciplines with high intensity may not necessarily continue sports activities after retirement. The habit of sports activities after retiring from athletics may have more influence on mortality than the static/dynamic intensity of each sports discipline.


Inhibition of ATM Kinase Reduces Cellular Senescence and SASP in Progeroid Mice

Progeroid mice with DNA repair deficiencies exhibit an accelerated formation of senescent cells and manifestation of age-related conditions. This class of animal model has been used in research relating to cellular senescence in order to cost-effectively demonstrate that targeted removal of senescent cells is beneficial. However, one still needs to be careful when drawing conclusions based on their peculiar biochemistry. Progeria of this nature is quite unlike normal aging at the detail level.

Cells become senescent in response to reaching the Hayflick limit, tissue injury, molecular damage, or a toxic environment. A senescent cell ceases replication and generates senescence-associated secretory phenotype (SASP), a mix of signals that encourages both tissue remodeling and an immune response to destroy the senescent cell. In young people near all senescent cells are efficiently destroyed soon after their creation, but in older people senescent cells linger to cause chronic inflammation and tissue dysfunction.

A great deal of effort is going into deeper explorations of the biochemistry of cellular senescence these days. This is in no small part because any new discovery might have the potential to become a therapy that can treat numerous age-related conditions, and even aging itself, by alleviating the burden of senescent cells and their inflammatory, harmful signaling.

Researchers here identify an important regulator gene linking DNA damage with cellular senescence. Suppressing it in progeroid mice that exhibit high levels of DNA damage reduces markers of cellular senescence. However, it isn't clear that interfering in this process is a good idea. Removing or at least halting replication for cells with meaningful damage to their DNA is necessary to reduce the risk of cancer. Allowing damaged cells to continue replication is a poor strategy. It is far better to allow cells to become senescent in response to circumstances that carry an elevated risk of cancer, and then destroy them with periodic application of senolytic therapies.

ATM is a key driver of NF-κB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging

DNA damage is known to increase with aging as demonstrated by an increase in DNA damage foci and oxidative DNA lesions. Intriguingly, persistent DNA damage response (DDR) signaling mediated by ATM activation has been reported to contribute to cellular senescence and the senescence-associated secretory phenotype (SASP). In vitro, SASP is dependent on ATM activation, suggesting a molecular link between ATM and NF-κB. However, it is still unclear if aberrant DNA damage-induced activation of ATM in vivo exacerbates the cellular stress response to increase NF-κB, senescence, SASP and subsequently aging.

To address the role of ATM in driving NF-κB mediated senescence and aging, we used Ercc1-/Δ mice that model a human progeroid syndrome caused by impaired repair of DNA damage. The mice express only 5% of the normal level of the DNA repair endonuclease ERCC1-XPF that is required for nucleotide excision, interstrand crosslink, and repair of some double-strand breaks. As a consequence, the Ercc1-/Δ mice spontaneously and rapidly develop progressive age-related diseases, including osteoporosis, sarcopenia, intervertebral disc degeneration, glomerulonephropathy, neurodegeneration, peripheral neuropathy, and loss of cognition.

Here, we demonstrate that ATM and downstream effectors are persistently elevated in Ercc1-/∆ and naturally aged mice, concomitant with hyperactive NF-κB signaling. Reducing ATM activity either genetically or pharmacologically reduced cellular senescence and downregulated NF-κB activation in cell culture. Importantly, Ercc1-/Δ mice heterozygous for Atm exhibited significantly reduced NF-κΒ activity, reduced cellular senescence, improved muscle-derived stem cell and progenitor cell function and attenuated age-related bone and intervertebral disc pathologies, leading to an extension of healthspan. Similarly, inhibiting ATM in Ercc1-/∆ mice by treatment with the ATM inhibitor KU-55933 reduced senescence and SASP marker expression. These results demonstrate a key role for ATM in aging and suggest that it is a therapeutic target for delaying or improving numerous age-related diseases.

AQP1 and Cellular Senescence in the Aging of Tendons

Researchers here show that declining expression of the aquaporin AQP1 influences the age-related increase in cellular senescence in tendon stem cells and progenitor cells. Since the more general acceptance in the research community of senescent cell accumulation as an important cause of degenerative aging, there has been an increased interest in deeper investigations of the biochemistry of cellular senescence. It isn't clear that preventing cells from becoming senescent is always going to be beneficial, however. Does the method of prevention work by reducing cell damage and dysfunction that provokes senescence, which is a good thing, or does it work by holding back senescence in damaged and dysfunctional cells? That latter option may cause more problems than it solves.

