The Damaraland mole-rat (DMR; Fukomys damarensis) is a long-lived (~ 20 years) Bathyergid rodent that diverged 26 million years ago from its close relative, the naked mole-rat (NMR). While the properties of NMR cultured fibroblasts have been extensively studied and have revealed several unusual features of this cancer-resistant, long-lived species, comparative DMR studies are extremely limited. We optimized conditions for successfully culturing primary DMR skin fibroblasts and also established immortalized DMR cells using simian virus 40 early region expression. Like NMRs, DMR fibroblasts are more resistant than mice to various cytotoxins including heavy metals, DNA-damaging agents, oxidative stressors, and proteasome inhibitors. DMR genome sequencing analyses revealed the presence of premature stop codons in the master regulator genes of necroptosis, an inflammatory programmed cell death-receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), although these mutations have different locations to those found in the NMR. DMR cells, like NMR cells, did not show significantly increased cell death in response to necroptosis induction. Our data suggest that both Bathyergid species require species-specific cell culture conditions for optimized growth, display similar resistance to cytotoxins, and show loss-of-function mutations abrogating the ability to employ necroptosis. These shared traits may contribute to their evolved adaptations to their subterranean lifestyle and prolonged longevity. These convergent insights and valuable resource may be pertinent to biomedical research.

Dietary restriction (DR) extends lifespan in diverse species, from yeast to mammals. However, its underlying mechanisms are not well understood. In this study, through using the tractable model Caenorhabditis elegans, we show a role for the DEAD-box RNA helicase, DDX-23 (homologous to mammal DDX23) as a regulator of healthspan in response to dietary restriction. Meanwhile, DDX-23 is also required for heat and oxidative stress response in C. elegans. Intriguingly, DDX-23 functions in the germline during adult to regulate dietary restriction-induced longevity. We then find that PHA-4/FOXA acts downstream of DDX-23 to mediate the transcriptional response of SOD-related genes and consequently the lifespan of the animals. Furthermore, we find that the DEAD-box RNA helicase, DDX-23 negatively regulates the healthy lifespan extension by up-regulating the expression of miR-231, and resulting in suppressing the activation of FOXO transcription factor DAF-16. Our work shows a newly discovered for DEAD-box RNA helicase DDX-23 in the regulation of dietary restriction-mediated longevity in C. elegans and reveals the downstream transcriptional regulation mechanisms.

There is a critical need to generate age- and sex-specific survival curves to characterize chronological aging consistently across nonhuman primates (NHP) used in biomedical research. Sex-specific Kaplan-Meier survival curves were computed in 12 translational aging models: baboon, bonnet macaque, chimpanzee, common marmoset, coppery titi monkey, cotton-top tamarin, cynomolgus macaque, Japanese macaque, pigtail macaque, rhesus macaque, squirrel monkey, and vervet/African green. After employing strict inclusion criteria, primary results are based on 12,269 NHPs that survived to adulthood and died of natural/health-related causes. A secondary analysis was completed for 32,616 NHPs that died of any cause. Results show a pattern of reduced male survival among catarrhines (African and Asian primates), especially macaques, but not platyrrhines (Central and South American primates). For many species, median lifespans were lower than previously reported. An important consideration is that these analyses may offer a better reflection of healthspan than lifespan since research NHPs are typically euthanized for humane welfare reasons before their natural end of life. This resource represents the most comprehensive characterization of sex-specific lifespan and age-at-death distributions for 12 biomedically relevant species, to date. These results clarify relationships among NHP ages and provide a valuable resource for the aging research community, improving human-NHP age equivalencies, informing investigators of expected survival rates, providing a metric for comparisons in future studies, and contributing to understanding of factors driving lifespan differences within and among species.

Non-essential amino acids are often overlooked in biomedical research; however, they are crucial components of organismal metabolism. One such metabolite that is integral to physiological function is serine. Serine acts as a pivotal link connecting glycolysis with one-carbon and lipid metabolism, as well as with pyruvate and glutathione syntheses. Interestingly, increasing evidence suggests that serine metabolism may impact the aging process, and supplementation with serine may confer benefits in safeguarding against aging and age-related disorders. This review synthesizes recent insights into the regulation of serine metabolism during aging and its potential to promote healthy lifespan and mitigate a spectrum of age-related diseases.

