Fight Aging! Newsletter, March 9th 2020

Methuselah Fund has found success by investing in the longevity field when almost no one else was willing to take the risk. The M Fund has proved to be an invaluable resource in our efforts to put mission first and money second. M Fund continues to perform well and plans for a second Fund are currently underway. The Methuselah Fund hopes to continue to guide investments into the winners of the longevity field for many years to come.

New Parts for People: Volumetric

Incubated by the Methuselah Foundation, Volumetric was featured on the cover of Science Magazine in May 2019 due to their technological breakthrough. Their proprietary light-based 3D bio-printers and bio-inks represent a 10x leap in speed and 5x leap in resolution compared to legacy bio-printing methods, making them a leading competitor for the NASA Tissue Engineering Prize. In September 2019, the Vascular Tissue Challenge, managed by the Methuselah Foundation for NASA, entered its final phase of the project. By the end of September 2019, a total of 21 teams successfully completed their trial applications after the last Vascular Tissue Challenge Summit hosted at NASA Ames Research Center. Three teams have been approved to move into the final Trial Phase of the Challenge. Another five teams are in a second round of review with the judging committee and are nearing approval of their trials. We are looking forward to an exciting year with upcoming Vascular Tissue Challenge trials taking place to mark a renaissance in bioengineering of thick tissues.

Curing Alzheimer's disease: Leucadia Study Results

Leucadia Therapeutics discovered a major trigger for Alzheimer's disease pathology and has developed a patented strategy to correct that condition. Leucadia's patented Arethusta technology restores cerebrospinal fluid flow across the cribriform plate, improving the clearance of toxic metabolites from the earliest regions of the brain to be affected by Alzheimer's disease. Leucadia will soon pursue FDA approval for clinical trials. Before that, however, they will finish a 2,000-person cribriform plate imaging study called Project Cribrose by mid-2020. One exciting and more immediate development is that Leucadia has created a diagnostic algorithm using sophisticated machine learning technology. Project Cribrose will optimize and enhance this algorithm so that Leucadia can start providing a diagnostics tool to find out a patient's likelihood of developing Alzheimer's.

Anti-aging Drugs: Oisín Bio Human Trial

Oisín is pursuing a scientifically sound, targeted approach that may have the potential to address the biological underpinnings of some of society's most devastating age-related diseases by addressing the damage created by the aging process itself. In preclinical studies, the company's investigational therapeutics have significantly reduced senescent cell burden in naturally aged mice and extended median lifespan by more than 20% even when the treatment was started in old age. The company has advanced studies in non-human primates and has spun out another company, OncoSenX to prove the platform and technology in one indication, cancer. This company was formed with an original grant from the Methuselah Foundation to use Oisín Bio's technology to target prostate cancer. The project was a success and the company is targeting starting phase 1 trials in Canada this year.

Reversing Aging by Reprogramming Cells: Turn Bio

Turn Bio has developed a technology capable of safely reprogramming how DNA functions epigenetically, which is paving the way to a potentially translatable strategy for in-vivo and ex-vivo cell rejuvenation treatments. They are also developing interventions that are focused on treating skin rejuvenation, osteoarthritis, and muscular diseases. Turn Bio has successfully achieved in situ delivery in multiple tissues, such as in muscles, eyes, and brain. They are also in the process of testing systemic delivery. Currently, their most promising approach is based on nanotechnology developed to deliver a mRNA cocktail to tissues - accurately and without invoking an adverse immune response.

Accelerating Drug Discovery: Viscient

Founded and staffed by a team composed of former Organovo and Ardea Biosciences scientists and entrepreneurs, Viscient is driving a revolution in drug discovery, overthrowing the old paradigm of discovery in animal models. It is our conviction that the age of using mice as the primary research model for biotech is rapidly passing, to be replaced by tissue engineered organoids. Methuselah's goal in investing in Viscient is to drive the actual use of human 3D printed tissue models, and through their demonstrated vast superiority to animal models, lead in reorienting the industry and the regulators away from animal testing and to more efficient, effective, and relevant models of disease.