Previous studies have demonstrated that tendon aging is closely associated with the functional changes of tendon stem/progenitor cells (TSPCs). TSPCs express classical stem cell markers and typical tendon-lineage genes. Studies have demonstrated the vital role of TSPCs in tendon repair, regeneration, and homeostasis maintaining. However, TSPCs premature entry into senescence during tendon aging, senescent TSPCs exhibit reduced self-renewal, migration, and tenogenic differentiation capacity compared with young cells, and these age-related changes in TSPCs would impair tendon healing and regeneration capacity.

Aquaporins (AQPs) are a family of small water-transporting membrane proteins. Previous studies also indicated that AQP1 is involved in tissue aging. In aged skin tissue, AQP1 level was decreased, which might be correlated with aging-related skin dryness. In addition, recent studies have indicated that AQP1 is also involved in the regulation of stem cells. Although studies have indicated the important role of AQP1 in tissue aging and regulation of stem cell, there were no studies focused on the role of AQP1 in TSPCs senescence.

In the present study, we investigated the AQP1 expression profile of TSPCs isolated from rats at different ages. We demonstrated that AQP1 expression declines with age in TSPCs, and AQP1 plays a vital role in TSPCs senescence. Decreased AQP1 was associated with activation of JAK-STAT signaling pathways in aged TSPCs. Furthermore, overexpression of AQP1 restored the age-related reduction of self-renewal, migration, and tenogenic differentiation in TSPCs. Our results collectively indicated that AQP1 could be an ideal target for antagonizing tendon aging.


Fasting Accelerates Wound Healing in Mice

This study delves into the mechanisms by which a short period of fasting can accelerate wound healing. Fasting triggers many of the same cellular stress responses, such as upregulated autophagy, as occur during the practice of calorie restriction. It isn't exactly the same, however, so it is always worth asking whether any specific biochemistry observed in either case does in fact occur in both situations. In particular, the period of refeeding following fasting appears to have beneficial effects that are distinct from those that occur while food is restricted.

Multiple forms of therapeutic fasting have been reported regarding their efficacy to improve health (decreasing body fat and blood pressure, promoting stem cell function and regeneration, reversing immunosuppression, suppressing inflammation, etc.), delay aging and extend life span. Recently, it was shown that fasting for 48 h during a 4-day observation period after stroke was able to augment angiogenesis in ischemic brain and alleviate cerebral ischemic injury in mice; periodic fasting (a 48-hour period of fasting per week for one month) resulted in reduced cortical atrophy and long-term neurobehavioral improvement. Nonetheless, the regulatory molecular mechanism through which fasting affects angiogenesis remains unclear.

Here, we generated full-thickness excisional or burn skin wounds in streptozotocin-induced diabetic mice and normal mice, respectively, and determined whether fasting prior to or after wound injury for a certain period of time can promote angiogenesis and speed up the process of wound healing. In vitro, we evaluated the effects of fasting and refeeding on the proliferation, migration and angiogenic tube formation of endothelial cells. To further explore the molecular mechanism, transcriptome sequencing of fasting and non-fasting endothelial cells was conducted to screen the differentially expressed angiogenesis-related genes and the role of the candidate genes in the fasting-induced promotion of angiogenesis was demonstrated.

Two times of 24-h fasting in a week after but especially before wound injury efficiently induced faster wound closure, better epidermal and dermal regeneration, less scar formation and higher level of angiogenesis in mice with diabetic or burn wounds. Transcriptome sequencing revealed that fasting itself, but not the following refeeding, induced a prominent upregulation of a variety of pro-angiogenic genes, including SMOC1 and SCG2. Immunofluorescent staining confirmed the increase of SMOC1 and SCG2 expression in both diabetic and burn wounds after fasting treatment. When the expression of SMOC1 or SCG2 was down-regulated, the fasting/refeeding-induced pro-angiogenic effects were markedly attenuated.


