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.

Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

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.

Secreted meningeal chemokines, but not VEGFA, modulate the migratory properties of medulloblastoma cells.

Leptomeningeal metastasis is a cause of morbidity and mortality in medulloblastoma, but the understanding of molecular mechanisms driving this process is nascent. In this study, we examined the secretory chemokine profile of medulloblastoma cells (DAOY) and a meningothelial cell line (BMEN1). Conditioned media (CM) of meningothelial cells increased adhesion, spreading and migration of medulloblastoma. VEGFA was identified at elevated levels in the CM from BMEN1 cells (as compared to DAOY CM); however, recombinant VEGFA alone was insufficient to enhance medulloblastoma cell migration. In addition, bevacizumab, the VEGFA scavenging monoclonal antibody, did not block the migratory phenotype induced by the CM. These results reveal that paracrine factors secreted by meningothelial cells can influence migration and adherence of medulloblastoma tumor cells, but VEGFA may not be a specific target for therapeutic intervention in this context.

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.

Chondroblastoma of the talus.

We report the case of a 13-year-old male child who presented with a painful left ankle. On imaging (radiography and computed tomography scan with 3-dimensional reconstruction views), an osteolytic lesion in the body of the talus was revealed. Open biopsy, curettage, and fibular bone grafting were done, and the specimen was sent for histopathologic examination. The histopathologic report confirmed the specimen to be chondroblastoma of the talus bone. Chondroblastoma is a rare benign cartilaginous neoplasm that accounts for approximately 1% of all bone tumors and characteristically arises in the epiphysis of a long bone, particularly the humerus, tibia, and femur. Chondroblastoma can affect people of all ages. It is, however, most common in children and young adults aged 10 to 20 years. Chondroblastoma in a tarsal bone is a rare entity. Managing chondroblastoma of the talus with curettage and bone grafting has shown good outcomes.

Rb1 gene inactivation expands satellite cell and postnatal myoblast pools.

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.

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.

18F-FDG microPET imaging detects early transient response to an IGF1R inhibitor in genetically engineered rhabdomyosarcoma models.

BACKGROUND: Alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) are among the most common and most treatment resistant soft tissue sarcomas of childhood. Here, we evaluated the potential of (18)F-Fluorodeoxyglucose (FDG) as a marker of therapeutic response to picropodophyllin (PPP), an IGF1R inhibitor, in a conditional mouse model of ARMS and a conditional model of ERMS/undifferentiated pleomorphic sarcoma (UPS). PROCEDURE: Primary tumor cell cultures from Myf6Cre,Pax3:Fkhr,p53 and Pax7CreER,Ptch1,p53 conditional models of ARMS and ERMS/UPS were found to be highly sensitive to PPP (IC(50) values 150 and 200 nM, respectively). Animals of each model were then treated with 80 mg/kg/day PPP by intraperitoneal injection for 12 days and imaged by (18)F-FDG microPET. RESULTS: Tumor volumes on day 4 for PPP-treated ARMS and ERMS mice were lower than untreated control mouse tumor volumes, although treated tumors were larger than day 0. However, tumor FDG uptake was significantly reduced on day 4 for PPP-treated mice compared to pretreatment baseline or untreated control mice on day 4 (P < 0.05). Nevertheless, by day 12 tumor volumes and FDG uptake for treated mice had increased significantly, indicating rapidly evolving resistance to therapy. CONCLUSIONS: (18)F-FDG PET imaging is a potential imaging biomarker of molecular susceptibility to targeted agents early in treatment for this aggressive form of sarcoma, but may find best use serially for Phase I/II studies where chemotherapy and targeted agents are combined to cytoreduce tumors and abrogate Igf1r inhibitor resistance.

Preclinical testing of tandutinib in a transgenic medulloblastoma mouse model.

Overexpression of platelet-derived growth factor receptor alpha (PDGFR-A) has been documented in association with primary tumors and metastasis in medulloblastoma. Tumors from our genetically engineered sonic hedgehog-driven medulloblastoma mouse model overexpress PDGFR-A in primary tumors and thus this mouse model is a good platform with which to study the role of PDGFR-A in this central nervous system malignancy. We hypothesized that inhibition of PDGFR-A in medulloblastoma can slow or inhibit tumor progression in living individuals. To test our hypothesis, we targeted PDGFR-A mediated tumor growth in vitro and in vivo using the tyrosine kinase inhibitor, tandutinib (MLN-518), which strongly inhibits PDGFR-A. Although PDGFR-A inhibition by this agent resulted in reduced mouse tumor cell growth and increased apoptosis in vitro, and reduced tumor cell proliferation in vivo, tandutinib did reduce tumor volume at the doses tested (360 mg/kg) in vivo. Thus, tandutinib may be an agent of interest for sonic hedgehog-driven medulloblastoma if a synergistic drug combination can be identified.

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.

Craniorachischisis and omphalocele in a stillborn cynomolgus monkey (Macaca fascicularis).

Nonhuman primates have been a common animal model to evaluate experimentally induced malformations. Reports on spontaneous malformations are important in determining the background incidence of congenital anomalies in specific species and in evaluating experimental results. Here we report on a stillborn cynomolgus monkey (Macaca fascicularis) with multiple congenital anomalies from the colony maintained at the Southwest National Primate Research Center at the Texas Biomedical Research Institute, San Antonio, Texas. Physical findings included low birth weight, craniorachischisis, facial abnormalities, omphalocele, malrotation of the gut with areas of atresia and intussusception, a Meckel diverticulum, arthrogryposis, patent ductus arteriosus, and patent foramen ovale. The macaque had normal male external genitalia, but undescended testes. Gestational age was unknown but was estimated from measurements of the limbs and other developmental criteria. Although cytogenetic analysis was not possible due to the tissues being in an advanced state of decomposition, array Comparative Genomic Hybridization analysis using human bacterial artificial chromosome clones was successful in effectively eliminating aneuploidy or any copy number changes greater than approximately 3-5 Mb as a cause of the malformations. Further evaluation of the animal included extensive imaging of the skeletal and neural tissue defects. The animal's congenital anomalies are discussed in relation to the current hypotheses attempting to explain the frequent association of neural tube defects with other abnormalities.

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.