The number of Americans who are age 65 or older is projected to double to more than 90 million in 2060, translating to nearly 25% of the population, due to the natural aging of the Baby Boomer generation. Today, approximately 80% of older adults have at least two chronic diseases, such as heart disease, cancer, stroke, or diabetes. This trend creates a need to solve the projected onslaught of health problems we face and is fueling scientists to dive into the molecular causes of aging and find medicines that help people live long, healthy lives.

Clusters of chromatin - the mix of DNA and protein normally found in the cell nucleus - leak out to the cytoplasm in senescent cells, triggering inflammatory signals that can promote a number of undesirable health conditions. Researchers set out to find what prompts the formation of chromatin clusters in the first place, embarking on a series of experiments using a human lung cell model of senescence. They found that mitochondria were the culprits driving the formation of pro-inflammatory cytoplastic chromatin and did so through a retrograde communication path to the nucleus.

The scientists also found that an HDAC inhibitor, an FDA-approved drug currently used to treat certain cancers, transformed senescent cells from a large and flat form to a healthier and more visually youthful condition. The HDAC inhibitor-treated cells also had better mitochondrial function, less cytoplasmic chromatin and produced less inflammatory signals. The scientists observed similar beneficial effects when examining the livers of mice in which senescence was induced through radiation or high doses of acetaminophen. However, the side effects of HDAC inhibitors - which include fatigue, nausea and more - make the drugs too toxic for use in preventing disease in healthy individuals.

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown.

Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction.

Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP.

Despite the fundamental requirement for lifelong establishment and maintenance of an overall effective and adequate defense against pathogens, the function of the immune system deteriorates with age, affecting both innate and adaptive immune responses (immunosenescence). The thymus, which reaches its maximal size and T cell output during early postnatal life, exhibits early thymic involution, a phenomenon that becomes even more prominent with advancing age. Although the size of the human thymus seems to remain unchanged throughout life under normal conditions, in other vertebrates, it declines during aging. Nevertheless, in almost all vertebrates having a thymus, thymic cellularity is progressively decreased and replaced by adipose tissue over time, resulting in perturbation of the normal tissue architecture. Since T cell production is proportional to thymic epithelial tissue mass, thymic involution results in significant loss of its capability for de novo generation of immunocompetent T cells. The net outcome is a decline in frequency and function of naïve T cells, leading to a restricted T cell repertoire in the periphery.

Even though age-associated thymic regression represents one of the most recognizable features of the aging immune system, the underlying mechanisms are not well understood. Several candidates have been proposed, suggesting that thymic regression involves the interplay of various and different mechanisms; interestingly, there are lines of evidence that in this complex process, the thymic stroma and especially the TECS are the most sensitive compartment. A number of studies reported that sex steroid hormones, and especially androgens, contribute to age-associated thymic involution. This notion was based on the observations (a) that thymic involution, although beginning in early postnatal life, is more pronounced with the onset of puberty when sex steroid levels increase and (b) that high doses of sex steroid administration cause degeneration of the thymus.

Numerous studies have also implicated the growth hormone- (GH-) insulin-like growth factor- (IGF-) I axis in thymus regression. Both hormones promote thymic growth, and lately, GH has been used as an alternative strategy to rejuvenate the thymus in certain immunodeficiency disorders associated with thymic atrophy. GH and IGF-I have been also considered as regulators of age-associated thymic involution, since GH production declines with age. However, the effects of hormone treatment on thymus size in older mice are limited, implying that there are other factors that prevent thymic atrophy.

The phenomenon of infection-induced inflammation and consequently thymus regression has also been reported; in human studies as well as in animal experimental models, infections with pathogens led to thymic atrophy, although the underlying mechanisms have not been extensively studied. Lately, a new player suggested to be involved in accelerated thymus involution and dysfunction with age is oxidative stress. Notwithstanding that the generation of reactive oxygen metabolites is an integral feature of normal cellular metabolism, the accumulation of such genotoxic and proteotoxic oxygen-derived by-products seems to exert detrimental effects on thymic tissue. Contrariwise, genetic or biochemical enhancement of antioxidant activity has been proven to ameliorate thymic atrophy.