The Longevity 2020 Online Conference, to be Held April 27th to May 1st 2020

All of the longevity industry and gerontology conferences in coming months have been cancelled or rescheduled as a result of the present mix of COVID-19 pandemic and hysteria. An equal mix of both, perhaps. The best data to date puts the mortality rate at 0.66% or so, meaning about six yearly flu seasons worth of risk, and even that number is most likely still overstating the risk, as it misses the presently unknown count of infections that result in only minor symptoms. An article from earlier in the month on the uncertainties in all published numbers remains one of the more sane pieces written on the topic.

Still, when life hands you lemons, set up an online conference instead of merely mourning the end of gathering in person. So the Longevity.Technology team is assembling Longevity 2020, a four day online event of presentations for scientists, entrepreneurs, and investors in the longevity industry. The online conference will run from April 27th to May 1st, just four weeks away. Since the restrictions on productive economic activity look to last to the end of April at the very least in the US, we will all still have time on our hands by that point. If you have a company to pitch, research to present, or something sensible to say about human longevity and the treatment of aging as a medical condition, then it isn't too late to reach out and secure a place in the program.

Longevity 2020

You're at home. So are most of the experts in longevity. So, let's get together. Learn, share knowledge, build our networks, and take a break from Netflix. This difficult period will pass. It all begins with an idea. We're the team behind Longevity.Technology and, like you, we're disappointed that events have been cancelled, funding rounds postponed, and new knowledge is going unshared. So we thought: everyone's at home, even world experts, so let's get together and keep the Longevity sector on track. Boom.

We're using a new and interactive online events platform that enables you to interact with speakers directly, see full media presentations, meet with like-minded people, and build lasting relationships. Once we're happy that we have our killer programme of speakers and subjects we'll switch ticketing to live. Meanwhile, please pre-register so that you can stay updated: the button's up-top.

Defining Biological Aging

Addressing the need for candidate definitions from different, biological, clinical, logical and computational angles to adopt a consensus definition for biological aging. How are traditional biomarkers like body composition, muscle strength and cognition complimented by the techniques of methylation, inflammation, and epigenetics? With so many opinions and options: how can scientists prove their technologies and investors invest with confidence?

Rejuvenation Therapies

What progress is being made in senolytics, gene editing, immunotherapy, mitochondrial restoration, indication expansion, nutraceuticals, peptides, stem cells, and more? What companies are moving through the drug development pipeline towards in-human studies and ultimately the approval to market true rejuvenation therapies? With the toning-down of the '1000 year lifespan' rhetoric, there is growing confidence in the sciences of healthspan and lifespan: what should we expect within our lifetimes?

AI and Longevity

How is AI accelerating longevity and where: molecule identification, pre-clinical validation, digital health, lifestyle interventions, finance. With pre-clinical discovery reduced from months down to days, what are the implications for new longevity therapies in the new AI world? With apps directing us to live better and exercise more and people needing to live independently for longer - we explore the latest exciting AI applications.

Longevity Investing

It's all about growing the investment category: risk management, going public, investment platforms, emerging tech: 3D bioprinting, neuroceuticals, organ growth, agetech, and more. As markets are challenged by COVID-19 and investors run to safe havens, who are the key players in Longevity investing and where are they putting their money? The global longevity economy is projected to reach $27 trillion in 2026. With such a wide field, where are the market opportunities and which companies are innovating and disrupting?

Longevity: Start Now

What can you be doing to address your Longevity now: supplements, metformin, mTOR, NAD+, rapamycin, biohacking, fasting, biomarker tracking, monitoring apps. There are many pharmacological and non-pharmacological therapies that people talk about - but which ones show the most evidence in managing the hallmarks of aging? With many beginning to prescribe and self-prescribe drugs off-label - what are the risks and where is the proof of efficacy?

Debating the Direction of Causation Between Physical Decline and Cognitive Decline in Aging

Researchers here suggest that the direction of causation between physical decline and cognitive decline is largely the opposite of the present consensus. Most of the evidence of recent decades points to physical decline, and associated lack of activity, having a negative impact the brain. Certainly there are any number of studies showing exercise to have a beneficial effect on cognitive function. Here, however, researchers propose that declines in cognitive function lead the declines in physical function in aging.

Someone dies somewhere in the world every 10 seconds owing to physical inactivity - 3.2 million people a year. From the age of 50, there is a gradual decline not just in physical activity but also in cognitive abilities since the two are correlated. But which of them influences the other? Does physical activity impact on the brain or is it the other way around?

