Fight Aging! Newsletter, May 8th 2017

Our understanding of the role of cellular senescence in different biological contexts has been impeded in part by the difficulty of detecting their presence within tissues. Such detection is currently performed mainly by evaluation of senescence-associated beta-galactosidase (SA-β-gal). However, SA-β-gal activity alone is not enough to allow us to conclude with confidence that cells are senescent, as positive staining can also occur in other biological contexts. Therefore, SA-β-gal staining is usually combined with staining for additional markers such as γH2AX-a marker for activation of DNA damage response. In addition, negative markers that should be absent in senescent cells can be used to exclude the cells that are not senescent. These markers indicate cell proliferation, like Ki67 or BrdU incorporation, or proteins ubiquitously present in the cell nuclei, but secreted from senescent cells and thus absent in their nucleus, like HGMB1.

The SA-β-gal and each of the markers are usually evaluated separately in consecutive sections. This procedure is not only laborious and expensive but also does not allow multiple senescence biomarkers to be detected within the same cells, limiting the possibility of quantitative evaluation of senescent cells derived from tissues. Alternatively, SA-β-gal activity within cells can be quantified by flow cytometry using 5-dodecanoylaminofluorescein di-β-D-galactopyranoside as a substrate. However, this method can be performed only on intact cells and therefore does not allow identification of intracellular markers in the same cells. Altogether, current methods do not allow detection and quantification of senescent cells in tissues based on combination of markers that is essential for their confident identification.

Conventional SA-β-gal staining fails to distinguish between different cell types that can be a source of senescent cells within complex tissues, limiting our understanding of the underlying biological phenomena. In an attempt to overcome the limitations of current methods for identification of senescent cells, we utilized ImageStreamX, an advanced imaging flow cytometer capable of producing multiple high-resolution, fluorescent and bright-field (BF) images of every cell directly in flow. Our approach combines the quantitative power of flow cytometry with high-content image analysis. We modified the traditional SA-β-gal assay to meet the requirements of the ImageStreamX and performed the assay in a single-cell suspension. Using this method, we identified and quantified senescent cells in tumors, fibrotic tissues, and normal tissues of young and aged mice.

In this study, we evaluated several biomarkers of cellular senescence and found a significant correlation between SA-β-gal staining and the lack of nuclear HMGB1 staining in vitro. This combination might allow more reliable identification of senescent cells, compared to SA-β-gal assay alone. Therefore, it provides significant advantage over existing techniques, including the use of fluorescent β-gal substrate, which does not allow combination staining with any intracellular molecular markers. Accordingly, it seems possible to take advantage of this method to screen for new senescence biomarkers that correlate with SA-β-gal activity in vivo, and would consequently open the way to a deeper understanding of the senescent state in vivo. Furthermore, the use of senescence biomarkers will potentially yield greater biological insight by allowing protein localization and colocalization to be monitored and compared between senescent and nonsenescent cells.

Through its use of cell-type-specific biomarkers, our protocol can successfully determine which cell types undergo cellular senescence and which do not. Importantly, in the experiments with mice of different age, SA-β-gal staining was performed for 12 hours in all tissues to ensure consistency. We suggest that in future studies SA-β-gal staining time has to be calibrated for each tissue and in some circumstances even different cell population, to achieve the most accurate results. Moreover, staining of the cells for live-dead markers immediately following tissue dissociation will allow quantification of SA-β-gal-positive cells specifically from the live cell population. This is particularly pertinent since the dissociation of cells from tissues might result in a certain amount of cell death. We showed that about 96% of the cells are viable following tumor dissociation, but this percentage can diverse greatly depending on the tissue examined.

Developing endurance means being able to sustain an aerobic activity for longer periods of time. As people become more fit, their muscles shift from burning carbohydrates (glucose) to burning fat. So researchers assumed that endurance is a function of the body's increasing ability to burn fat, though details of the process have been murky. Previous work into a gene called PPAR delta (PPARD) offered intriguing clues: mice genetically engineered to have permanently activated PPARD became long-distance runners who were resistant to weight gain and highly responsive to insulin - all qualities associated with physical fitness. The team found that a chemical compound called GW1516 (GW) similarly activated PPARD, replicating the weight control and insulin responsiveness in normal mice that had been seen in the engineered ones. However, GW did not affect endurance (how long the mice could run) unless coupled with daily exercise, which defeated the purpose of using it to replace exercise.

In the current study, researchers gave normal mice a higher dose of GW, for a longer period of time (8 weeks instead of 4). Both the mice that received the compound and mice that did not were typically sedentary, but all were subjected to treadmill tests to see how long they could run until exhausted. Mice in the control group could run about 160 minutes before exhaustion. Mice on the drug, however, could run about 270 minutes - about 70 percent longer. For both groups, exhaustion set in when blood sugar (glucose) dropped to around 70 mg/dl, suggesting that low glucose levels (hypoglycemia) are responsible for fatigue.