Similarly, oxidative damage is also a well-documented inducer of cellular senescence, a state of permanent cell cycle arrest. The accumulation of senescent cells maintains an inflammatory milieu (inflammaging) that causes tissue remodeling, affects the regenerative potential and proper function of tissues/organs due to exhaustion of progenitor and stem cells, and, ultimately, promotes aging and age-related pathologies. Considering that (1st) oxidative stress has been linked to both the induction of cellular senescence and thymic involution and (2nd) aging is characterized by accumulation of senescent cells as well as a decline in thymus function, it is not unreasonable to assume that cellular senescence may exert a critical role in the induction of thymic involution, with oxidative stress being the common denominator. Indeed, recent evidence, from human and animal studies, supports this notion.

A new study shows that microglia - the resident immune cells of the brain - initially protect the blood-brain barrier from damage due to "systemic inflammation," a condition of chronic inflammation associated with factors like smoking, ageing, and diabetes, and leading to an increased risk of neurodegenerative disorders. However, these same microglia can change their behavior and increase the blood-brain barrier permeability, thereby damaging it.

A key point of interest was the systemic inflammation induced by injecting the mice with an inflammation-inducing substance. Such injections resulted in the movement of microglia to the blood vessels and increased the permeability of the blood-brain barrier within a few days. Then, the microglia initially acted to protect the blood-brain barrier and limit increases in permeability, but as inflammation progressed, the microglia reversed their behavior by attacking the components of the blood-brain barrier, thus increasing the barrier's permeability. The subsequent leakage of molecules into the brain had the potential to cause widespread inflammation in the brain and consequent damage to neurons.

Uncontrolled inflammatory responses in the brain can cause a range of cognitive disorders and adverse neurological effects, and drugs that target microglia may help patients avoid such problems by preserving the integrity of the blood-brain barrier. More studies are required to understand more about the processes underlying the microglial behaviors observed in this study. Nevertheless, the study's results offer hope for the development of therapies that could "force" microglia to promote blood-brain barrier integrity and prevent microglia from transitioning to behaviors that damage the barrier.

Microglia are active surveyors of brain parenchyma with important roles in sculpting and coordinating neural circuits in healthy brains that respond rapidly to form a range of reactive phenotypes in brain infection and damage. Activated microglia play roles in a range of acute and neurodegenerative diseases, where they can help clear neuronal damage by phagocytosis, but can also contribute to disease progression by releasing molecules that can initiate a neuroinflammatory states. Microglia can also respond to peripheral inflammatory diseases.

A key question is how microglia change phenotypes when the primary pathological insult resides in the peripheral organs and systemic circulation. Determining how these systemic and neuronal inflammatory responses are linked may help reduce the deleterious impact of systemic immune activation and inflammation on cognitive function and susceptibility to brain disease. The blood-brain barrier (BBB) represents a major pathway by which systemic inflammation and immune responses potentially interact with the brain microenvironment.

The goal of this study was to examine the role of microglia in responding to systemic infection and inflammation, and its contribution to BBB integrity. Using two different models of peripheral inflammation, MRL/lpr mice and mice treated for 7 days with lipopolysaccharide, we demonstrated that resident brain microglia migrate to cerebral vessels during systemic inflammation in response to the release of the chemokine CCL5 from endothelial cells. This triggers microglial cells to express CLDN5 and to infiltrate through the neurovascular unit, thus contacting endothelial cells and forming tight junctions to maintain BBB integrity. Consistently, partial microglial ablation or blocking CCL5 signaling, actually increased BBB permeability during the early stages of inflammation.

Mitochondrial dysfunction, including a decreased oxidative phosphorylation capability and increased reactive oxygen species (ROS) production, is substantially responsible for aging and age-related features. Studies in various organisms, such as nematodes, Drosophila, rodents, and humans, have strongly supported that aging is closely associated with mitochondrial dysfunction. Thus, protection of the mitochondrial structure or stimulation of mitochondrial function is considered as practical ways in anti-aging. However, since most of the mitochondrial damage is irreversible during aging process, the agents can always provide limited protection.