Alzheimer's Disease (AD) is a complex polygenic neurodegenerative disorder. Its genetic risk's relationship with all-cause dementia may be influenced by the plasma proteome. Up to 40,139 UK Biobank participants aged ≥ 50y at baseline assessment (2006-2010) were followed-up for ≤ 15 y for dementia incidence. Plasma proteomics were performed on a sub-sample of UK Biobank participants (k = 1,463 plasma proteins). AD polygenic risk scores (PRS) were used as the primary exposure and Cox proportional hazards models were conducted to examine the AD PRS-dementia relationship. A four-way decomposition model then partitioned the total effect (TE) of AD PRS on dementia into an effect due to mediation only, an effect due to interaction only, neither or both. The study found that AD PRS tertiles significantly increased the risk for all-cause dementia, particularly among women. The study specifically found that AD PRS was associated with a 79% higher risk for all-cause dementia for each unit increase (HR = 1.79, 95% CI: 1.70-1.87, P < 0.001). Eighty-six plasma proteins were significantly predicted by AD PRS, including a positive association with PLA2G7, BRK1, the glial acidic fibrillary protein (GFAP), neurofilament light chain (NfL), and negative with TREM2. Both GFAP and NfL significantly interacted synergistically with AD PRS to increase all-dementia risk (> 10% of TE is pure interaction), while GFAP was also an important consistent mediator in the AD PRS-dementia relationship. In summary, we detected significant interactions of NfL and GFAP with AD PRS, in relation to dementia incidence, suggesting potential for personalized dementia prevention and management.

Cognitive aging has become a public health concern as the mean age of the population is ever-increasing. It is a naturalistic and common process of degenerative and compensatory changes that may result in neurocognitive disorders. While heterogeneous, cognitive aging mostly affects executive functions that may be associated with functional losses during activities of daily living. Cognition-oriented treatments like cognitive training and transcranial direct current stimulation (tDCS) have garnered considerable attention in the past few decades while the exact picture regarding their efficacy in healthy older adults has not been determined yet. The present paper aimed to evaluate the effects of a 3-month intervention of tDCS over the dorsolateral prefrontal cortex (DLPFC) with multimodal cognitive training on the Stroop test and Trail Making Tests A and B performance. One hundred and ninety-three healthy older adults from 2 sites were administered repeated sessions of active/sham tDCS with cognitive training. Baseline, post-intervention, and 1-year performance results between groups were compared using multiple linear regressions. Active tDCS resulted in better Stroop test performance at post-intervention (p = 0.033) but not at 1-year follow-up while no differences between groups were observed in Trail Making Tests A & B performance. The present results may correspond to a modest improvement in conflict monitoring, potentially due to modulation of prefrontal regions, without changing shifting performance. Further investigation is warranted to draw an interference regarding the subdomain-specific impact of repeated tDCS with multimodal cognitive training on executive functions.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and progressive neuronal damage. Recent research has highlighted the significant roles of the gut microbiota and microRNAs (miRNAs) in the pathogenesis of AD. This review explores the intricate interaction between gut microbiota and miRNAs, emphasizing their combined impact on Alzheimer's progression. First, we discuss the bidirectional communication within the gut-brain axis and how gut dysbiosis contributes to neuroinflammation and neurodegeneration in AD. Changes in gut microbiota composition in Alzheimer's patients have been linked to inflammation, which exacerbates disease progression. Next, we delve into the biology of miRNAs, focusing on their roles in gene regulation, neurodevelopment, and neurodegeneration. Dysregulated miRNAs are implicated in AD pathogenesis, influencing key processes like inflammation, tau pathology, and amyloid deposition. We then examine how the gut microbiota modulates miRNA expression, particularly in the brain, potentially altering neuroinflammatory responses and synaptic plasticity. The interplay between gut microbiota and miRNAs also affects blood-brain barrier integrity, further contributing to Alzheimer's pathology. Lastly, we explore therapeutic strategies targeting this gut microbiota-miRNA axis, including probiotics, prebiotics, and dietary interventions, aiming to modulate miRNA expression and improve AD outcomes. While promising, challenges remain in fully elucidating these interactions and translating them into effective therapies. This review highlights the importance of understanding the gut microbiota-miRNA relationship in AD, offering potential pathways for novel therapeutic approaches aimed at mitigating the disease's progression.