To understand what was happening at the molecular level, the team compared gene expression in a major muscle of mice. They found 975 genes whose expression changed in response to the drug, either becoming suppressed or increased. Genes whose expression increased were ones that regulate breaking down and burning fat. Surprisingly, genes that were suppressed were related to breaking down carbohydrates for energy. This means that the PPARD pathway prevents sugar from being an energy source in muscle during exercise, possibly to preserve sugar for the brain. Activating fat-burning takes longer than burning sugar, which is why the body generally uses glucose unless it has a compelling reason not to - like maintaining brain function during periods of high energy expenditure. Although muscles can burn either sugar or fat, the brain prefers sugar, which explains why runners who "hit the wall" experience both physical and mental fatigue when they use up their supply of glucose.

Interestingly, the muscles of mice that took the exercise drug did not exhibit the kinds of physiological changes that typically accompany aerobic fitness: additional mitochondria, more blood vessels and a shift toward the type of muscle fibers that burn fat rather than sugar. This shows that these changes are not exclusively driving aerobic endurance; it can also be accomplished by chemically activating a genetic pathway. In addition to having increased endurance, mice who were given the drug were also resistant to weight gain and more responsive to insulin than the mice who were not on the drug.

In endurance sport competitions such cycling, marathon runs, race walking, and cross-country skiing, "hitting the wall" is a dramatic demonstration of sudden and complete exhaustion. It is thought to be due to the depletion of liver and muscle glycogen and can be averted by training that promotes mitochondrial biogenesis, increased type I fibers, and enhanced fatty acid burning. In this study, we show that PPARδ expression correlates with endurance, and its activation by exercise mimetics, such as GW, is sufficient to increase running time by ∼100 min without changes in either muscle fiber type or mitochondrial biogenesis. Thus, the foundational core of endurance enhancement appears to be purely metabolic. Furthermore, even though the GW impact appears to be achieved via increased fatty acid metabolism, the strongest correlation to endurance is maintenance of blood glucose above 70 mg/dL.

This work identifies PPARδ as both the master regulator and key executor of adaptive changes in energy substrate use in skeletal muscle. Notably, pharmacologic activation of PPARδ replicates the exercise-induced changes in substrate utilization to preserve systemic glucose and thereby delay the onset of hypoglycemia, or "hitting the wall." While exercise-induced muscle remodeling is well documented, the health benefits have been largely attributed to mitochondrial biogenesis and fiber-type transformation. In contrast, pharmacophores that activate PPARδ promote endurance through preserving glucose, essentially "pushing back the wall," without affecting mitochondrial biogenesis or fiber-type transformation. This ability to chemically activate energetic circuits regulated by PPARδ has the potential to confer health benefits in a variety of human diseases.

Caloric or dietary restriction has been shown to increase life span in a wide variety of species. A number of proposed mechanisms for the phenomena have been suggested including: retardation of growth, decreased fat content, reduced inflammation, reduced oxidative damage, body temperature, and insulin signaling, and increase in physical activity and autophagy. However, no coherent mechanistic explanation has been generally accepted for this widely observed phenomenon that caloric restriction extends life span across the species. Yet, an obvious metabolic change associated with caloric restriction is ketosis. Increased ketone body concentrations occur during caloric restriction in widely different species ranging from Caenorhabditis elegans to Drosophila to man where ketone bodies are produced in liver from free fatty acids released from adipose tissue.

Ketone bodies were first found in the urine of subjects with diabetes creating in physicians the thought that their presence was pathological. However, it was shown that ketone bodies were the normal result from fasting in man, where they could be used in man in most extrahepatic tissue including brain. The ketone bodies, D-β-hydroxybutyrate (D-βHB) and its redox partner acetoacetate are increased during fasting, exercise, or by a low carbohydrate diet. Originally ketone bodies were thought to be produced by a reversal of the β-oxidation pathway of fatty acids. However, it was definitively and elegantly shown that the β-hydroxybutyrate of the β oxidation pathway was of the L form while that produced during ketogenesis was the D form. This fundamental difference in the metabolism of the D and L form of ketone bodies has profound metabolic effects.

Recently, it was shown that administration of D-βHB to C. elegans caused an extension of life span resulting in that ketone body to be presciently labeled as "an anti-aging ketone body". In the same experiment, L-β-hydroxybutyrate failed to extend life span. If it is accepted that the ketone body, D-βHB is an "anti-aging" compound, this could account for the widespread observation that caloric restriction, and its resultant ketosis, leads to life span extension. Many aging-induced changes, such as the incidence of malignancies in mice, the increases in blood glucose and insulin caused by insulin resistance, and the muscular weakness have been shown to be decreased by the metabolism of ketone bodies, a normal metabolite produced from fatty acids by liver during periods of prolonged fasting or caloric restriction.

In addition to ameliorating a number of diseases associated with aging, the general deterioration of cellular systems independent of specific disease seems related to reactive oxygen species toxicity and the inability to combat it. In contrast increases in life span occur across a number of species with a reduction in function of the insulin signaling pathway and/or an activation of the FOXO transcription factors, inducing expression of the enzymes required for free radical detoxification. In C. elegans, these results have been accomplished using RNA interference or mutant animals. Similar changes should be able to be achieved in higher animals, including humans, by the administration of d-βHB itself or its esters.