Mitochondrial therapy (mitotherapy) is to transfer functional exogenous mitochondria into mitochondria-defective cells for recovery of the cell viability and consequently, prevention of the disease progress. Accumulating evidence has indicated that exogenous mitochondria can directly enter animal tissue cells for disease therapy following local and intravenous administration. In our recent reports, systemic injection of isolated mitochondria could reduce liver injury induced by acetaminophen and high-fat diet through improving hepatocyte energy supply and decreasing oxidative stress. Therefore, we assumed that the mitochondria isolated from young animals (young mitochondria) into aged ones might play a role in anti-aging.

In this study, we intravenously administrated the young mitochondria into aged mice to evaluate whether energy production increase in aged tissues or age-related behaviors improved after the mitochondrial transplantation. The results showed that heterozygous mitochondrial DNA of both aged and young mouse coexisted in tissues of aged mice after mitochondrial administration, and meanwhile, ATP content in tissues increased while reactive oxygen species (ROS) level reduced. Besides, the mitotherapy significantly improved cognitive and motor performance of aged mice. Our study, at the first report in aged animals, not only provides a useful approach to study mitochondrial function associated with aging, but also a new insight into anti-aging through mitotherapy.

A new handheld 3D printer can deposit sheets of skin to cover large burn wounds - and its "bio ink" can accelerate the healing process. The device covers wounds with a uniform sheet of biomaterial, stripe by stripe. The bio ink dispensed by the roller is composed of mesenchymal stromal cells (MSCs) - stem cells that differentiate into specialized cell types depending on their environment. In this case, the MSC material promotes skin regeneration and reduces scarring. The team unveiled the first prototype of the skin printer in 2018. The device was believed to be the first device of its kind to form tissue in situ, depositing and setting in place in two minutes or less. "Previously, we proved that we could deposit cells onto a burn, but there wasn't any proof that there were any wound-healing benefits - now we've demonstrated that."

The current method of care for burns is autologous skin grafting, which requires transplantation of healthy skin from other parts of the body onto the wound. But large, full-body burns pose a greater challenge. Full-thickness burns are characterized by the destruction of both the outermost and innermost layers of the skin; these burns often cover a significant portion of the body.

Since 2018, the printer has gone through 10 redesigns, as the team moves towards a design they envision surgeons using in an operating room. The current prototype includes a single-use microfluidic printhead to ensure sterilization, and a soft wheel that follows the track of the printhead, allowing for better control for wider wounds. The researchers believe that the handheld skin printer could be seen in a clinical setting within the next five years. "Once it's used in an operating room, I think this printer will be a game changer in saving lives. With a device like this, it could change the entirety of how we practice burn and trauma care."

Dietary restriction (DR) extends health and life span across taxa, from baker's yeast to mice, with very few exceptions. The reduction in total calories - or restriction of macronutrients, such as protein - extends life span reliably. Although the precise universal mechanisms that connect DR to ageing remain elusive, translation of DR's health benefits to human medicine is deemed possible. The widespread assumption of DR's translational potential originates from the notion that DR's beneficial effects are facilitated by shared evolutionary conserved mechanisms, as beneficial effects of DR are observed across taxa. Experiments on our close evolutionary relatives, rhesus monkeys (Macaca mulatta), have demonstrated that DR could be translational. Still, the mechanisms by which these benefits are accrued physiologically may differ between species, as no single genetic or pharmaceutical manipulation mimicking the benefits of DR across model organisms exists.

Shared universal mechanisms can only be inferred from the ubiquity of the DR longevity response in the animal kingdom, when the selection pressures responsible for such evolutionary conservation are understood. The DR response itself may have evolved once, and mechanisms might be conserved. Alternatively, DR could have undergone convergent evolution, either using similar mechanisms - or by adopting alternative ones. Only if the DR response is rooted in ancient physiology (i.e., evolved once or through convergent evolution) can possible translation of mechanistic research on model organisms be confidently inferred.