"Correlations have been established between these two factors, particularly in terms of memory, but also regarding the growth and survival of new neurons. But we have never yet formally tested which comes first: does physical activity prevent a decline in cognitive skills or vice versa? That's what we wanted to verify. Earlier studies based on the correlation between physical activity and cognitive skills postulated that the former prevent the decline of the latter. But what if this research only told half the story? That's what recent studies suggest, since they demonstrate that our brain is involved when it comes to engaging in physical activity."

Researchers tested the two possible options formally using data from the SHARE survey (Survey of Health, Aging and Retirement in Europe), a European-wide socio-economic database covering over 25 countries. The cognitive abilities and level of physical activity of 105,206 adults aged 50 to 90 were tested every two years over a 12-year period. Researchers employed this data in three separate statistical models. In the first, they looked at whether physical activity predicted the change in cognitive skills over time; in the second, whether cognitive skills predicted the change in physical activity; and in the third, they tested the two possibilities bidirectionally.

The researchers found that the second model adjusted the most precisely to the data of the participants. The study demonstrates, therefore, that cognitive capacities mainly influence physical activity and not vice versa, as the literature to date had postulated. "Obviously, it's a virtuous cycle, since physical activity also influences our cognitive capacities. But, in light of these new findings, it does so to a lesser extent."


Oral Administration of IAP Slows Aging in Mice by Reducing Gut Inflammation

The work here might be taken as an indication of the importance to aging of chronic inflammation and breakdown of the intestinal barrier generated in part by changes in the gut microbiome. Oral supplementation with the enzyme intestinal alkaline phosphatase (IAP) reduces both of these issues, and the result is mice that exhibit a slowed aging and lesser degrees of age-related frailty.

It's now accepted that gut-barrier dysfunction and gut-derived chronic inflammation play a role in human aging, but how that process is regulated is still largely a mystery. Studying mice and fruitflies, researchers found that the enzyme intestinal alkaline phosphatase (IAP) helped prevent intestinal permeability and gut-derived systemic inflammation, resulting in less frailty and extended life span. "Oral IAP supplementation in older mice significantly preserved gut barrier function and was associated with preserving the homeostasis of the gut microbiota during aging. In other words, the enzyme maintained the composition of the gut bacteria and controlled the low-grade chronic inflammation that can happen with aging."

Because the scientists were using animal models, they were able to test blood from the portal venous system, which goes from the GI tract into the liver and then on through the rest of the body. This gave a more direct measure of what is passing across the gut barrier than blood from a human arm would provide. Previous research suggests that IAP blocks an endotoxin called lipopolysaccharide (LPS). Because IAP is a naturally occurring enzyme that almost entirely remains in the gut rather than traveling throughout the system, researchers believe it should prove nontoxic to humans, and those who are found to have low levels, especially as they age, will simply be able to supplement.


Transplanting Gut Microbes from Long-Lived Humans into Mice to Assess the Outcomes

It is well known that the gut microbiome is influential on long-term health, and undergoes detrimental changes with advancing age. Beneficial species decline, while inflammatory and otherwise unhelpful species prosper. The reasons for these changes are not well understood, but probably involve a combination of many factors, such as diet, immune dysfunction, and so forth. There is a growing interest in the research community in assessing the contribution of gut microbiome changes to degenerative aging, and finding ways to reverse those changes.

The study noted here is less interesting for the presented data, and more interesting for demonstrating that one can in fact transplant gut microbes from a human to a mouse and expect to see results that mimic the quality of the human microbiome. Thus transplants from long-lived humans - with what is assessed via other measures to be a better, more youthful, more diverse gut microbiome - leads to healthier mice than is the case for transplants from an average older person with a more degraded gut microbiome. That this can be accomplished might lead to faster progress towards treatments that adjust the gut microbiome to a more beneficial state.

The most direct, blunt approach to therapy is some form of fecal microbiota transplant from young donor to old individual. In short-lived animal species, this resets the gut microbiome to a more youthful state and extends healthy life. In human medicine, fecal microbiota transplants are already carried out for severe conditions in which the gut is overtaken by pathological microbes. The challenges in implementation largely involve screening out undesirable microbes that a young donor can keep suppressed but an old recipient would struggle with. A possibly better approach would be a probiotic strategy of some sort, in which large volumes of desirable microbes are provided orally, encapsulated in a way that allows for their survival into the gut. These classes of therapy are close to practical realization, at present only lacking the will and the funding to move ahead.