In summary, decreased signaling through the insulin/IGF-1 receptor pathway increases life span. Decreased insulin/IGF-1 receptor activation leads to a decrease in PIP3, a decrease in the phosphorylation and activity of phosphoinositide-dependent protein kinase (PDPK1), a decrease in the phosphorylation and activity of AKT, and a subsequent decrease in the phosphorylation of FOXO transcription factors, allowing them to continue to reside in the nucleus and to increase the transcription of the enzymes of the antioxidant pathway. In mammals, many of these changes can be brought about by the metabolism of ketone bodies. The metabolism of ketones lowers the blood glucose and insulin thus decreasing the activity of insulin signaling and its attendant changes in the pathway described above. However, in addition ketone bodies act as a natural inhibitor of class I HDACs, inducing FOXO gene expression stimulating the synthesis of antioxidant and metabolic enzymes. An added important factor is that the metabolism of ketone bodies in mammals increases the reducing power of the NADP system providing the thermodynamic drive to destroy oxygen free radicals which are a major cause of the aging process.

We all grow old. We all die. For Aubrey de Grey, a biogerontologist and chief science officer of the SENS Research Foundation, accepting these truths is, well, not good enough. He decided in his late twenties (he's currently 54) that he "wanted to make a difference to humanity" and that battling age was the best way to do it. His life's work is now a struggle against physics and biology, the twin collaborators in bodily decay. He calls it a "war on age." de Grey considers aging an engineering problem. The human body is a machine, he told me in the following interview, and like any machine, it can be maintained for as long as we want. This is not an isolated view. There is a broader anti-aging movement afoot, which seems to be growing every day.

de Grey's work is particularly interesting. For too long, he argues, scientists have been looking for solutions in all the wrong places. There is no monocausal explanation for aging. We age because the many physical systems that make up our body begin to fail at the same time and in mutually detrimental ways. So he's developed what he calls a "divide-and-conquer strategy," isolating the seven known causes of aging and tackling them individually. Whether it's cell loss or corrosive mitochondrial mutations, de Grey believes each problem is essentially mechanical, and can therefore be solved.

Sean Illing: Is there a simple way to describe theoretically what the anti-aging therapies you're working on will look like - what they'll do to or for the body?

Aubrey de Grey: Oh, much more than theoretically. The only reason why this whole approach has legs is because 15 or 17 or so years ago, I was actually able to go out and enumerate and classify the types of damage. We've been studying it for a long time, so when I started out in this field in the mid-'90s so I could learn about things, I was gratified to see that actually aging was pretty well understood. Scientists love to say that aging is not well understood because the purpose of scientists is to find things, out so they have to constantly tell people that nothing is understood, but it's actually bullshit. The fact is, aging is pretty well understood, and the best of it is that not only can we enumerate the various types of damage the body does to itself throughout our lives, we can also categorize them, classify them into a variable number of categories. We know how people age; we understand the mechanics of it. More importantly, for each category there is a generic approach to fixing it, to actually performing the maintenance approach that I'm describing, repairing the damage.

Sean Illing: Can you give me an example of one of these categories and what the approach to fixing it looks like?

Aubrey de Grey: One example is cell loss. Cell loss simply means cells dying and not being automatically replaced by the division of other cells, so that happens progressively in a few tissues in the body and it definitely drives certain aspects of aging. Let's take Parkinson's disease. That's driven by the progressive loss of a particular type of neuron, the dopaminergic neuron, in a particular part of the brain. And what's the generic fix for cell loss? Obviously it's stem cell therapy. That's what we do. We preprogram cells in the laboratory into a state where you can inject them into the body and they will divide and differentiate to replace themselves that the body is not replacing on its own. And stem cell therapy for Parkinson's disease is looking very promising right now.

Sean Illing: Is it best to think of aging as a kind of engineering problem that can be reversed or stalled? You're not trying to solve the problem of death or even aging, really. It's more about undoing the damage associated with aging.

Aubrey de Grey: Absolutely. It's a part of technology. The whole of medicine is a branch of technology. It's a way of manipulating what would otherwise happen, so this is just one part of medicine. Certainly the goal is to undo the damage that accumulates during life, and whether you call that "solving aging" is up to you.

Sean Illing: What do you say to those who see this as a quixotic quest for immortality, just the latest example of humanity trying to transcend its condition?

Aubrey de Grey: Sympathy, mainly. I understand it takes a certain amount of guts to aim high, to actually try to do things that nobody can do, that nobody's done before. Especially things that people have been trying to do for a long time. I understand most people don't have that kind of courage, and I don't hate them for that. I pity them. Of course, the problem is that they do get in my way, because I need to bring money in the door and actually get all this done. Luckily, there are some people out there who do have courage and money, and so we're making progress.

Sean Illing: Are there any ethical questions or reservations that give you pause at all?

Aubrey de Grey: Not at all. Once one comes to the realization that this is just medicine, then one can address the entire universe of potential so-called ethical objections in one go. Are you in favor of medicine or not? In order to have any so-called ethical objection to the work we do, the position that one has to take is the position that medicine for the elderly is only a good thing so long as it doesn't work very well, and that's a position no one wants to take.