Guided by the conviction that DR evolved as an adaptive, pro-longevity physiological response to food scarcity, biomedical science has interpreted DR as an activator of pro-longevity molecular pathways. Current evolutionary theory predicts that organisms invest in their somatic tissues during DR, and thus, when resource availability improves, should outcompete rich-fed controls in survival and/or reproduction. Testing this prediction in Drosophila melanogaster (more than 66,000 individuals across 11 genotypes), our experiments revealed substantial, unexpected mortality costs when flies returned to a rich diet following DR. The physiological effects of DR should therefore not be interpreted as intrinsically pro-longevity, acting via somatic maintenance. We suggest DR could alternatively be considered an escape from costs incurred under nutrient-rich conditions, in addition to costs associated with DR.

Many researchers now view senescence as a beneficial process that evolved as a developmental and cancer prevention mechanism, but one that came with a tradeoff of the damage senescent cells can cause as they accumulate with age. There are still many unanswered questions about how these cells function, but it is already clear to scientists in the field that senescent cells influence a range of age-related pathologies, at least in rodents. Genetic ablation of senescent cells reduces the number of atherosclerotic plaques in mice, improves cartilage development in mouse models of osteoarthritis, boosts bone strength in murine models of osteoporosis, and even staves off neurodegenerative symptoms in models of Alzheimer's disease. These findings have a number of scientists thinking: If clearing senescent cells had such beneficial effects on health, could drugs be developed to do just that?

James Kirkland's group at the Mayo Clinic had started to search for senolytic agents long before the scientific community was convinced of senescent cells' role in aging, but it took him years to work out a good strategy to identify them. In the mid-2000s, his team tried developing toxins or antibodies that target senescent cells, but none of these approaches succeeded in killing senescent cells while sparing non-senescent ones. In 2013, it occurred to Kirkland's team to target the molecular machinery known to be used by senescent cells to defy death. The cells must have those mechanisms in place to avoid undergoing the apoptotic processes that would typically follow exposure to the high levels of harmful proteins they are producing, the team reasoned. Using a bioinformatics approach, the researchers identified several anti-apoptotic pathways that are upregulated in senescent cells, including certain pathways used by malignant B cells to avoid apoptosis and cause lymphoma.

They then screened for approved drugs and natural products that targeted those pathways and thus selectively killed senescent cells. To the group's surprise, two compounds appeared very effective in killing senescent cells in vitro as well as in mice: dasatinib, approved in the US to treat certain leukemias and lymphomas, and quercetin, which is used as a nutritional supplement. "I thought we'd have to screen millions of compounds to get drugs that regulate senescence." But it took fewer than 50 drugs to get the first hits.

The brain depends on the continuous delivery of oxygen and energy substrates due to its high-energy demand and the lack of long-term energy storage. The cerebral vasculature is well suited for this purpose, where regional cerebral blood flow (rCBF) is tightly regulated and can adapt to match the local energy demands of the nervous tissue. While the topology, anatomy and signalling cascades of the cerebral vasculature is unique to serve its specialized functions, its vascular bed is connected to the general circulation of the body. Thus, any change in blood content (e.g., as part of haemostasis, inflammation or infection) or haemodynamic and biomechanical changes of central blood vessel will affect the cerebral vasculature. Conversely, alterations of the cerebral vessels might have systemic effects. We know that many brain diseases are associated with vascular dysfunctions.

Alzheimer's disease (AD) impairs cognition, memory, and language and causes dementia. AD is defined by deposition of fibrillar amyloid-β (Aβ) plaques and neurofibrillary tangles of hyperphospohorylated tau and neurodegeneration. Accumulating evidence has shown that cerebrovascular disease is a common comorbidity in the presence of AD - and can on its own cause cognitive impairment and dementia (known as vascular dementia) - that contributes additively to its symptomatology and lowers the threshold for the development of dementia. However, given the marked structural changes of the microvasculature, an alternative hypothesis has been proposed stating that vascular dysfunction causes AD-related neuropathology and cognitive impairment (the "vascular hypothesis of AD"). Today, the picture seems more complex and far from complete. It is believed that neuropathological and vascular pathways interact synergistically and feedback to each other to potentiate AD symptoms.