Transplant of microbiota from long-living people to mice reduces aging-related indices and transfers beneficial bacteria

The interactions between gut microbiota and their host have become a popular topic in research. There is growing evidence to suggest that a close relationship exists between gut microbiota and aging. Age-related changes in gut microbiota occur widely among animals, with evidence of this ranging from insects to mammals. Human-based studies have revealed a trend in age-related microbiota features, which shows an increase in gut microbiota diversity from infants to adults, followed by a decrease as adults age. Researchers found signatures of extreme longevity in gut microbiota composition that were related to extreme aging. Others found 11 features shared among long-living Chinese and Italian people, including higher alpha diversity and operational taxonomic units (OTUs); they also showed that long-living people had greater gut microbiota diversity than a younger group among Chinese and Japanese populations.

High microbiota diversity has been associated with good health in general. Early research on the gut microbiota of elderly people has indicated that healthier subjects have significantly greater gut microbiota diversity than those in long-term residential care. Overall, the information obtained from studies such as these suggests that long-living people can serve as an acceptable model to investigate whether gut microbiota is a feasible target for promoting healthy aging. However, the exact roles that the microbiota play still require investigation.

Studies in animal subjects have shown that age-related microbiota can affect the lifespan of the host. Ten-day-old and 30-day-old Drosophila were used as microbiota donors for 10-day-old Drosophila. The lifespan of the 10-day-old transplant group lived significantly longer than the 30-day-old transplant group, and had a decreased frequency in intestinal barrier dysfunction. Subsequently, researchers transplanted the gut contents of young and old African turquoise killifish to old fish. Consistent with the results from Drosophila, fish transplanted with feces from young donors had a longer lifespan and were significantly more active. These results suggest that the gut microbiota of young individuals can slow host aging and prolong the lifespan of the tested species.

In the current study, the hypothesis that the gut microbiota of long-living people has the ability to delay host aging compared with those of average lifespan, is tested. To test this hypothesis, the gut microbiota of long-living (L group) and typical aging elderly people were transplanted into antibiotic-treated mice, which were then analyzed for differences in gut microbiota and aging indices. L group mice demonstrated greater microbiota diversity and beneficial bacteria, such as probiotic genera and short-chain fatty acid producers. Importantly, aging-related indices, such as lipofuscin and β-galactosidase accumulation, were less in the L group. Our experiment provided primary evidence that the gut microbiota of long-living people has the ability to delay host aging.

p53 in Cellular Senescence

Cellular senescence is one of the contributing causes of aging, in the sense that senescent cells accumulate in old tissues. Even when only a tiny fraction of all cells are senescent, their signaling causes chronic inflammation and disruption of tissue function. Senescence is, however, a helpful program when these cells exist only temporarily and are promptly destroyed. The signaling that is so harmful when maintained over the long term aids in wound healing and suppression of cancer when present for a short time only. Since the comparatively recent acceptance of senescent cells as an important cause of aging, the research community has spent a great deal of effort in better understanding the biochemistry of cellular senescence. The open access paper here is a representative example of the output of these scientific initiatives.

The classical depiction of senescence as a static, uniform, and irreversible cellular state has been progressively reconsidered, and senescence is now envisioned as a dynamic and multistep process. During the initiation of the senescence, which is also called as "primary senescence", the stressed cells may be still able to repair and/or eliminate the cause of the damage and then can escape from cell cycle arrest. For example, it was demonstrated that a small proportion of senescent embryonic fibroblasts were capable of reentering the cell cycle when p53 expression was suppressed through RNA interference.

However, these rare cases are considered to take place only in the early stage of senescence establishment. Moreover, persistent exposure to a damaging environment leads the cells to the next stage of senescence, known as "developing senescence", where cells are poised to demonstrate full-featured senescence. Nevertheless, if senescent conditions continue for extended periods of time, as it happens, for instance, in the aging process, the cells enter a third phase of senescence, known as "late senescence", in which they may be characterized by heterogeneous phenotypes such as flattened and enlarged cell shape, expanded lysosomal compartment and vacuoles, increased metabolic rate and reactive oxygen species (ROS) production, senescence-associated secretory phenotype (SASP) secretion.