Sean Illing: When will the therapies you're developing be ready for human experimentation?

Aubrey de Grey: That will happen incrementally over the next 20 years. Each component of the SENS panel will have standalone value in addressing one or another disease of old age, and some of them are already in clinical trials. Some of them are a lot harder, and the full benefit will only be seen when we can combine them all, which is a long way out.

The National Eye Institute (NEI), part of the National Institutes of Health, has opened the first stage of a federal prize competition designed to generate miniature, lab-grown human retinas. The retina is the light- sensitive tissue in the back of the eye. Over the next three years pending availability of funds, NEI plans to offer more than 1 million in prize money to spur development of human retina organoids. "None of the model systems currently available to researchers match the complex architecture and functionality of the human retina. We are looking for new ideas to create standardized, reproducible 3-D retina organoids that can speed the discovery of treatments for diseases such as age-related macular degeneration and diabetic eye disease, both leading causes of blindness."

Research models are more valuable the more closely they mimic human tissue. Researchers hope to use retina organoids to study how retinal cells interact under healthy and diseased conditions, and to test potential therapies. The ideation stage of the 3-D Retina Organoid Challenge aims to generate innovative ideas that can later be turned into concrete concepts. Running until August 1, 2017, the total prize purse for the ideation stage is 100,000. "We're looking for creative insights and application of new technology to unleash the full potential of retinal organoids. Our goal is for researchers to be able to generate or obtain retinal organoids easily so that they can be widely used for understanding diseases and testing drugs. To do this, we are encouraging entries from diverse teams of participants."

The development stage of the challenge will require demonstration of a functional retina organoid prototype. This stage is planned to launch in fall 2017 and expected to offer 1 million in prize money. Full details of the 3-D Retina Organoid Challenge prize competition are available online.

The team used cartilage cells harvested from patients who underwent knee surgery, and these cells were then manipulated in a laboratory, causing them to rejuvenate and revert into "pluripotent" stem cells, i.e. stem cells that have the potential to develop into many different types of cells. The stem cells were then expanded and encapsulated in a composition of nanofibrillated cellulose and printed into a structure using a 3D bioprinter. Following printing, the stem cells were treated with growth factors that caused them to differentiate correctly, so that they formed cartilage tissue.

Most of the team's efforts had to do with finding a procedure so that the cells survive printing, multiply and a protocol that works that causes the cells to differentiate to form cartilage. "We investigated various methods and combined different growth factors. Each individual stem cell is encased in nanocellulose, which allows it to survive the process of being printed into a 3D structure. We also harvested mediums from other cells that contain the signals that stem cells use to communicate with each other so called conditioned medium. In layman's terms, our theory is that we managed to trick the cells into thinking that they aren't alone." A key insight gained from the team's study is that it is necessary to use large amounts of live stem cells to form tissue in this manner.

The cartilage formed by the stem cells in the 3D bioprinted structure is extremely similar to human cartilage. Experienced surgeons who examined the artificial cartilage saw no difference when they compared the bioprinted tissue to real cartilage, and have stated that the material has properties similar to their patients' natural cartilage. Just like normal cartilage, the lab-grown material contains Type II collagen, and under the microscope the cells appear to be perfectly formed, with structures similar to those observed in samples of human-harvested cartilage.

Chronic inflammatory diseases such as arthritis are characterized by dysregulated responses to pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α). Pharmacologic anti-cytokine therapies are often effective at diminishing this inflammatory response, but due to the pleiotropic roles of TNF-α and IL-1 and their involvement in tissue homeostasis, the use of such therapies may have significant side effects, including increased susceptibility to infection as well as to autoimmune diseases. Moreover, excess inhibition of these cytokines can interfere with tissue regeneration and repair. Therefore, methods to dynamically deliver precisely calibrated doses of anti-inflammatory biologic therapies could improve treatments by combating cytokine-mediated pain and degeneration while spatially and temporally regulating the production of anti-cytokine drugs.

Here, we propose a regenerative medicine approach to the treatment of chronic inflammatory diseases by engineering cells that execute real-time, programmed responses to environmental cues, including pro-inflammatory cytokines. We used genome editing with the CRISPR/Cas9 system to create stem cells that antagonize IL-1- and TNF-α-mediated inflammation in an autoregulated manner. To achieve this, we selected to overtake the chemokine (C-C) ligand 2 (Ccl2) gene, which is also known as macrophage chemoattractant protein-1 (Mcp-1). The Ccl2 gene product regulates trafficking of monocytes/macrophages, basophils, and T lymphocytes. TNF-α and IL-1 serve as two of the most potent stimulators of Ccl2 expression; however, the persistence of Ccl2 expression depends on continued exposure to inflammatory cues, so resolution of inflammation results in rapid decay of Ccl2 transcripts.