Parkinson's disease (PD) is a prevalent neurodegenerative disease, characterized by movement disorders and non-motor symptoms like cognitive impairment and depression. Degeneration of dopaminergic neurons in the substantia nigra and Lewy bodies have long been considered as main neuropathological changes. However, recent magnetic resonance imaging (MRI) studies have shown that white matter lesions (WMLs) were present in PD patients. WMLs are characterized by loss or impairment of myelin sheath in central nerve fibers, which are closely correlated with motor and cognitive dysfunction in PD. WMLs alterations precede nigrostriatal neuronal losses and can independently affect the clinical severity or characteristics of motor coordination in PD patients. Currently, the exact mechanism of WMLs involvement in the occurrence and development of PD remains unclear. It is speculated that WMLs may participate in the pathogenesis of PD by disrupting important connections in brain or promoting axonal degeneration. In this review, we will discuss the pathological changes and mechanisms of WMLs, elaborate the impact of WMLs on the progression of PD, clarify the importance of WMLs in PD pathogenesis, and thus provide novel targets for PD treatments.

The lifestyle patterns of top athletes are highly disciplined, featuring strict exercise regimens, nutrition plans, and mental preparation, often beginning at a young age. Recently, it was shown that physically active individuals exhibit slowed epigenetic aging and better age-related outcomes. Here, we investigate whether the extreme intensity of physical activity of Olympic champions still has a beneficial effect on epigenetic aging. To test this hypothesis, we examined the epigenetic aging of 59 Hungarian Olympic champions and of the 332 control subjects, 205 were master rowers. We observed that Olympic champions exhibit slower epigenetic aging, applying seven state-of-the-art epigenetic aging clocks. Additionally, male champions who won any medal within the last 10 years showed slower epigenetic aging compared to other male champions, while female champions exhibited the opposite trend. We also found that wrestlers had higher age acceleration compared to gymnasts, fencers, and water polo players. We identified the top 20 genes that showed the most remarkable difference in promoter methylation between Olympic champions and non-champions. The hypo-methylated genes are involved in synaptic health, glycosylation, metal ion membrane transfer, and force generation. Most of the hyper-methylated genes were associated with cancer promotion. The data suggest that rigorous and long-term exercise from adolescence to adulthood has beneficial effects on epigenetic aging.

With a growing elderly population in the European Union, age-related diseases associated with unhealthy aging pose increasing public health challenges, including a loss of independence and heightened societal burdens. The Semmelweis Study, a prospective occupational cohort study in Hungary, seeks to identify determinants of unhealthy aging, focusing on the complex relationship between lifestyle, environmental, occupational factors, and the development of chronic age-associated diseases, including age-related vascular cognitive impairment (VCI). The primary objective of this pilot study was to establish a robust, high-throughput assessment methodology to comprehensively evaluate both peripheral and cerebrovascular health to provide a solid foundation for the forthcoming Semmelweis Study framework. The study involved 49 participants aged 23 to 87 years, and it assessed multi-domain cognitive performance through an automated battery of tests (CANTAB). Vascular health was comprehensively evaluated using laser speckle contrast imaging (LSCI), flow-mediated dilation (FMD), static and dynamic retinal vessel analysis (SVA, DVA), and measurements of vascular stiffness. The retinal microvasculature, which closely mirrors the cerebral circulation in anatomy, physiology, and pathophysiology, provided a unique window for examination. Optical imaging through SVA and DVA enables the identification of structural and functional changes in the central nervous system's microcirculation, which are highly relevant to the pathogenesis of VCI. Subsequently, the collected measures were integrated into vascular health indices using principal component analysis (PCA) and the relationship to the age and cognitive status of study participants was explored. These comprehensive vascular health indices demonstrated a correlation not only with age but also with cognitive performance. This methodology holds promise for providing novel insights into the intricate interplay between vascular and cognitive health within the context of the Semmelweis Study.

