Seven knowledge gaps in modern biogerontology.

About a year ago, members of the editorial board of Biogerontology were requested to respond to a query by the editor-in-chief of the journal as to what one question within their field of ageing research still needs to be asked and answered. This editorial is inspired by the wide range and variety of questions, ideas, comments and suggestions received in response to that query. The seven knowledge gaps identified in this article are arranged into three main categories: evolutionary aspects of longevity, biological survival and death aspects, and heterogeneity in the progression and phenotype of ageing. This is not an exhaustive and exclusive list, and may be modified and expanded. Implications of these knowledge gaps, especially in the context of ongoing attempts to develop effective interventions in ageing and longevity are also discussed.

Nutrition, Food and Diet in Health and Longevity: We Eat What We Are.

Nutrition generally refers to the macro- and micro-nutrients essential for survival, but we do not simply eat nutrition. Instead, we eat animal- and plant-based foods without always being conscious of its nutritional value. Furthermore, various cultural factors influence and shape our taste, preferences, taboos and practices towards preparing and consuming food as a meal and diet. Biogerontological understanding of ageing has identified food as one of the three foundational pillars of health and survival. Here we address the issues of nutrition, food and diet by analyzing the biological importance of macro- and micro-nutrients including hormetins, discussing the health claims for various types of food, and by reviewing the general principles of healthy dietary patterns, including meal timing, caloric restriction, and intermittent fasting. We also present our views about the need for refining our approaches and strategies for future research on nutrition, food and diet by incorporating the molecular, physiological, cultural and personal aspects of this crucial pillar of health, healthy ageing and longevity.

Perspectives on using bacteriophages in biogerontology research and interventions.

With the development of materials engineering, gerontology-related research on new tools for diagnostic and therapeutic applications, including precision and personalised medicine, has expanded significantly. Using nanotechnology, drugs can be precisely delivered to organs, tissues, cells, and cell organelles, thereby enhancing their therapeutic effects. Here, we discuss the possible use of bacteriophages as nanocarriers that can improve the safety, efficiency, and sensitivity of conventional medical therapies. Phages are a new class of targeted-delivery vectors, which can carry high concentrations of cargo and protect other nontargeted cells from the senescent cell killing effects of senolytics. Bacteriophages can also be subjected to chemical and/or genetic modifications that would acquire novel properties and improve their ability to detect senescent cells and deliver senolytics. Phage research in experimental biogerontology will also develop strategies to efficiently deliver senolytics, target senescent cells, activate extrinsic apoptosis pathways in senescent cells, trigger immune cells to recognise senescent cells, induce autophagy, promote cell and tissue regeneration, inhibit senescence-associated secretory phenotype (SASP) by senomorphic activity, stimulate the properties of mild stress-inducing hormetic agents and hormetins, and modulate the gut microbiome.

Female Mice Reaching Exceptionally High Old Age Have Preserved 20S Proteasome Activities.

Oxidized, damaged and misfolded proteins accumulate during aging and contribute to impaired cell function and tissue homeodynamics. Damaged proteins are degraded by cellular clearance mechanisms like the 20S proteasome. Aging relates to low 20S proteasome function, whereas long-lived species show high levels. However, contradictory results exist depending on the tissue or cell type and it is unknown how the 20S proteasome functions in exceptionally old mice. The aim of this study was to investigate two proteasome activities (caspase-like and chymotrypsin-like) in several tissues (lung, heart, axillary lymph nodes, liver, kidney) and cells (peritoneal leukocytes) from adult (28 ± 4 weeks, n = 12), old (76 ± 4 weeks, n = 9) and exceptionally old (128 ± 4 weeks, n = 9) BALB/c female mice. The results show different age-related changes depending on the tissue and the activity considered, so there is no universal decline in proteasome function with age in female mice. Interestingly, exceptionally old mice displayed better maintained proteasome activities, suggesting that preserved 20S proteasome is associated with successful aging.

Naive extrapolations, overhyped claims and empty promises in ageing research and interventions need avoidance.

Most proclamations about another wonder breakthrough and another imminent miracle treatment of ageing are usually overhyped claims and empty promises. It is not that the experimental science behind those claims is totally wrong or fake. But it is often a case of being ahistorical and ignoring the cumulated knowledge and understanding of the evolutionary and biological principles of ageing and longevity. Furthermore, remaining stuck to the body-as-a-machine viewpoint reduces ageing and its associated health challenges to a mere problem of engineering and design. However, highly dynamic nature of the living systems with properties of interaction, interdependence, tolerance, adaptation and constant remodelling requires wholistic and interactive modes of understanding and maintaining health. The physiological relevance and significance of progressively accumulating molecular damage remains to be fully understood. As for ageing interventions, the three pillars of health-food, physical activity, and social and mental engagement-which actually show health-promoting effect, cannot simply be reduced to a single or a limited number of molecular targets with hopes of creating an exercise pill, a fasting pill, a happiness pill and so on. If we want to increase the credibility and socio-political-economic support of ageing research and interventions, we need to resist the temptation to overhype the claims or to make far-fetched promises, which undermine the theoretical and practical significance of new discoveries in biogerontology.

