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BACKGROUND: Cellular senescence has been associated with cancer as either a barrier mechanism restricting autonomous cell proliferation or a tumour-promoting microenvironmental mechanism that secretes proinflammatory paracrine factors. With most work done in non-human models and the heterogeneous nature of senescence, the precise role of senescent cells in the development of cancer in humans is not well understood. Furthermore, more than 1 million non-malignant breast biopsies are taken every year that could be a major resource for risk stratification. We aimed to explore the clinical relevance for breast cancer development of markers of senescence in mammary tissue from healthy female donors. METHODS: In this retrospective cohort study, we applied single-cell deep learning senescence predictors, based on nuclear morphology, to histological images of haematoxylin and eosin-stained breast biopsy samples from healthy female donors at the Komen Tissue Bank (KTB) at the Indiana University Simon Cancer Center (Indianapolis, IN, USA). All KTB participants (aged ≥18 years) who underwent core biopsies for research purposes between 2009 and 2019 were eligible for the study. Senescence was predicted in the epithelial (terminal duct lobular units [TDLUs] and non-TDLU epithelium), stromal, and adipose tissue compartments using validated models, previously trained on cells induced to senescence by ionising radiation (IR), replicative exhaustion (or replicative senescence; RS), or antimycin A, atazanavir-ritonavir, and doxorubicin (AAD) exposures. To benchmark our senescence-based cancer prediction results, we generated 5-year Gail scores-the current clinical gold standard for breast cancer risk prediction-for participants aged 35 years and older on the basis of characteristics at the time of tissue donation. The primary outcome was estimated odds of breast cancer via logistic modelling for each tissue compartment based on predicted senescence scores in cases (participants who had been diagnosed with breast cancer as of data cutoff, July 31, 2022) and controls (those who had not been diagnosed with breast cancer). FINDINGS: 4382 female donors (median age at donation 45 years [IQR 34-57]) were eligible for the study. As of data cutoff (median follow-up of 10 years [7-11]), 86 (2·0%) had developed breast cancer a mean of 4·8 years (SD 2·84) after date of donation and 4296 (98·0%) had not received a breast cancer diagnosis. Among the 86 cases, we found significant differences in adipose-specific IR and AAD senescence prediction scores compared with controls. Risk analysis showed that individuals in the upper half (above the median) of scores for the adipose tissue IR model had higher odds of developing breast cancer (odds ratio [OR] 1·71 [95% CI 1·10-2·68]; p=0·019), whereas the adipose AAD model revealed a reduced odds of developing breast cancer (OR 0·57 [0·36-0·88]; p=0·013). For the other tissue compartments and the RS model, no significant associations were found (except for stromal tissue via the IR model, had higher odds of developing breast cancer [OR 1·59, 1·03-2·49]). Individuals with both of the adipose risk factors had an OR of 3·32 (1·68-7·03; p=0·0009). Participants with 5-year Gail scores above the median had an OR for development of cancer of 2·33 (1·46-3·82; p=0·0012) compared with those with scores below the median. When combining Gail scores with our adipose AAD risk model, we found that individuals with both of these predictors had an OR of 4·70 (2·29-10·90; p<0·0001). When combining the Gail score with our adipose IR model, we found that individuals with both predictors had an OR of 3·45 (1·77-7·24; p=0·0002). INTERPRETATION: Assessment of senescence-associated nuclear morphologies with deep learning allows prediction of future cancer risk from normal breast biopsy samples. The combination of multiple models improved prediction of future breast cancer compared with the current clinical benchmark, the Gail model. Our results suggest an important role for microscope image-based deep learning models in predicting future cancer development. Such models could be incorporated into current breast cancer risk assessment and screening protocols. FUNDING: Novo Nordisk Foundation, Danish Cancer Society, and the US National Institutes of Health.

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Biomarkers of aging (BOA) are quantitative parameters that predict biological age and ideally its changes in response to interventions. In recent years, many promising molecular and omic BOA have emerged with an enormous potential for translational geroscience and improving healthspan. However, clinical translation remains limited, in part due to the gap between preclinical research and the application of BOA in clinical research and other translational settings. We surveyed experts in these areas to better understand current challenges for the translation of aging biomarkers. We identified six key barriers to clinical translation and developed guidance for the field to overcome them. Core recommendations include linking BOA to clinically actionable insights, improving affordability and availability to broad populations and validation of biomarkers that are robust and responsive at the level of individuals. Our work provides key insights and practical recommendations to overcome barriers impeding clinical translation of BOA.