Thus, we performed targeted gene addition of IL-1 and TNF-α antagonists at the Ccl2 locus to confer cytokine-activated and feedback-controlled expression of biologic therapies. These programmed stem cells were then used to engineer articular cartilage tissue to establish the efficacy of self-regulated therapy toward protection of tissues against cytokine-induced degeneration. We hypothesized that this approach of repurposing normally inflammatory signaling pathways would allow for transient, autoregulated production of cytokine antagonists in direct response to cytokine stimulation. This type of approach could provide an effective "vaccine" for the treatment of chronic diseases while overcoming limitations associated with delivery of large drug doses or constitutive overexpression of biologic therapies.

Two immune responses are important for recovery after a heart attack - an acute inflammatory response that attracts leukocyte immune cells to remove dead tissue, followed by a resolving response that allows healing. Failure of the resolving response can allow a persistent, low-grade nonresolving inflammation, which can lead to progressive acute or chronic heart failure. Using a mouse heart attack model, researchers have shown that knocking out one particular lipid-modifying enzyme, along with a short-term dietary excess of a certain lipid, can improve post-heart attack healing and clear inflammation.

Why are lipids and lipid-modifying enzymes important in inflammation and resolving inflammation? Three key lipid modifying enzymes in the body change the lipids into various signaling agents. Some of these signaling agents regulate the triggering of inflammation, and others promote the reparative pathway. The lipids modified by the enzymes are two types of essential fatty acids that come from food, since mammals cannot synthesize them. One is n-6 or omega-6 fatty acids, and the other type is n-3 or omega-3 fatty acids. The balance of these two types is important. The three main lipid-modifying enzymes compete with each other to modify whatever fatty acids are available from the diet. So, researchers asked, what will happen if we knock out one of the key enzymes, the 12/15 lipoxygenase? They reasoned that this would increase the metabolites produced by the other two main enzymes, cyclooxygenase and cytochrome P450 because they no longer had to compete with 12/15 lipoxygenase for lipids to modify. This might be a benefit because those signaling lipids produced through the cyclooxygenase and cytochrome P450 pathways were already known to lead to major resolution promotion factors for post-heart attack healing.

The researchers found that knocking out the 12/15 lipoxygenase and feeding the mice a short-term excess of polyunsaturated fatty acids led to increased leukocyte clearance after experimental heart attack, meaning less chronic inflammation. It also improved heart function, increased the levels of bioactive lipids during the reparative phase of healing, and led to higher levels of reparative cytokine markers. Additionally, the heart muscle showed less of the fibrosis that is a factor in heart failure. Besides congestive heart failure, persistent inflammation aggravates a vicious cycle in many cardiovascular diseases. Further mechanistic studies are warranted to develop novel targets for treatment and to find therapies that support the onset of left ventricle healing and prevent heart failure pathology.

In March 2017, the Second Interventions in Aging Conference was held in Cancun, Mexico. The meeting, similar to the earlier event in 2015, was focused on interventional strategies. One notable difference was that this year's meeting was much more directed toward potential interventions to target human aging. The field has been very successful over the last decade in identifying interventions that extend lifespan and healthspan in animal models such as yeast, flies, worms, mice and, to some extent, primates. However, the primary goal is to employ knowledge from basic aging research to develop novel medical strategies aimed at extending human healthspan. Aging is the biggest risk factor for a wide range of chronic diseases that, to date, medical strategies have treated as separate entities, and as they arise. Yet, aging is driven by a limited number of coordinated pathways that can be modulated, and evidence suggests that interventions delaying aging will protect against multiple age-related diseases simultaneously. Discoveries in basic aging research thus point towards a broad-spectrum, preventative, medical strategy for aging-related disease.

There were seven research topics each addressed thematically at the meeting. All were chosen because they embody different strategies to target human aging. Each session combined talks from Platform speakers with those chosen from submitted abstracts. The first and largest theme was targeted toward Organismal Aging, or understanding the intrinsic pathways that govern aging of the entire organism. The interesting aspect of these presentations is that they address strategies to modify aging that touch back to research from the early days of aging research while simultaneously pointing to novel strategies for future interventions: new mechanisms linking growth hormone signaling to aging; using mammalian models to re-evaluate the role of reactive oxygen species; new evidence for links between progeria and normal aging, interpreting these strategies in the context of possible interventions that may affect both normal and "premature" aging; linking NFκB signaling to sarcopenia, a major driver of frailty in aging; mechanisms linking calorie restriction to lifespan extension in primates; strategies to examine the impact of aging pathways in elderly human populations.

The second theme was focused on using Stem Cells to target aging, with exciting presentations on aging of epithelial stem cells in flies and mice, on links between metabolism, autophagy and aging in the hematopoietic system, and on how adult stem cells self-organize into functional configurations. The third theme, addressing Cellular Mechanisms of Longevity Assurance, focused on pathways suspected to modulate aging, including autophagy, mitochondrial function and aging with emphasis on the role of small mitochondrial peptides, and the hypoxia pathway. Theme 4 centered on Epigenetics, which is not only becoming a target for intervention in aging, but is rapidly becoming a leading candidate for providing biomarkers of biological age: mechanisms leading to transgenerational inheritance of epigenetic marks that impact lifespan; links between the epigenome and activation of somatic retrotransposons, and how this activation may drive senescence and aging; further promoting the epigenetic clock as a marker of accelerated and delayed aging.