Biphasic Dose-Response and Hormetic Effects of Stress Hormone Hydrocortisone on Telomerase-Immortalized Human Bone Marrow Stem Cells In Vitro.

Although high levels of stress hormones are associated with well-known negative health outcomes, their low levels can have health-promoting effects by virtue of the phenomenon of mild stress-induced hormesis. We have studied the effects of a wide range (between 100 nmol/L and 150 µmol/L) of hydrocortisone (HC) on human bone marrow stem cells in vitro. Telomerase-immortalized human mesenchymal stem cells (hTERT-MSCs) were exposed to various doses of HC for different durations (1-6 days) and analyzed for survival and metabolic activity by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, for cell migratory ability by a wound-healing assay and for osteoblastic and adipogenic differentiation abilities in vitro. Our findings indicate that hTERT-MSCs exposed to HC resulted in a biphasic hormetic dose-response in some measures but not all. Although the mitochondrial and metabolic MTT activity assay clearly showed low-level stimulatory (between 0.1 and 1 µmol/L) and high-level inhibitory effects (from about 10 µmol/L onward), the cytostatic and differentiation-inducing effects were mostly linear at concentrations between 1 and 100 µmol/L. Further long-term studies will elucidate whether chronic or intermittent exposure of human cells to stress hormones has physiologically beneficial hormetic effects.

Re-analysis of herbal extracts data reveals that inflammatory processes are mediated by hormetic mechanisms.

Using data from Schink et al. (2018), a large number of herbal extracts were assessed for their capacity to induce pro- and anti-inflammatory effects based on TLR4 expression normalized for cell viability in two immune cell models (i.e., HeLa-TLR4 transfected reporter cell line, and THP-1 monocytes) applying seven concentrations (0.01-3.0%). The analysis revealed that 70-80% of the extracts satisfying the a priori entry criteria also satisfied a priori evaluative criteria for hormetic concentration responses. These findings demonstrate that a large proportion of herbal extracts display hormetic dose responses in immune cells, indicating that hormetic mechanisms mediate pro- and anti-inflammatory processes and may provide a means to guide optimal dosing strategies. The identification of doses eliciting only anti-inflammatory therapeutic activity as well as the use of dose-variable herbal extracts in the treatment of inflammatory diseases will be challenging.

Phytochemicals Rosmarinic Acid, Ampelopsin, and Amorfrutin-A Can Modulate Age-Related Phenotype of Serially Passaged Human Skin Fibroblasts in vitro.

One of the aims of the EU-funded Research and Innovation Action (RIA), titled "Ageing with Elegans" (AwE) is to enhance better understanding of the factors causing health and disease in aging and develop evidence-based preventive, diagnostic, therapeutic, and other strategies. The work package-5 of this project is focused on testing the effects of phytochemicals of natural and synthetic origin on aging, longevity, and health of human cells in vitro, after the initial screening using the animal model systems of nematodes and rats and mice. Accordingly, the first series of three compounds, rosmarinic acid (ROSM), ampelopsin (AMPEL), and amorfrutin-A (AMOR), were selected to test for their short-term and long-term effects on human skin fibroblasts undergoing aging and senescence in vitro. The lifelong modulatory effects of these compounds were tested individually at two doses (0.5 and 1.0 µM), selected after a short-term dose response check of a 20,000-fold range (0.01-200 µM). The results show that these compounds do have some beneficial effects in terms of supporting the long-term lifelong growth and enhanced stress tolerance of serially passaged cells. These effects seem to be achieved by reducing the extent of loss of telomeres, of 5-methyl-cytosine (5-mC) and of 5-hydroxymethyl-cytosine (5-hmC), by reducing the accumulation of oxidative DNA damage product 8-OHdG. There is also some indication that these compounds induce at least one of the stress responses in terms of the increased synthesis of heat shock protein Hsp70. Thus, these phytochemicals may be potential hormetins, which bring about their health beneficial effects by the phenomenon of mild stress-induced hormesis.

Plant-Derived Molecules α-Boswellic Acid Acetate, Praeruptorin-A, and Salvianolic Acid-B Have Age-Related Differential Effects in Young and Senescent Human Fibroblasts In Vitro.