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Gene drives are genetic modifications designed to propagate efficiently through a population. Most applications rely on homologous recombination during sexual reproduction in diploid organisms such as insects, but we recently developed a gene drive in herpesviruses that relies on co-infection of cells by wild-type and engineered viruses. Here, we report on a viral gene drive against human herpes simplex virus 1 (HSV-1) and show that it propagates efficiently in cell culture and during HSV-1 infection in mice. We describe high levels of co-infection and gene drive-mediated recombination in neuronal tissues during herpes encephalitis as the infection progresses from the site of inoculation to the peripheral and central nervous systems. In addition, we show evidence that a superinfecting gene drive virus could recombine with wild-type viruses during latent infection. These findings indicate that HSV-1 achieves high rates of co-infection and recombination during viral infection, a phenomenon that is currently underappreciated. Overall, this study shows that a viral gene drive could spread in vivo during HSV-1 infection, paving the way toward therapeutic applications.

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Epigenetic clocks are age predictors that use machine-learning models trained on DNA CpG methylation values to predict chronological or biological age. Increases in predicted epigenetic age relative to chronological age (epigenetic age acceleration) are connected to aging-associated pathologies, and changes in epigenetic age are linked to canonical aging hallmarks. However, epigenetic clocks rely on training data from bulk tissues whose cellular composition changes with age. Here, we found that human naive CD8+ T cells, which decrease in frequency during aging, exhibit an epigenetic age 15-20 years younger than effector memory CD8+ T cells from the same individual. Importantly, homogenous naive T cells isolated from individuals of different ages show a progressive increase in epigenetic age, indicating that current epigenetic clocks measure two independent variables, aging and immune cell composition. To isolate the age-associated cell intrinsic changes, we created an epigenetic clock, the IntrinClock, that did not change among 10 immune cell types tested. IntrinClock shows a robust predicted epigenetic age increase in a model of replicative senescence in vitro and age reversal during OSKM-mediated reprogramming.

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The cultivation of pineapple (Ananas comosus) is threatened worldwide by mealybug wilt disease of pineapple (MWP), whose etiology is not yet fully elucidated. In this study, we characterized pineapple mealybug wilt-associated ampeloviruses (PMWaVs, family Closteroviridae) from a diseased pineapple plant collected from Reunion Island, using a high-throughput sequencing approach combining Illumina short reads and Nanopore long reads. Reads co-assembly resulted in complete or near-complete genomes for six distinct ampeloviruses, including the first complete genome of pineapple mealybug wilt-associated virus 5 (PMWaV5) and that of a new species tentatively named pineapple mealybug wilt-associated virus 7 (PMWaV7). Short reads data provided high genome coverage and sequencing depths for all six viral genomes, contrary to long reads data. The 5' and 3' ends of the genome for most of the six ampeloviruses could be recovered from long reads, providing an alternative to RACE-PCRs. Phylogenetic analyses did not unveil any geographic structuring of the diversity of PMWaV1, PMWaV2 and PMWaV3 isolates, supporting the current hypothesis that PMWaVs were mainly spread by human activity and vegetative propagation.

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BACKGROUND: Evidence links lifestyle factors with Alzheimer's disease (AD). We report the first randomized, controlled clinical trial to determine if intensive lifestyle changes may beneficially affect the progression of mild cognitive impairment (MCI) or early dementia due to AD. METHODS: A 1:1 multicenter randomized controlled phase 2 trial, ages 45-90 with MCI or early dementia due to AD and a Montreal Cognitive Assessment (MoCA) score of 18 or higher. The primary outcome measures were changes in cognition and function tests: Clinical Global Impression of Change (CGIC), Alzheimer's Disease Assessment Scale (ADAS-Cog), Clinical Dementia Rating-Sum of Boxes (CDR-SB), and Clinical Dementia Rating Global (CDR-G) after 20 weeks of an intensive multidomain lifestyle intervention compared to a wait-list usual care control group. ADAS-Cog, CDR-SB, and CDR-Global scales were compared using a Mann-Whitney-Wilcoxon rank-sum test, and CGIC was compared using Fisher's exact test. Secondary outcomes included plasma Aß42/40 ratio, other biomarkers, and correlating lifestyle with the degree of change in these measures. RESULTS: Fifty-one AD patients enrolled, mean age 73.5. No significant differences in any measures at baseline. Only two patients withdrew. All patients had plasma Aß42/40 ratios <0.0672 at baseline, strongly supporting AD diagnosis. After 20 weeks, significant between-group differences in the CGIC (p= 0.001), CDR-SB (p= 0.032), and CDR Global (p= 0.037) tests and borderline significance in the ADAS-Cog test (p= 0.053). CGIC, CDR Global, and ADAS-Cog showed improvement in cognition and function and CDR-SB showed significantly less progression, compared to the control group which worsened in all four measures. Aß42/40 ratio increased in the intervention group and decreased in the control group (p = 0.003). There was a significant correlation between lifestyle and both cognitive function and the plasma Aß42/40 ratio. The microbiome improved only in the intervention group (p <0.0001). CONCLUSIONS: Comprehensive lifestyle changes may significantly improve cognition and function after 20 weeks in many patients with MCI or early dementia due to AD. TRIAL REGISTRATION: Approved by Western Institutional Review Board on 12/31/2017 (#20172897) and by Institutional Review Boards of all sites. This study was registered retrospectively with clinicaltrials.gov on October 8, 2020 (NCT04606420, ID: 20172897).