Aquaporin-1 (AQP1) is a highly conserved water-channel protein, found to be expressed by astrocytes in adult humans and non-human primates (NHPs). Upregulation of cortical AQP1 expression occurs with cancer, injury, and neurodegenerative disease, but minimal information is available about the effects of normative aging on AQP1 expression. This study leverages tissues from the oldest-old rhesus macaques, some greater than 40 years of age, from the National Institute on Aging longitudinal study of caloric restriction (CR). We tested whether AQP1 levels are altered in the NHP brain as a function of diet group, sex, and age. Sections of formaldehyde-fixed prefrontal (PFC) and temporal (TC) cortices from 36 rhesus macaques (both sexes, 22 to 44 years, + / - CR) were immunochemically stained for AQP1, then the percent area of AQP1 staining was regionally measured using ImageJ free-ware. Results showed age-related regional increases of AQP1 expression, with no effect of diet group or sex. Specifically, in the PFC, AQP1 positively-stained area increased with age in multiple subregions. For the TC subregions, AQP1 area coverage was not affected by age, despite having average levels that were greater than in the PFC. The peak expression of AQP1 in astrocytes appeared in clusters across cortical layers in a subgroup of animals 30 + years old. Astrocytic AQP1 dysregulation may contribute to progressive risk of neuropathology with aging.

Life satisfaction predicts lower risk of adverse health outcomes, including morbidity and mortality. Research on life satisfaction and risk of dementia has been limited by a lack of comprehensive clinical assessments of dementia. This study builds on previous research examining life satisfaction and clinically ascertained cognitive impairment and dementia. Participants (N = 23070; Mean = 71.83, SD = 8.80) from the National Alzheimer's Coordinating Center reported their satisfaction with life at baseline. Incident dementia was ascertained through clinical assessment over up to 18 years. Life satisfaction was associated with about 72% lower risk of all-cause of dementia, an association that remained significant accounting for demographic (age, sex, race, ethnicity, education, marital and living status), psychological (depression), clinical (obesity, diabetes, hypertension), behavioral (current and former smoking), and genetic risk (APOE ϵ4) factors. The association was not moderated by demographics, depression, and APOE ε4 status groups. The association was similar when cases occurring in the first five years were excluded, reducing the likelihood of reverse causality. Life satisfaction was also linked to specific causes of dementia, with a reduced risk ranging from about 60% to 90% for Alzheimer's disease and vascular dementia to > 2-fold lower risk of Lewy Body and frontotemporal dementia. Older adults who were satisfied with their lives were also at 61% lower risk of incident mild cognitive impairment and at 22% lower risk of converting from mild cognitive impairment to dementia. Being satisfied with one's life is associated with a lower risk of dementia. Improving life satisfaction could promote better cognitive health and protect against dementia.