Cerebral vascular abnormalities are highly prevalent in AD patients and can result in cognitive impairment and dementia, and thus can add to the symptomatology caused by AD pathology. However, many vascular processes directly affect and modulate, and often proceed AD neuropathology, the most important one being blood-brain barrier (BBB) impairment and hemodynamic dysfunction. This implicates vascular dysfunction as an integral part of AD etiology and pathophysiology. The interaction is bidirectional, where AD neuropathology can also lead to changes in vascular function. In addition, many changes observed to occur at the cerebral vasculature are related to systemic vascular abnormalities, which occur during aging and can be accelerated and aggravated by cardiovascular diseases. Thus, the cerebral vasculature is the locus where multiple pathogenic processes converge and contribute to cognitive impairment. Strategies that promote vascular health by managing vascular risk factors, changes in life style, and medication can significantly reduce the prevalence of AD, which has been demonstrated in some smaller studies.

When data from the Systolic Blood Pressure Intervention Trial (SPRINT) were published in 2015, the medical community responded enthusiastically to the news that reducing blood pressure lower than the normal targets could reduce overall death rates by 27 percent for adults at high cardiovascular risk. Investigators now describe how aggressively lowering blood pressure levels can extend a person's life expectancy. They report that having a blood pressure target of less than 120 mm Hg - rather than the standard 140 mm Hg - can add six months to three years to a person's lifetime, depending upon how old they are when they begin intensive blood pressure control.

By applying age-based methods to the data from SPRINT, the team could estimate the long-term benefits of intensive blood pressure control. The SPRINT study enrolled more than 9,000 adults who were 50 years or older, were at high cardiovascular risk but did not have diabetes, and had a systolic blood pressure between 130- and 180-mm Hg (130 mm Hg or higher is considered high blood pressure). Participants were randomized to intensive (at least 120 mm Hg) or standard (at least 140 mm Hg) systolic blood pressure targets. Participants were given antihypertensive therapies, free of cost, to achieve their blood pressure targets and were followed for an average of a little over three years.

Researchers estimated that if people had continued taking their antihypertensive therapies for the remainder of their lives, those with the intensive blood pressure target could add six months to three years to their life expectancy, compared to those with the standard blood pressure target. This span depended upon the person's age - for someone who began antihypertensive medications at 50 years old, they predicted a difference of 2.9 years; for someone 65 years old, a difference of 1.1 years; and for someone 80 years old, a difference of nine months.

To what extent microvascular rarefaction contributes to impaired skeletal muscle function remains unknown. Our understanding of whether pathological changes in the microcirculation can be reversed remains limited by a lack of basic physiological data in otherwise healthy tissue. The principal objectives here were to: (1) quantify the effect of random microvascular rarefaction on limb perfusion and muscle performance, and (2) determine if these changes could be reversed. We developed a novel protocol in rats whereby microspheres injected into the femoral artery allowed a unilateral reduction in functional capillary density in the extensor digitorum longus (EDL), and assessed acute and chronic effects on muscle function.

Simultaneous bilateral EDL force and hindlimb blood flow measurements were made during electrical stimulation. Following functional capillary rarefaction there was an acute microsphere dose-dependent reduction in muscle fatigue resistance, despite preserved femoral artery perfusion. Histological analysis of EDL samples taken from injected animals confirmed a positive correlation between the proportion of functional capillaries and fatigue resistance. Such impaired performance persisted for at least 2 weeks.

Concomitant mechanical overload improved both perfused capillary density and fatigue resistance, confirming that the capacity for muscle remodelling was retained following chronic distributed ischaemia, and that the impact of capillary rarefaction could be alleviated. These results demonstrate that loss of functional capillaries is detrimental to muscle function, even in otherwise healthy tissue, independent of arterial perfusion. Restoration of muscle performance following a mechanical overload stimulus indicates that angiogenic treatments to alleviate microvascular rarefaction may be key to restoring exercise tolerance.

Aging is the highest risk factor for many human diseases, including cancer, dementia, diabetes, and metabolic syndrome. Caloric restriction has been shown in animal models to be one of the most effective interventions against these age-related diseases. And although researchers know that individual cells undergo many changes as an organism ages, they have not known how caloric restriction might influence these changes.