Testing and screening of plant-derived molecules on normal human cells in vitro is a widely used approach for discovering their eventual health beneficial effects for human ageing and longevity. As little is known about age-associated differential effects of such molecules, here we report that young (<25% replicative lifespan completed) and near-senescent (>90% replicative lifespan completed) human skin fibroblasts exposed for 1-15 days to a wide range of concentrations (0.1-100 µM) of the three selected phytochemicals, namely α-boswellic acid acetate (ABC), praeruptorin-A (PTA), and salvianolic acid-B (SAB) had age-related differential effects. The parameters studied were the metabolic activity (MTT assay), cellular morphological phenotype, one-step growth characteristics, expression of genes involved in the cell cycle regulation and cytokine network genes, protein levels of p53, cytosolic superoxide dismutase (SOD1) and microtubule-associated protein 1A/1B-light chain 3 (LC3), and the extent of protein carbonylation and protein aggregation as a sign of oxidative stress. All three compounds showed biphasic hormetic dose response by stimulating cell growth, survival and metabolic activity at low doses (up to 1 µM), while showing inhibitory effects at high doses (>10 µM). Furthermore, the response of early passage young cells was different from that of the late passage near-senescent cells, especially with respect to the expression of cell cycle-related and inflammation-related genes. Such studies have importance with respect to the use of low doses of such molecules as health-promoting and/or ageing-interventions through the phenomenon of hormesis.

Biogerontology: research status, challenges and opportunities.

Biogerontology is the study of the biological basis of ageing and age-related diseases. The phenomenon and the process of ageing are well understood in evolutionary and biological terms; and a conceptual framework has been established within which general principles of ageing and longevity can be formulated. The phenotype of ageing in terms of progressive loss of physical function and fitness is best seen during the period of survival after the evolution-determined essential lifespan (ELS) of a species. However, the ageing phenotype is highly heterogenous and individualistic at all levels from the whole body to the molecular one. Most significantly, the process and the progression of ageing are not determined by any specific gerontogenes. Ageing is the result of imperfect maintenance and repair systems that allow a progressive shrinkage of the homeodynamic space of an individual. The challenge is to develop and apply wholistic approaches to the complex trait of ageing for maintaining and/or improving health. One such approach is that of mild stress-induced physiological hormesis by physical, mental and nutritional hormetins. Biogerontological research offers numerous opportunities for developing evidence-based novel biomedical technologies for maintaining and improving health, for preventing the onset of age-related diseases, and for extending the health-span.

A preliminary attempt to establish multiple stress response profiles of human skin fibroblasts exposed to mild or severe stress during ageing in vitro.

Optimal stress response (SR) is an essential aspect of the property of dynamic homeostasis of all biological systems, including cells in culture. Whereas severe stress can induce the so-called stress-induced premature senescence (SIPS), a model developed by Olivier Toussaint, mild stress can strengthen homeodynamics and can postpone senescence through the phenomenon of hormesis. We have attempted to establish multiple stress response profiles (SRP) of early passage young and late passage senescent human facial skin fibroblasts, FSF-1, exposed to either mild (41°C) and severe (43°C) heat shock for 1h, or to mild (2%) and severe (0%) serum deprivation for up to 48h. The results obtained show that FSF-1 cells exposed to two different intensities of stress from two different stressors separately have differential SRP to mild and severe stress, which also vary significantly between young and senescent cells. Establishing multiple and differential SRP to mild and severe stress may facilitate distinguishing between the mild stress-induced beneficial hormetic effects and the harmful effects of severe stress.

Chronic exposure to rapamycin and episodic serum starvation modulate ageing of human fibroblasts in vitro.

Mild stress-induced activation of stress response (SR) pathways, such as autophagy, heat shock response, oxidative SR, DNA damage response, and inflammatory response, can be potentially health beneficial. Using the model system of cellular ageing and replicative senescence in vitro, we have studied the ageing modulatory effects of the two conditions, rapamycin and serum starvation. Chronic exposure to 0.1, 1 and 10 nM rapamycin positively modulated the survival, growth, morphology, telomere length, DNA methylation levels, 8-oxo-dG level in DNA, N6-methyl-adenosine level in RNA, and ethanol stress tolerance of serially passaged normal human skin fibroblasts. Furthermore, episodic (once a week) serum starvation of human skin fibroblasts extended their replicative lifespan by about 22%, along with the maintenance of early passage youthful morphology even in late passage cultures. Although the results of this study may be considered preliminary, it can be inferred that intermittent and episodic induction of SR, rather than chronic up-regulation of SR, is more effective and applicable in the practice of hormesis for healthy ageing and longevity.

Ageing with elegans: a research proposal to map healthspan pathways.