The impact of metabolic dysfunction-associated steatotic liver disease (MASLD), the preferred nomenclature for NAFLD, on cardiovascular health and mortality among older adults is uncertain. As such, we aimed to identify whether MASLD increases the risk of Major Adverse Cardiovascular Events (MACE) (a composite of fatal coronary heart disease [excluding heart failure], nonfatal myocardial infarction, or fatal or nonfatal ischemic stroke), Atrial Fibrillation (AF), or all-cause mortality in older adults, and whether aspirin attenuates these risks in individuals with MASLD. This is a non-prespecified post-hoc analysis of the ASPREE (ASPirin in Reducing Events in the Elderly) randomized trial. Participants were community dwelling well adults aged ≥ 70 years without a history of atherosclerotic cardiovascular disease or AF. Fatty Liver Index (FLI) was used to identify MASLD at baseline. FLI is a composite of anthropometric and biochemical markers used in epidemiologic studies to rule in and rule out hepatic steatosis. MACE and cause of death were adjudicated by clinical experts; AF was assessed by previously defined algorithm in ASPREE. 9,097 participants were stratified into groups according to FLI. In univariate analysis, prevalent MASLD (FLI ≥ 60 with evidence of metabolic dysfunction; n = 2,998 [33.0%]) was associated with an increased risk of MACE (HR 1.47 [95% CI 1.22-1.78]) and AF (HR 1.50 [95% CI 1.19-1.88] but not all-cause mortality (HR 1.04 [95% CI 0.91-1.19]). After adjusting for cardiovascular disease risk factors, only the association between MASLD and AF remained significant (HR 1.46 [95% CI 1.11-1.93]). Aspirin did not reduce the risk of MACE, death, or AF in the MASLD group. MASLD was associated with an increased hazard of incident AF, but not of MACE or all-cause mortality, in community dwelling older adults. Primary prevention with aspirin does not ameliorate these risks in older adults with MASLD.

Obesity is a significant modifiable risk factor for dementia. This study aims to quantify the global impact of obesity on dementia burden and examine how national strategies for managing overweight/obesity and dietary factors influence dementia prevalence and mortality, with a focus on sex-specific differences. We used data from the Global Burden of Disease (GBD) and World Health Organization (WHO) to evaluate the association between obesity age-standardized prevalence rate (ASPR) and dementia age-standardized mortality rate (ASMR) and ASPR across 161 countries. A two-step multivariate analysis adjusted for socioeconomic and lifestyle factors was performed. Temporal trends in dementia were analyzed based on the presence of national obesity management strategies and varying dietary scores. A 1% increase in national obesity prevalence was associated with a 0.36% increase in dementia mortality (OR: 1.0036; 95% CI: 1.0028-1.0045) in males and 0.12% in females (OR: 1.0012; 95% CI: 1.0007-1.0018). A 1% increase in national obesity ASPR was associated with an increase in ASPR of dementia by 0.26% for males (OR: 1.0026, 95% CI: 1.0024-1.0028) and 0.05% for females (OR: 1.0005, 95% CI: 1.0004-1.0006). Males exhibited a higher susceptibility to obesity-related dementia. Countries with national obesity management strategies showed a significantly greater reduction in dementia mortality, particularly among females (P = 0.025). Higher dietary scores were associated with a more significant decrease in dementia prevalence across both sexes. Rising obesity prevalence is linked to increased dementia burden globally, with males being more vulnerable to this relationship. National management of overweight/obesity and healthier dietary habits may help mitigate the dementia burden, emphasizing the need for integrated public health interventions.

Cardiovascular disease (CVD) represents a major public health issue, claiming numerous lives. This study aimed to demonstrate the advantages of employing artificial intelligence (AI) models to improve the prediction of CVD risk using a large cohort of relatively healthy adults aged 70 years or more. In this study, deep learning (DL) models provide enhanced predictions (DeepSurv: C-index = 0.662, Integrated Brier Score (IBS) = 0.046; Neural Multi-Task Logistic Regression (NMTLR): C-index = 0.660, IBS = 0.047), as compared to the conventional (Cox: C-index = 0.634, IBS = 0.048) and machine learning (Random Survival Forest (RSF): C-index = 0.641, IBS = 0.048) models. The risk scores generated by the DL models also demonstrated superior performance. Moreover, AI models (NMTLR, DeepSurv, and RSF) were more effective, requiring the treatment of only 9 to 10 patients to prevent one CVD event, compared to the conventional model requiring treatment of nearly four times higher number of patients (NNT = 38). In summary, AI models, particularly DL models, possess superior predictive capabilities that can enhance patient treatment in a more cost-effective manner. Nonetheless, AI tools should serve to complement and assist healthcare professionals, rather than supplant them. The DeepSurv model, selected due to its relatively superior performance, is deployed in the form of web application locally, and is accessible on GitHub ( https://github.com/Robidar/Chuchu_Depl ). Finally, as we have demonstrated the benefit of using AI for reassessment of an existing CVD risk score, we recommend other infamous risk scores undergo similar reassessment.