Theme 5 was designed to take a Systems Aging viewpoint. Such a holistic understanding of the aging process is in a sense the ultimate goal of the research. Is it possible to understand such a complex process as aging not just one gene and pathway at a time but in totality? The final theme centered on Signaling and Metabolism, hitting the major metabolic pathways that are linked to aging and that can be targeted with interventional strategies. These include the mTOR pathway and rapalogs; dietary restriction and links through mTOR to regulation of mRNA splicing; NAD metabolism and sirtuins; mitochondrial roles in regulating aging and metabolism.

A team of scientists has identified an enzyme that could help in the continuous battle against mid-life obesity and fitness loss. They used mice to test the potentially key role this enzyme plays in obesity and exercise capacity. They administered an inhibitor that blocked the enzyme in one group being fed high-fat foods, but withheld it in another. The result was a 40 percent decrease in weight gain in the group that received the inhibitor. Researchers have known for years that losing weight and maintaining the capacity to exercise tend to get harder beginning between ages 30 to 40 - the start of midlife. Scientists have developed new therapies for obesity, including fat-fighting pills. However, many of those therapies have failed because of a lack of understanding about the biological changes that cause middle-aged people to gain weight, particularly around their abdomen.

Researchers searched for biochemical changes that occurred in middle-aged animals (human equivalent of 45 years). They found that an enzyme called DNA-dependent protein kinase, or DNA-PK, increases in activity with age. Further work showed that DNA-PK promotes conversion of nutrients to fat and decreases the number of mitochondria, tiny organelles in the cells that turn fat into energy to fuel the body. Mitochondria can be found in abundance among young people, but the numbers drop considerably in older people. Researchers know that decreased mitochondria can promote obesity as well as loss of exercise capacity. The researchers theorized that reducing DNA-PK activity may decrease fat accumulation and increase mitochondria number as well as promote fat burning. The researchers tested their theory by orally administering a drug that inhibits DNA-PK and found that, in addition to preventing weight gain in the mice, the inhibitor drug boosted mitochondrial content in skeletal muscle, increased aerobic fitness in obese and middle aged mice, and reduced the incidence of obesity and type-2 diabetes. The study opens the door to the development of a new type of weight-loss medication that could work by inhibiting DNA-PK activity, however DNA-PK inhibitors have yet to be tested this way in humans.

People with no major heart disease risk factors in middle age stay healthy and live longer, according to a 40-year study. Compared to those who had two or more high risk factors in middle age, those who reached age 65 without a chronic illness lived an average 3.9 years longer and survived 4.5 years longer before developing a chronic illness, researchers found. They also spent 22 percent fewer of their senior years with a chronic illness - 39 percent compared to 50 percent - and saved almost 18,000 in Medicare costs.

Researchers examined data from the Chicago Health Association study, which included initial health assessments in the late 1960s/early 1970s and has followed participants on an ongoing basis using Medicare health records. Researchers determined how many participants had favorable factors such as non-smokers, free of diabetes, normal weight, blood pressure and cholesterol levels versus those with elevated risk factors or high risk factors. Looking solely at heart disease in 18,714 participants who reached age 65 without having a heart attack, stroke or congestive heart failure, those with all favorable risk factors lived 6.9 years longer without heart disease and spent 46.5 percent fewer of their senior years with heart disease.

"We need to think about cardiovascular health at all stages of life. The small proportion of participants with favorable levels in their 40s is a call for all of us to maintain or adopt healthy lifestyles earlier in life. But risk factors and their effects accumulate over time, so even if you have risks it's never too late to reduce their impact on your later health by exercising, eating right, and treating your high blood pressure, cholesterol and diabetes." The data is even more grim than a 2011-12 national survey suggesting only 8.9 percent of U.S. adults age 40-59 had five or more "ideal" health factors.

Researchers at have successfully developed a method to grow inner ear tissue from human stem cells - a finding that could lead to new platforms to model disease and new therapies for the treatment of hearing and balance disorders. "The inner ear is only one of few organs with which biopsy is not performed and because of this, human inner ear tissues are scarce for research purposes. Dish-grown human inner ear tissues offer unprecedented opportunities to develop and test new therapies for various inner ear disorders."

The research builds on the team's previous work with a technique called three-dimensional culture, which involves incubating stem cells in a floating ball-shaped aggregate, unlike traditional cell culture in which cells grow in a flat layer on the surface of a culture dish. This allows for more complex interactions between cells, and creates an environment that is closer to what occurs in the body during development. By culturing human stem cells in this manner and treating them with specific signaling molecules, the investigators were able to guide cells through key processes involved in the development of the human inner ear. This resulted in what the scientists have termed inner ear "organoids," or three-dimensional structures containing sensory cells and supporting cells found in the inner ear.