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Lysine malonylation is a protein posttranslational modification. We present a protocol to generate stable gene-knockdown K562 cell lines through lentiviral infection of a CRISPR interference (CRISPRi) system followed by lysine malonylation measurement using mass spectrometry (MS). We detail guide RNA (gRNA) vector cloning, lentiviral infection, cell line purification, protein digestion, malonyl-lysine enrichment, desalting, and MS acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Zhang et al.1 and Bons et al.2.

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In 2014, Physostegia chlorotic mottle virus (PhCMoV) was discovered in Austria in Physostegia virginiana. Subsequent collaborative efforts established a link between the virus and severe fruit symptoms on important crops such as tomato, eggplant, and cucumber across nine European countries. Thereafter, specific knowledge gaps, which are crucial to assess the risks PhCMoV can pose for production and how to manage it, needed to be addressed. In this study, the transmission, prevalence, and disease severity of PhCMoV were examined. This investigation led to the identification of PhCMoV presence in a new country, Switzerland. Furthermore, our research indicates that the virus was already present in Europe 30 years ago. Bioassays demonstrated PhCMoV can result in up to 100% tomato yield losses depending on the phenological stage of the plant at the time of infection. PhCMoV was found to naturally infect 12 new host plant species across eight families, extending its host range to 21 plant species across 15 plant families. The study also identified a polyphagous leafhopper (genus Anaceratagallia) as a natural vector of PhCMoV. Overall, PhCMoV was widespread in small-scale diversified vegetable farms in Belgium where tomato is grown in soil under tunnels, occurring in approximately one-third of such farms. However, outbreaks were sporadic and were associated at least once with the cultivation in tomato tunnels of perennial plants that can serve as a reservoir host for the virus and its vector. To further explore this phenomenon and manage the virus, studying the ecology of the vector would be beneficial.

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With geroscience research evolving at a fast pace, the need arises for human randomized controlled trials to assess the efficacy of geroprotective interventions to prevent age-related adverse outcomes, disease, and mortality in normative aging cohorts. However, to confirm efficacy requires a long-term and costly approach as time to the event of morbidity and mortality can be decades. While this could be circumvented using sensitive biomarkers of aging, current molecular, physiological, and digital endpoints require further validation. In this review, we discuss how collecting real-world evidence (RWE) by obtaining health data that is amenable for collection from large heterogeneous populations in a real-world setting can help speed up validation of geroprotective interventions. Further, we propose inclusion of quality of life (QoL) data as a biomarker of aging and candidate endpoint for geroscience clinical trials to aid in distinguishing healthy from unhealthy aging. We highlight how QoL assays can aid in accelerating data collection in studies gathering RWE on the geroprotective effects of repurposed drugs to support utilization within healthy longevity medicine. Finally, we summarize key metrics to consider when implementing QoL assays in studies, and present the short-form 36 (SF-36) as the most well-suited candidate endpoint.

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The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.

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The search for biomarkers that quantify biological aging (particularly 'omic'-based biomarkers) has intensified in recent years. Such biomarkers could predict aging-related outcomes and could serve as surrogate endpoints for the evaluation of interventions promoting healthy aging and longevity. However, no consensus exists on how biomarkers of aging should be validated before their translation to the clinic. Here, we review current efforts to evaluate the predictive validity of omic biomarkers of aging in population studies, discuss challenges in comparability and generalizability and provide recommendations to facilitate future validation of biomarkers of aging. Finally, we discuss how systematic validation can accelerate clinical translation of biomarkers of aging and their use in gerotherapeutic clinical trials.