The diagnosis of dehydration among older patients with oropharyngeal dysphagia (OD) remains a clinical challenge. We aimed to assess the prevalence of dehydration in hospitalized older adults with OD and its relationship with other comorbidities. This is a cross-sectional study involving hospitalized older patients (≥ 70 years) with OD, assessed for OD using the Volume-Viscosity Swallowing Test. Treatment for impaired safety of swallow was based on xanthan gum thickened fluids with appropriate bolus viscosity (250 mPa·s or 800 mPa·s). Hydration status was evaluated using three different methodologies: (a) osmolarity equation (> 295 mmol/L), (b) blood urea nitrogen to creatinine ratio (BUN/Cr > 20 and (c) bioimpedance analysis (BIA): phase angle (PA) < 4.5. Nutritional status (Mini Nutritional Assessment-short form), functionality (Barthel Index), frailty (Fried Index), and sarcopenia (European Working Group on Sarcopenia in Older People) were also assessed. We included 218 hospitalized (9.1 ± 7.2 days) patients with OD (87.4 ± 5.5 years), 85.3% with safety impairments. On admission, (a) up to 58.3% needed fluid thickening for safe swallowing (90.6% of them at 250 mPa·s) and 93.6% textured modified diets, (b) According to BUN/Cr ratio, 78.9% were dehydrated and to osmolarity, 81.2% and (c) 61.1% of participants had values of PA < 4.5, and (d) hydration status was significantly worse in patients with lower functional status, frailty, malnutrition, and sarcopenia. Dehydration is a highly prevalent condition among hospitalized older patients with OD. Assessment of hydration status, and promotion and monitoring of safe fluid intake with multimodal hydration interventions, is mandatory in patients with OD.

Complex lipids, essential components in biological processes, exhibit conserved age-related changes that alter membrane properties and cellular functions and are implicated as biomarkers and contributors to longevity and age-related diseases. While physical activity alleviates age-related comorbidities and physical impairments, comprehensive exploration of the underlying biological mechanisms, particularly at the level of complex lipids, remains limited. However, clinical studies suggest that physical activity may counteract these age-related lipidomic changes, presenting a promising avenue for intervention. We performed lipidomic profiling of plasma from an extensively characterized cohort of young and aged individuals. Annotating 1446 unique lipid species across 24 lipid classes, we found the most prominent difference in older adults was an accumulation of triacylglycerols (TGs), with lower physical activity levels associated with higher TG levels in plasma and reduced physical functionality. Remarkably, lipid species in the TG class did not accumulate uniformly. Rather, our study unveiled a negative correlation between higher physical activity levels and TGs with shorter chain lengths and more double bonds in this demographic. Overall, our research highlights that plasma TG length and saturation level can help mark healthy aging groups in humans. These findings deepen our understanding of how aging affects complex lipids and the influence of physical activity on this process.

Identifying early predictors of cognitive decline and at-risk individuals is essential for timely intervention and prevention of dementia. This study aimed to detect neurobiological changes and factors related to cognitive performance in the Metropolit 1953 Danish male birth cohort. We analyzed data from 582 participants, aged 57-68 years, using machine learning techniques to group cognitive trajectories into four clusters differentiating high- and low-performing groups. These clusters were then evaluated with MRI, EEG, and lifestyle/familial risk factors to identify predictors of cognitive decline. Low education and occupation, alcohol consumption, and type 2 diabetes were associated with lower cognitive performance. Declines in neocortical volume and increases in frontotemporal alpha and temporoparietal gamma activity preceded clinical symptoms of cognitive decline. Neocortical atrophy and disruptions in network activity were prominent in lower-performing groups, with higher education and IQ scores and a lower prevalence of lifestyle factors moderating cognitive decline.