"This is essentially a recipe for how to make human inner ears from stem cells. After tweaking our recipe for about a year, we were shocked to discover that we could make multiple inner ear organoids in each pea-sized cell aggregate." The researchers used CRISPR gene editing technology to engineer stem cells that produced fluorescently labeled inner ear sensory cells. Targeting the labeled cells for analysis, they revealed that their organoids contained a population of sensory cells that have the same functional signature as cells that detect gravity and motion in the human inner ear. "We also found neurons, like those that transmit signals from the ear to the brain, forming connections with sensory cells. This is an exciting feature of these organoids because both cell types are critical for proper hearing and balance."

Aging is a complex and multi-factorial process characterized by increased risk of adverse health outcomes. Understanding the intimate mechanisms of aging is therefore instrumental for contrasting its negative correlates. As initially proposed in the "mitochondrial theory of aging", mitochondria are deeply involved in the aging process mainly through respiratory dysfunction and oxidant generation. Although unique as fueling systems within the cell, mitochondria participate in other essential functions, including heme metabolism, regulation of intracellular calcium homeostasis, modulation of cell proliferation, and integration of apoptotic signaling. It is therefore crucial that a pool of healthy and well-functioning organelles is maintained within the cell. To this aim, a comprehensive set of adaptive quality control processes operates via interrelated systems, including pathways pertaining to protein folding and degradation, mitochondrial biogenesis, dynamics, and autophagy (mitophagy). The activation of individual mitochondrial quality control (MQC) pathways depends on the degree of mitochondrial damage. Due to these vital responsibilities, disruption of the MQC axis is invoked as a major pathogenic mechanism in a number of disease conditions.

Together with mitochondrial dysfunction, chronic inflammation is another hallmark of both aging and degenerative diseases. Interestingly, emerging evidence suggests that the two phenomena are related to one another. In particular, circulating cell-free mitochondrial DNA (mtDNA), one of the cell damage-associated molecular patterns (DAMPs), has been proposed as a functional link between mitochondrial damage and systemic inflammation. Indeed, mtDNA, which is released as a result of cellular stress, contains hypomethylated CpG motifs resembling those of bacterial DNA and is therefore able to induce an inflammatory response. These regions bind and activate membrane or cytoplasmic pattern recognition receptors (PRRs), such as the Toll-like receptor (TLR), the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR), and the cytosolic cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) DNA sensing system-mediated pathways. The mechanisms responsible for the generation of mitochondrial DAMPs as well as their contribution to the inflammatory milieu that characterizes aging and its associated conditions are not completely understood.

Population aging poses a tremendous burden on the society. This has instigated intense research on the mechanisms that make the elderly more susceptible to diseases and disability. Several processes have been identified. Among these, inflamm-aging, a condition of chronic inflammation that develops independent of infections, has gained special attention. The cellular mechanisms responsible for inflamm-aging are not fully understood. However, recent studies suggest that a danger cellular-driven response may represent a relevant player. The coexistence of oxidative stress resulting from mitochondrial dysfunction and sterile inflammation has been summarized in the concept of oxy-inflamm-aging that merges the role of inflammation and oxidative stress in the aging process. Specific "danger molecules" generated in an oxidative milieu have been proposed to contribute to inflamm-aging. From this perspective, aging may be envisioned as the result of an "autoimmune-like" process. Given the role played by mitochondrial DAMPs in the activation of sterile inflammation, the mechanisms favoring organelle damage, in particular failing MQC processes, represent a relevant matter to be addressed by future investigations.

Like all the other organs, there are structural and functional changes in the liver during aging, including diminished functions. Notably, a decrease in regenerative capacity in aging liver has been observed in old patients who had severe viral and toxic injury. In addition, studies on liver transplantation in human patients showed lower graft and recipient survival if the donor was in advanced age. Similar results were also observed after liver transplantation in rats. Therefore, investigating the mechanisms of declined regeneration in liver is critical to understand age-associated hepatic pathologies and diseases.

Decreased tissue regeneration and homeostasis are frequently associated with impaired stem cell function, implicating alterations of stem cells within tissues and organs during aging. As recently reported, aging-associated phenotypical and functional variations have been observed for adult stem cells or progenitor cells in various tissues, including epidermis, muscle, blood and brain. Age-related decrements in stem-cell functionality may occur at different levels, including cell-autonomous dysfunction, altered niche where stem cells reside, systemic milieu and the external environment. Liver is an organ with low turnover in homeostasis, but high regenerative capacity under acute injury. However, little is known about the changes of stem cells within liver responsible for liver regeneration upon liver injury during aging.

Liver progenitor cells (LPCs), also known as 'oval cells', are a stem cell population within the liver. Upon massive liver injury, LPCs may be activated. LPC expansion occurs in many human liver diseases and experimental animal models, and treatment with LPCs could prevent liver injury in rodents. Therefore, LPCs play an important role in maintaining the homeostasis and regeneration of the liver. Characterizing biological properties LPCs during aging will be important to gain insight into age-associated liver pathologies and disease.

