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.

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.

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.

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.

Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro.

The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

Healthy ageing, but what is health?

Ageing occurs in spite of complex pathways of maintenance and repair. There is no "enemy within", which has the specific evolution-selected function to cause ageing and death. This understanding of ageing should transform our approach towards interventions from therapeutic "anti-ageing" to maintaining health. But what is health? Ideally, health is a state of complete physical and mental independence in activities of daily living. But in pragmatic terms, health is a state of adequate physical and mental independence in activities of daily living. In order to identify a set of measurable, evidence-based and demonstratable parameters of health, robustness and resilience at various levels, the concept of homeodynamic space can be a useful one. Age-related health problems for which there are no clear-cut causative agents, except the complex process of ageing, may be better tackled by focusing on health mechanisms and their maintenance, rather than disease management and treatment. Continuing the disease-oriented research approaches are economically, socially and psychologically unsustainable as compared with health-oriented and preventive strategies, such as hormesis. Supporting health-oriented research is the urgency of our time.

Biogerontology: from here to where? The Lord Cohen Medal Lecture-2011.

Ageing is a progressive shrinkage of the homeodynamic space and, at the molecular level, it is associated with the stochastic occurrence and progressive accumulation of molecular damage. Imperfection of the maintenance and repair systems results in the failure of homeodynamics characterized by increased molecular heterogeneity, altered cellular functioning, reduced stress tolerance and reduced remodeling and adaptation, which lead to increased probability of diseases and eventual death. Although, several types of molecular damages have been shown to accumulate and increase molecular heterogeneity during ageing, its relevance and significance with respect to the physiology, survival and longevity remains to be determined. Such studies are essential for establishing biomarkers of health, frailty, remodeling and adaptation, and for developing effective methods for the prevention and reversion of age-related changes. A promising strategy for ageing intervention and modulation is that of strengthening the homeodynamics through repeated mild stress-induced hormesis by physical, biological and nutritional hormetins. Because a number of ethical, social, and personal implications emerge by the development and use of anti-ageing and life-extending technologies, biogerontologists should incorporate these elements while developing their research agenda in biogerontology.

Age-related and sex-specific differences in proteasome activity in individual Drosophila flies from wild type, longevity-selected and stress resistant strains.

We have measured the caspase-like proteasome activity in individual male and female Drosophila flies by using a non-denaturing lysing technique that consistently extracts total protein. The mean proteasome activity in control C1 females was more than two times higher as compared with that in C1 males. However, in longevity-selected LS1 flies the proteasome activity was significantly lower compared to C1 flies, but the sex differences were maintained to some extent. Five other stress resistant lines also had significantly reduced proteasome activity in both sexes. During ageing, there was a progressive decrease in proteasome activity in C1 females, but not in C1 males. This age-related decline in proteasome activity observed in C1 females was not observed in LS1 flies. We conclude that the proteasome activity in control male and female flies is significantly different from each other and that increased lifespan and stress resistance lead to a reduction in proteasome activity and recession of the age-related decline observed in control females.

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.

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.

Curcumin induces stress response and hormetically modulates wound healing ability of human skin fibroblasts undergoing ageing in vitro.

Wound healing becomes impaired in several diseases and during ageing. A commonly used model for the study of wound healing is a scratched monolayer of cells in vitro, which is convenient for the analysis of the cellular and molecular changes occurring during the two phases of wound healing, namely cell migration and cell proliferation. Cell migration, which is the primary event to occur during initial wound healing, is inversely dependent on the number of focal adhesions (FA) that attach cells to the extracellular matrix. Here we report that the number of FA, measured by determining the levels of FA-proteins paxillin and talin, increase with increasing population doubling level of the serially passaged normal adult skin fibroblasts, and that this increase may account for the age-related slowing down of wound healing in vitro. We also report that curcumin, a component of the widely used spice turmeric, modulates wound healing in vitro in a biphasic dose response manner, being stimulatory at low doses (between 1 and 5 µM), and inhibitory at higher doses. Furthermore, our results show that the hormetic effects of low levels of curcumin are achieved by virtue of it being a hormetin in terms of the induction of stress response pathways, including Nrf2 and HO-1 in human cells.

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.

Longevity mutants do not establish any "new science" of ageing.

The biological reasons for ageing are now well known, so it is no longer an unsolved problem in biology. Furthermore, there is only one science of ageing, which is continually advancing. The significance and importance of the mutations that lengthen the lifespan of invertebrates can be assessed only in relationship to previous well-established studies of ageing. The mutant strains of model organisms that increase longevity have altered nutrient signalling pathways similar to the effects of dietary restriction, and so it is likely that there is a shift in the trade-off between reproduction and maintenance of the soma. To believe that the isolation and characterisation of a few invertebrate mutations (as well as those in yeast) will "galvanise" the field and provide new insights into human ageing is an extreme point of view which does not recognize the huge progress in ageing research that has been made in the last 50 years or so.

Synthesis, modification and turnover of proteins during aging.

Iterations in the rate and extent of protein synthesis, accuracy, post-translational modifications and turnover are among the main molecular characteristics of aging. A decline in the cellular capacity through proteasomal and lysosomal pathways to recognize and preferentially degrade damaged proteins leads to the accumulation of abnormal proteins during aging. The consequent increase in molecular heterogeneity and impaired functioning of proteins is the basis of several age-related pathologies, such as cataracts, sarcopenia and neurodegerative diseases. Understanding the proteomic spectrum and its functional implications during aging can facilitate developing effective means of intervention, prevention and therapy of aging and age-related diseases.

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.