Human longevity continues to increase world-wide, often accompanied by decreasing birth rates. As a larger fraction of the population thus gets older, the number of people suffering from disease or disability increases dramatically, presenting a major societal challenge. Healthy ageing has therefore been selected by EU policy makers as an important priority ( http://www.healthyageing.eu/european-policies-and-initiatives ); it benefits not only the elderly but also their direct environment and broader society, as well as the economy. The theme of healthy ageing figures prominently in the Horizon 2020 programme ( https://ec.europa.eu/programmes/horizon2020/en/h2020-section/health-demographic-change-and-wellbeing ), which has launched several research and innovation actions (RIA), like "Understanding health, ageing and disease: determinants, risk factors and pathways" in the work programme on "Personalising healthcare" ( https://ec.europa.eu/research/participants/portal/desktop/en/opportunities/h2020/topics/693-phc-01-2014.html ). Here we present our research proposal entitled "ageing with elegans" (AwE) ( http://www.h2020awe.eu/ ), funded by this RIA, which aims for better understanding of the factors causing health and disease in ageing, and to develop evidence-based prevention, diagnostic, therapeutic and other strategies. The aim of this article, authored by the principal investigators of the 17 collaborating teams, is to describe briefly the rationale, aims, strategies and work packages of AwE for the purposes of sharing our ideas and plans with the biogerontological community in order to invite scientific feedback, suggestions, and criticism.

Low doses of nanodiamonds and silica nanoparticles have beneficial hormetic effects in normal human skin fibroblasts in culture.

Nanodiamonds (ND) and silica nanoparticles (SiO2-NP) have been much investigated for their toxicity at high doses, little is known about their biological activity at low concentrations. Here we report the biphasic dose response of ND and SiO2-NP in modulating normal human facial skin fibroblasts (FSF1) in culture. ND and SiO2-NP at low concentration (up to 0.5 µg/ml) had beneficial effects on FSF1 in terms of increasing their proliferation and metabolic activity. Exposure of FSF1 cells to low levels of NP enhanced their wound healing ability in vitro and slowed down aging during serial passaging as measured by maintenance of youthful morphology, reduction in the rate of loss of telomeres, and the over all proliferative characteristics. Furthermore, NP treatment induced the activation of Nrf2- and FOXO3A-mediated cellular stress responses, including an increased expression of heme oxygenease (HO-1), sirtuin (SIRT1), and DNA methyltransferase II (DNMT2). These results imply that ND and SiO2-NP at low doses are potential hormetins, which exert mild stress-induced beneficial hormetic effects through improved survival, longevity, maintenance, repair and function of human cells.

Basal level of autophagy is increased in aging human skin fibroblasts in vitro, but not in old skin.

Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.

Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically, depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of nePTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues.

Biology of ageing: principles, challenges and perspectives.

Living systems owe their survival and health to a series of complex biochemical pathways of maintenance and repair. These defense systems create the homeodynamic space of an individual, which is characterized by stress tolerance, molecular damage control and continuous remodeling. Ageing, age-related diseases and eventual death are the consequences of a progressive shrinkage of the homeodynamic space, due to the failure of maintenance and repair. Whereas longevity assurance genes do affect the essential lifespan of a species, there are no ageing-specific gerontogenes to cause ageing and to limit the lifespan of an individual. The challenge of preventing, managing or treating age-related chronic diseases and other health problems requires abandoning the traditional "one-target, one-shot" biomedical approach. Wholistic methods incorporating lifestyle-based hormetic interventions, including food, physical activity and mental engagement, appear to be potentially more successful in maintaining health and in extending healthspan and longevity.

Differential translocation of heat shock factor-1 after mild and severe stress to human skin fibroblasts undergoing aging in vitro.

Repeated exposure to mild heat shock (HS) has been shown to induce a wide range of health promoting hormetic effects in various biological systems, including human cells undergoing aging in vitro. In order to understand how cells distinguish between mild and severe stress, we have investigated the extent of early and immediate HS response by analyzing the nuclear translocation of the transcription factor heat shock factor-1 (HSF1), in serially passaged normal adult human facial skin fibroblasts exposed to mild (41 °C) or severe (43 °C) HS. Cells respond differently when exposed to mild and severe HS at different passage levels in terms of the extent of HSF1 translocation. In early passage young cells there was a 5-fold difference between mild and severe HS in the extent of HSF1 translocation. However, in near senescent late passage cells, the difference between mild and severe stress in terms of the extent of HSF1 translocation was reduced to less than 2-fold. One of the reasons for this age-related attenuation of heat shock response is due to the fact there was a higher basal level of HSF1 in the nuclei of late passage cells, which is indicative of increased intrinsic stress during cellular aging. These observations are consistent with previously reported data that whereas repeated mild stress given at younger ages can slow down aging and increase the lifespan, the same level of stress given at older ages may not provide the same benefits. Therefore, elucidating the early and immediate steps in the induction of stress response can be useful in deciding whether a particular level of stress is potentially hormetically beneficial or not.