In most of the models studying senescent cells, p53 (as well as the DNA damage response proteins) seems to be involved in the earlier stages, and the time factor plays an important role in the entire process. p53 activity decreases with time, and this is consistent with the idea of p53 (and p21cip1) being a crucial factor for the induction of the senescence and a gate to an early phase and still reversible senescence. On the other hand, the subsequent increase of p16 activity would be responsible for a late senescent state characterized by a distinct and permanent senescence phenotype, which is no longer reversible through p53 inhibition.


Age-Slowing Interventions in the Context of Lung Aging

Researchers here consider a very conservative set of interventions known to modestly slow the progression of aging in laboratory species, largely by altering metabolism to upregulate beneficial cellular stress responses. The researchers look through the lens of lung aging, specifically, reviewing the evidence for these therapies to slow the deterioration in lung function and onset of lung disease in older individuals, or to be the basis for treating established lung disease.

To date, the most reliable, best-researched way to extend life span is through the practice of calorie restriction (CR), which involves reducing calorie intake while simultaneously maintaining good nutritional status. Although the effects of diet on lung aging per se has thus far been rarely studied, several studies suggest a significant role of dietary modulation on lung aging. When a study examined the effects of aging on lung epithelial cells and stem cells and the effect of CR on young and old lungs, CR was identified to induce several potentially beneficial changes in lung epithelial cells, even when it is initiated at an older age, including reversal of some aging-induced changes.

The growth hormone/IGF axis can be manipulated in animal models to promote longevity; IGF-related proteins have also been implicated in risk of aging-associated diseases in humans. Indeed, a recent study which evaluated lung function parameters in a large cohort of patients with acromegaly due to excess growth hormone, revealed that these patients showed signs of small airway obstruction. However, the idea of inhibiting the growth hormone/IGF-I axis for the management of chronic obstructive pulmonary disease (COPD) may not be straightforward. Previously, there were attempts to use recombinant human growth hormone treatment which has been proposed to improve nitrogen balance and to increase muscle strength in patients with COPD, although significant beneficial effects were not observed.

A series of studies showed that mTOR inhibitor rapamycin extended lifespan in yeast, nematodes, fruit flies and mice, firmly establishing mTOR signaling as a central, evolutionarily conserved regulator of longevity. Aging may affect adaptive responses to stress decreasing autophagy through activation of mTORC1 in lung fibroblasts, and this mTOR activation may contribute to the resistance to cell death in idiopathic pulmonary fibrosis (IPF) lung fibroblasts. In addition, a recent metaanalysis of genome-wide studies across three independent cohorts reported the importance of mTOR signaling in lung fibrosis.

Sirtuins (SIRTs) are well-known mediators of aging. Suppression of cellular senescence by SIRTs is mainly mediated through delaying age-related telomere attrition, sustaining genome integrity and promotion of DNA damage repair. A study suggested that accelerated epithelial senescence which can be antagonized by SIRT6 might play a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions. When the mRNA and protein levels of all seven known sirtuins (SIRT1-7) were assessed in primary lung fibroblasts from patients with IPF and systemic sclerosis-associated interstitial lung disease in comparison with lung fibroblasts from healthy controls, these unbiased tests revealed a tendency for all SIRTs to be expressed at lower levels in fibroblasts from patients compared with controls, but the greatest decrease was observed with SIRT7.

Metformin has been shown to increase lifespan and delay the onset of age-associated decline in several experimental models. In a bleomycin model of lung fibrosis in mice, metformin therapeutically accelerated the resolution of well-established fibrosis in an AMPK-dependent manner, further supporting a role for metformin to reverse established fibrosis by facilitating deactivation and apoptosis of myofibroblasts.

An age-associated increase in chronic, low-grade sterile inflammation termed "inflammaging" is a characteristic feature of mammalian aging that shows a strong association with occurrence of various age-associated diseases. Although it is not clearly defined whether the pulmonary environment becomes inflammatory with increasing age in humans, an in vivo study using a murine model organism suggests this possibility. The lungs of old mice have elevated levels of proinflammatory cytokines and a resident population of highly activated pulmonary macrophages.