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Senolytics, small molecules targeting cellular senescence, have emerged as potential therapeutics to enhance health span. However, their impact on epigenetic age remains unstudied. This study aimed to assess the effects of Dasatinib and Quercetin (DQ) senolytic treatment on DNA methylation (DNAm), epigenetic age, and immune cell subsets. In a Phase I pilot study, 19 participants received DQ for 6 months, with DNAm measured at baseline, 3 months, and 6 months. Significant increases in epigenetic age acceleration were observed in first-generation epigenetic clocks and mitotic clocks at 3 and 6 months, along with a notable decrease in telomere length. However, no significant differences were observed in second and third-generation clocks. Building upon these findings, a subsequent investigation evaluated the combination of DQ with Fisetin (DQF), a well-known antioxidant and antiaging senolytic molecule. After one year, 19 participants (including 10 from the initial study) received DQF for 6 months, with DNAm assessed at baseline and 6 months. Remarkably, the addition of Fisetin to the treatment resulted in non-significant increases in epigenetic age acceleration, suggesting a potential mitigating effect of Fisetin on the impact of DQ on epigenetic aging. Furthermore, our analyses unveiled notable differences in immune cell proportions between the DQ and DQF treatment groups, providing a biological basis for the divergent patterns observed in the evolution of epigenetic clocks. These findings warrant further research to validate and comprehensively understand the implications of these combined interventions.

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Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionally in vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHB's anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demonstrate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.

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Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

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In the past decade, severe epidemics of cucumber mosaic virus (CMV) have caused significant damage to Espelette pepper crops. This virus threatens the production of Espelette pepper, which plays a significant role in the local economy and touristic attractiveness of the French Basque Country, located in southwestern France. In 2021 and 2022, CMV was detected via double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) in Gorria pepper seed lots harvested from naturally infected fields scattered throughout the entire Espelette pepper production area. These seed lots were used in greenhouse grow-out tests to determine whether CMV could be transmitted to seedlings from contaminated seeds, using visual symptom assessment, DAS-ELISAs, and reverse transcription-polymerase chain reaction (RT-PCR). Despite the widespread occurrence of CMV in seeds of field samples, the grow-out experiments on a total of over 5000 seedlings yielded no evidence of seed transmission of local CMV isolates in Gorria pepper. Therefore, rather than seeds from infected pepper plants, sources of CMV inoculum in Espelette are more likely to be alternative hosts present in and around pepper fields that can allow for the survival of CMV during the off-season. These results have important epidemiological implications and will guide the choice of effective measures to control current epidemics.

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Despite remarkable progress, a cure for HIV-1 infection remains elusive. Rebound competent latent and transcriptionally active reservoir cells persevere despite antiretroviral therapy and rekindle infection due to inefficient proviral silencing. We propose a novel "block-lock-stop" approach, entailing long term durable silencing of viral expression towards an irreversible transcriptionally inactive latent provirus to achieve long term antiretroviral free control of the virus. A graded transformation of remnant HIV-1 in PLWH from persistent into silent to permanently defective proviruses is proposed, emulating and accelerating the natural path that human endogenous retroviruses (HERVs) take over millions of years. This hypothesis was based on research into delineating the mechanisms of HIV-1 latency, lessons from latency reversing agents and advances of Tat inhibitors, as well as expertise in the biology of HERVs. Insights from elite controllers and the availability of advanced genome engineering technologies for the direct excision of remnant virus set the stage for a rapid path to an HIV-1 cure.

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Mammalian female reproductive lifespan is typically significantly shorter than life expectancy and is associated with a decrease in ovarian NAD+ levels. However, the mechanisms underlying this loss of ovarian NAD+ are unclear. Here, we show that CD38, an NAD+ consuming enzyme, is expressed in the ovarian extrafollicular space, primarily in immune cells, and its levels increase with reproductive age. Reproductively young mice lacking CD38 exhibit larger primordial follicle pools, elevated ovarian NAD+ levels, and increased fecundity relative to wild type controls. This larger ovarian reserve results from a prolonged window of follicle formation during early development. However, the beneficial effect of CD38 loss on reproductive function is not maintained at advanced age. Our results demonstrate a novel role of CD38 in regulating ovarian NAD+ metabolism and establishing the ovarian reserve, a critical process that dictates a female's reproductive lifespan.