Cerebral pericytes are mural cells covering brain microvessels, organized as ensheathing, mesh and thin-strand pericytes. These latter two, together called capillary pericytes, have low levels of alpha smooth muscle actin (α-SMA), regulating basal vascular tone and applying a slow influence on cerebral blood flow. Pericytes are subject to alterations in ageing which may be even more pronounced in age-related pathologies, including microinfarcts, which usually affect a large number of vessels in the ageing brain. We modelled this condition by injecting 10 µm-size microspheres into the circulation of mice resulting in the occlusion of capillaries covered by ensheathing and mesh pericytes. We observed that α-SMA and Acta2, the gene encoding it, as well as TGF-β1/Tgfb1, the major regulator of α-SMA, decreased during ageing in cerebral microvessels. In the vicinity of the microspheres stalled in the capillaries, expression of α-SMA increased significantly in both ensheathing and especially in mesh pericytes, both in young (2 to 3 months of age) and old (24 months of age) mice. On the other hand, γ-actin was detected in endothelial cells, but not in pericytes, and decreased in microvessels of microsphere-containing hemispheres. Altogether, our data show that obstruction of cerebral microvessels increases α-SMA expression in pericytes in both age groups, but this does not compensate for the lower expression of the contractile protein in old animals. Increased α-SMA expression may lead to constriction of the obstructed vessels probably aggravating flow heterogeneity in the aged brain.

Epigenetic clocks, DNA methylation-based predictive models of chronological age, are often utilized to study aging associated biology. Despite their widespread use, these methods do not account for other factors that also contribute to the variability of DNA methylation data. For example, many CpG sites show strong sex-specific or cell-type-specific patterns that likely impact the predictions of epigenetic age. To overcome these limitations, we developed a multidimensional extension of the Epigenetic Pacemaker, the Multi-state Epigenetic Pacemaker (MSEPM). We show that the MSEPM is capable of accurately modeling multiple methylation-associated factors simultaneously, while also providing site-specific models that describe the per site relationship between methylation and these factors. We utilized the MSEPM with a large aggregate cohort of blood methylation data to construct models of the effects of age-, sex-, and cell-type heterogeneity on DNA methylation. We found that these models capture a large faction of the variability at thousands of DNA methylation sites. Moreover, this approach allows us to identify sites that are primarily affected by aging and no other factors. An analysis of these sites reveals that those that lose methylation over time are enriched for CTCF transcription factor chip peaks, while those that gain methylation over time are associated with bivalent promoters of genes that are not expressed in blood. These observations suggest mechanisms that underlie age-associated methylation changes and suggest that age-associated increases in methylation may not have strong functional consequences on cell states. In conclusion, the MSEPM is capable of accurately modeling multiple methylation-associated factors, and the models produced can illuminate site-specific combinations of factors that affect methylation dynamics.

Recently, a deep learning algorithm (DLA) has been developed to predict the chronological age from retinal images. The Retinal Age Gap (RAG), a deviation between predicted age from retinal images (Retinal Age, RA) and chronological age, correlates with mortality and age-related diseases. This study evaluated the reliability and accuracy of RA predictions and analyzed various factors that may influence them. We analyzed two groups of participants: Intravisit and Intervisit, both imaged by color fundus photography. RA was predicted using an established algorithm. The Intervisit group comprised 26 subjects, imaged in two sessions. The Intravisit group had 41 subjects, of whom each eye was photographed twice in one session. The mean absolute test-retest difference in predicted RA was 2.39 years for Intervisit and 2.13 years for Intravisit, with the latter showing higher prediction variability. The chronological age was predicted accurately from fundus photographs. Subsetting image pairs based on differential image quality reduced test-retest discrepancies by up to 50%, but mean image quality was not correlated with retest outcomes. Marked diurnal oscillations in RA predictions were observed, with a significant overestimation in the afternoon compared to the morning in the Intravisit cohort. The order of image acquisition across imaging sessions did not influence RA prediction and subjective age perception did not predict RAG. Inter-eye consistency exceeded 3 years. Our study is the first to explore the reliability of RA predictions. Consistent image quality enhances retest outcomes. The observed diurnal variations in RA predictions highlight the need for standardized imaging protocols, but RAG could soon be a reliable metric in clinical investigations.