In a new paper, researchers compared rats who ate 30 percent fewer calories with rats on normal diets. The animals' diets were controlled from age 18 months through 27 months. (In humans, this would be roughly equivalent to someone following a calorie-restricted diet from age 50 through 70). At both the start and the conclusion of the diet, the researchers isolated and analyzed a total of 168,703 cells from 40 cell types in the 56 rats. The cells came from fat tissues, liver, kidney, aorta, skin, bone marrow, brain, and muscle. In each isolated cell, the researchers used single-cell genetic-sequencing technology to measure the activity levels of genes. They also looked at the overall composition of cell types within any given tissue. Then, they compared old and young mice on each diet.

Many of the changes that occurred as rats on the normal diet grew older didn't occur in rats on a restricted diet; even in old age, many of the tissues and cells of animals on the diet closely resembled those of young rats. Overall, 57 percent of the age-related changes in cell composition seen in the tissues of rats on a normal diet were not present in the rats on the calorie restricted diet. Some of the cells and genes most affected by the diet related to immunity, inflammation, and lipid metabolism. The number of immune cells in nearly every tissue studied dramatically increased as control rats aged but was not affected by age in rats with restricted calories. In brown adipose tissue - one type of fat tissue - a calorie-restricted diet reverted the expression levels of many anti-inflammatory genes to those seen in young animals.

When the researchers homed in on transcription factors - essentially master switches that can broadly alter the activity of many other genes - that were altered by caloric restriction, one stood out. Levels of the transcription factor Ybx1 were altered by the diet in 23 different cell types. The scientists believe Ybx1 may be an age-related transcription factor and are planning more research into its effects.

Researchers recently developed a handheld 3D bioprinter that could revolutionize the way musculoskeletal surgical procedures are performed. The bioprinter enables surgeons to deposit scaffolds - materials to help support cellular and tissue growth - directly into the defect sites within weakened skeletal muscles. This allows proper filling of the cavity with fibrillar scaffolds in which fibers resemble the architecture of the native tissue. The scaffolds from the bioprinter adhere precisely to the surrounding tissues of the injury and mimic the properties of the existing tissue, eliminating the need for any suturing.

Current methods for reconstructive surgery have been largely inadequate in treating volumetric muscle loss. As a result, 3D printing technology has emerged as an up and coming solution to help reconstruct muscle. "A good solution currently does not exist for patients who suffer volumetric muscle loss. A customizable, printed gel establishes the foundation for a new treatment paradigm can improve the care of our trauma patients."

Existing 3D bioprinting technology is not without its problems. Implanting the hydrogel-based scaffolds successfully requires a very specific biomaterial to be printed that will adhere to the defect site. While 3D bioprinted scaffolds mimicking skeletal muscles have been created in vitro, they have not been successfully used on an actual subject. A handheld bioprinter approach fixes the problem. The bioprinter prints gelatin-based hydrogels - known as "bioink" - that have been proven to be effective in adhering to defect sites of mice with volumetric muscle loss injury. The mice showed a significant increase in muscle hypertrophy following the therapy.

Oxygen is key to many processes of life and is involved in all stages of wound healing in the skin, with many cells and pathways being reactive to changes in oxygen concentration. Following injury to the skin, disruption of the vasculature results in a hypoxic environment, which is further exacerbated by high oxygen consumption through the cells present at the edge of the wound. Hypoxia has been found to have myriad effects on cells and their function, such as inducing greater dermal fibroblast proliferation and production of TGF-β1. Furthermore, hypoxia has been shown to promote in vitro keratinocyte motility and leads to the secretion of several growth factors. These are but a few of the many roles acute hypoxia plays in the induction of skin healing, and although hypoxia is necessary for regeneration, a return to normoxic conditions is eventually required.

With hypoxia being of such importance to regeneration of the skin, the HIF pathways have drawn much attention. Hypoxia-inducible factors (HIFs) are pleiotropic key regulators of oxygen homeostasis. HIF-1 consists of two subunits: HIF-1α (or its analogs HIF-2α and HIF-3α) and HIF-1β, which bind to acquire the transcriptional capabilities that promote cell survival during hypoxia. Additionally, HIF-1 serves as a crucial modulator in the homeostatic processes during hypoxia by increasing vascularization and regulating anaerobic respiration.