According to the 'free-radical theory' of aging, endogenous oxidants could be generated in cells and resulted in cumulative damage. Those oxidants, free reactive oxygen species (ROS), are specific signaling molecules regulating biological processes under both physiological and pathophysiological conditions. Within a certain extent, the generation of ROS is essential to the maintenance of cellular homeostasis. However, excessive generation of ROS might lead to the damage of various cell components and the activation of specific signaling pathways, which will influence aging and the development of age-related diseases. Neutrophils can be recruited by a variety of cytokines or signals. Neutrophil-derived ROS are generated during the process of respiratory burst and are important for neutrophil bactericidal activity. Previous studies have found that spontaneous ROS production from neutrophils may increase with age and represent the different aspect of age-associated immune dysregulation.

Our findings demonstrate that liver regeneration and LPC activation are negatively regulated during aging. Impairment of liver regeneration in old mice might not be resulted from intrinsic changes of LPCs, but from changes of the stem cell niche including neutrophils and hepatic stellate cells. Based on our findings, we propose the following model. In old mice, upon induced liver injury, hepatic stellate cells produce CXCL7 to recruit neutrophils into liver. After neutrophils infiltrate into liver, they are activated and a neutrophil oxidative burst is induced. Then, neutrophil-derived excessive oxidative stress induces DNA double strand damage in LPCs and restricts LPC proliferation, leading to the impairment of liver regeneration. Our findings establish a mechanistic link between LPCs and the stem cell niche including neutrophils and hepatic stellate cells, during liver regeneration in old mice.

Measuring aging biologically rather than chronologically provides a personalized view to an optimal, rather than "normal" or "typical" health. Throughout the course of life, each of us gradually departs from the health trajectory defined by our individual genome. Even in the case of identical twins, substantial differences in the timing of the onset development of particular aging-associated symptoms are commonplace. Hence, an adult individual's rate of ageing depends primarily on lifestyle rather than genes. The newly introduced concept of anti-aging interventions enables individuals to actively modify their lifestyles or pharmacologically correct for accumulating biochemical or functional deficits. In order to properly evaluate relative efficiency of these interactions, objective measures of attained ageing are necessary.

At best, biological age can be reflected by overall resemblance of an aged individual to an average degree of age-associated changes observed in a given population at given age. In the frame of this definition, any departure from population-wide standard of aging stems from a combination of environmental and genetic factors that either promote or delay the development and subsequent involution of various physiological systems and their capability to adapt. Therefore, a positive or a negative difference between biological and chronological age, observed in a given individual, may be interpreted as either speeding up or slowing down the ageing process, thus, providing a measure for an evaluation of one or another anti-ageing intervention.

There is a long history of attempts to determine biological age and quantify the tempo of the process of ageing. Typically, age determination utilizes one or another molecular facet of ageing, for example, the degree of the damage to cell's DNA. Among more recently developed integrative biomarkers of aging is the GlycanAge index that profiles the structural details of sugar chains attached to the conserved N-glycosylation sites of three types of IgG molecules. This index reflects the level of systemic inflammation, predicts chronological age with standard deviation of 9.7 years, and is superior to age evaluation using telomere length. Peripheral blood mononuclear cells (PBMCs) mRNAs-based "transcriptome age" index predicts chronological age with mean absolute error of 7.8 years. Even more precise PBMCs-based "epigenetic age" relies the methylation of three CpG sites located in ITGA2B, ASPA and PDE4C genes with standard deviation of less than 5 years. An increase in the number of profiled CpG dinucleotides to 353 improves epigenetics-based age estimates by decreasing an error down to 2.9 years.

It should be noted that all the techniques described above require specialized equipment and skilled laboratory personnel, thus, limiting their clinical applicability. On another end of the spectrum are age-predicting models not specifically connected to any particular mechanism of aging, for example, deep neural networks (DNNs) modules evaluating common blood biochemistry and cell count tests. Though the accuracy of this model is quite high, the number of parameters in the model is also high. Since deep neural nets are, in a nutshell, "black boxes", the dissection of these models into mechanistic insights into the process of ageing is impossible. The majority of the techniques described above have not yet entered clinical practice. The major culprits causing this lack of translation to the clinic have been a high number of the parameters requiring evaluation, and the laboratory rather than clinical nature of tests being performed. From a clinical perspective, the most convenient estimate of biological age would be the one relying on a combination of biochemical and physiological parameters typically evaluated in course of annual physical exam.

In this study, we attempt the dissection of biochemical and clinical predictors of age, the development of a predictive model for biological age, and exploration of the deviation of these predictions from chronological age in a cohort of 303 individuals. We quantified 89 clinical and biochemical parameters, then selected the top five parameters with a highest Pearson's correlation with chronological age. Importantly, all five of these parameters reflect the functioning of the cardiovascular system. The outputs of the gender-specific linear regression models predicting chronological age were compared to actual age of the subjects. Substantially higher differences between the predicted age and the calendar age were noted for patients with Type 2 Diabetes Mellitus (T2D) as compared to non-T2D controls. We believe that the proposed gender-specific models, which we named Male and Female Arterial Indices, may serve as a good approximation for an elusive biological age. Importantly, the proposed age-approximation techniques rely on functional tests which do not require specialized laboratory equipment and, therefore, could be performed in hospitals and community healthcare settings.