While HIF-1 is undoubtedly important for wound healing, excessive expression of HIF-1 can lead to unwanted fibroproliferation in the form of keloids and hypertrophic scarring. Conversely, deficits in HIF-1 signaling can lead to inadequate wound healing. Chronic wounds are often characterized by a constant, nonresolving inflammatory phase, causing proinflammatory signaling cascades to persist. This results in higher levels of proteases that work to destroy extracellular matrix components and other molecules beneficial to wound healing, preventing the proliferation and tissue remodeling phases from advancing normally. Studies have identified HIF-1 signaling as one of the underlying causes behind many nonhealing wounds. Common examples of chronic wounds include diabetic wounds, pressure ulcers, and aged wounds.

The structure and pathophysiology of aged skin differs greatly from the skin in younger individuals. These discrepancies are responsible for the delayed healing found in aged skin, many of which are the result of irregular HIF-1 signaling. Over the past decade, extensive studies have examined the expression levels of HIF-1 in the aged compared with the young, and how regeneration was affected. One of the major observations of aged wound healing is that there is significantly lesser neovascularization following ischemia, impeding recovery. Neovascularization in humans occurs through both angiogenesis, the sprouting of new vessels from old ones, and vasculogenesis, the formation of new vessels by migration and aggregation of endothelial progenitor cells.

HIF-1α signaling has been shown to regulate both of these processes through transcription of cytokines such as SDF-1α. An improved understanding of the regulation of molecular mediators, such as HIF-1α and PHD, will allow for manipulation of the various factors underlying delayed wound healing in the aged. The findings highlighted in this may facilitate the development of potential therapeutic approaches involved in the alteration of cellular dynamics and aging.

It is already well known that exercise has many salutary effects, but= new findings hint at the possibility that exercise and fasting could also help reduce the risk of developing conditions associated with the accumulation of misfolded proteins, such as Alzheimer's and Parkinson's. That possibility, however, remains to be explored. In their experiments, the researchers analyzed the effects of exercise on cells obtained from the thigh muscles of four human volunteers before and after vigorous biking. Following exercise, the proteasomes of these cells showed dramatically more molecular marks of enhanced protein degradation, including greater levels of cAMP. The same changes were observed in the muscles of anesthetized rats whose hind legs were stimulated to contract repeatedly. Fasting - even for brief periods - produced a similar effect on the cells' protein-breakdown machinery. Fasting increased proteasome activity in the muscle and liver cells of mice deprived of food for 12 hours, the equivalent of an overnight fast.

In another round of experiments, the researchers exposed the liver cells of mice to glucagon, the hormone that stimulates production of glucose as fuel for cells and tissues during periods of food deprivation or whenever blood sugar levels drop. The researchers observed that glucagon exposure can stimulate proteasome activity and enhance the cells' capacity to destroy misfolded proteins. Exposure to the fight-or-flight hormone epinephrine produced a similar effect. Epinephrine, also known as adrenaline, is responsible for stimulating the liver and muscle to mobilize energy reserves to boost heart rate and muscle strength during periods of physiologic stress. Liver cells treated with epinephrine showed marked increases in cAMP, as well as enhanced 26S proteasome activity and protein degradation. Epinephrine exposure also boosted proteasome activity - a marker of protein degradation - in the hearts of living rats. Similarly, when researchers exposed mouse kidney cells to vasopressin - the antidiuretic hormone that helps the body retain water and prevents dehydration - they observed higher levels of protein degradation as well.

Taken together, these findings demonstrate that the rate of protein degradation can rise and fall swiftly in a variety of tissues in response to shifting conditions, and that such changes are mediated by fluctuations in hormone levels. This response was surprisingly rapid and short-lived, the scientists noted. For example, exposure to the antidiuretic hormone triggered protein breakdown in kidney cells within five minutes and subsided to pre-exposure levels within an hour, the experiments showed. The findings show that the diverse set of hormones that stimulate cAMP appear to share a common mechanism that alters the composition of cells. These have long been known to modify gene expression, but this latest research reveals they also play a critical role in cellular housecleaning by disposing of proteins that are no longer needed.