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The SARS-CoV-2 pandemic has reemphasized the urgent need to develop broad-spectrum antiviral therapies. Wedeveloped a computational pipeline that uses scRNA-Seq data to reconstruct the metabolic state of cells and tissuesduring viral infection. Using this pipeline, we investigated the cellular capacity to produce SARS-CoV-2 virions invarious tissues and disease conditions. Subsequently, we expanded our analysis to influenza A and dengue virus andidentified several metabolic targets and their inhibitors for broad-spectrum antiviral treatment. Phenformin, an inhibitorof NADH:ubiquinone oxidoreductase, suppressed SARS-CoV-2 and dengue virus replication. Using Atpenin A5 toblock the succinate dehydrogenase inhibited SARS-CoV-2, dengue virus, influenza A virus and respiratory syncytialvirus with superior therapeutic indices. Thus, our work establishes host metabolism as druggable for broad antiviraltherapy. Moreover, our pipeline, the identified targets, and inhibitors are invaluable tools for pandemic preparedness.

Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year's 8th Annual Aging Research and Drug Discovery (ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community., Aging, 14 (2), ISSN:1945-4589

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Aging is a natural process of organismal decay that underpins the development of myriad diseases and disorders. Extensive efforts have been made to understand the biology of aging and its regulation, but most studies focus solely on the host organism. Considering the pivotal role of the microbiota in host health and metabolism, we propose viewing the host and its microbiota as a single biological entity whose aging phenotype is influenced by the complex interplay between host and bacterial genetics. In this review we present how the microbiota changes as the host ages, but also how the intricate relationship between host and indigenous bacteria impacts organismal aging and life span. In addition, we highlight other microbiota-dependent mechanisms that potentially regulate aging, and present experimental animal models for addressing these questions. Importantly, we propose microbiome dysbiosis as an additional hallmark and biomarker of aging.

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention., Author summary The increasing number of people living with age-related diseases underscores the importance of ageing research to improve healthspan. Two well-studied evolutionary conserved interventions that extend lifespan and improve health are dietary restriction and down-regulation of nutrient sensing pathways, such as glucose sensing by insulin and amino acid sensing by the target-of-rapamycin signalling pathway. One common characteristic of these anti-ageing interventions is an increase in autophagy, a cellular pathway that degrades damaged proteins and organelles to supply essential building blocks and energy. To help provide a more direct link between autophagy and healthy ageing, we fine-tuned overexpression of Atg1 kinase, which is critical for autophagy induction, and measured its effect on longevity in the fruit fly Drosophila. Interestingly, we observed that a moderate increase in autophagy is beneficial in extending healthy lifespan, whereas strong autophagy up-regulation is detrimental and leads to progressive lipid loss and decreased lifespan. Moderate and stronger Atg1 overexpression displayed opposing transcriptional profiles of mitochondrial genes, being upregulated in long-lived and down-regulated in short-lived Atg1 over-expressing animals. Overall, we provide a detailed description of the phenotypes associated with varying degrees of autophagy up-regulation in vivo, demonstrating that autophagy enhancement delays ageing only when applied in moderation.

The nematode Caenorhabditis elegans is commonly used as a model organism in studies of the host immune response. The worm encodes twelve peroxidase-cyclooxygenase superfamily members, making it an attractive model in which to study the functions of heme peroxidases. In previous work, loss of one of these peroxidases, SKPO-1 (ShkT-containing peroxidase), rendered C. elegans more sensitive to the human, Gram-positive pathogen Enterococcus faecalis. SKPO-1 was localized to the hypodermis of the animals where it also affected cuticle development as indicated by a morphological phenotype called “dumpy.” In this work, a better understanding of how loss of skpo-1 impacts both sensitivity to pathogen as well as cuticle development was sought by subjecting a deletion mutant of skpo-1 to transcriptome analysis using RNA sequencing following exposure to control (Escherichia coli) and pathogenic (E. faecalis) feeding conditions. Loss of skpo-1 caused a general upregulation of genes encoding collagens and other proteins related to cuticle development. On E. faecalis, these animals also failed to upregulate guanylyl cyclases that are often involved in environmental sensing. Hoechst straining revealed increased permeability of the cuticle and atomic force microscopy exposed the misalignment of the cuticular annuli and furrows. These findings provide a basis for better understanding of the morphological as well as the pathogen sensitivity phenotypes associated with loss of SKPO-1 function.

Microbes are an integral part of life on this planet. Microbes and their hosts influence each other in an endless dance that shapes how the meta-organism interacts with its environment. Although great advances have been made in microbiome research over the past 20 years, the mechanisms by which both hosts and their microbes interact with each other and the environment are still not well understood. The nematode Caenorhabditis elegans has been widely used as a model organism to study a remarkable number of human-like processes. Recent evidence shows that the worm is a powerful tool to investigate in fine detail the complexity that exists in microbe-host interactions. By combining the large array of genetic tools available for both organisms together with deep phenotyping approaches, it has been possible to uncover key effectors in the complex relationship between microbes and their hosts. In this perspective, we survey the literature for insightful discoveries in the microbiome field using the worm as a model. We discuss the latest conceptual and technological advances in the field and highlight the strengths that make C. elegans a valuable biosensor tool for the study of microbe-host interactions.

Despite the widely acknowledged fact that the microbiota regulates many aspects of human health, the dynamics and factors that govern these interactions remain mostly unknown. Pharmacomicrobiomics is a new research frontier in pharmacology that studies the interaction between drugs and the microbiota. This discipline, by including the microbiota as a key regulator of host health, calls for a redefinition of what constitutes a drug target and ultimately what is a drug or drug therapy. This is supported by recent evidence showing that host physiology can no longer be studied in separation from its microbial ecology and the environmental factors that shape it, as the combination of these elements forms the physiological unit of study – the holobiont. Here we discuss both the novel challenges and untapped opportunities that this new framework creates. On one hand, a more complete understanding of the physiology of the host imposes the development/adaptation of new animal models to address these interactions. In particular, we focus on the advantages and disadvantages of Caenorhabditis elegans as a host organism. On the other hand – a complete understanding of the effects of the microbiota and xenobiotics (e.g. drugs and dietary metabolites) on host health opens new prospects for personalized therapy.

Genetic and environmental factors are key drivers regulating organismal lifespan but how these impact healthspan is less well understood. Techniques capturing biomechanical properties of tissues on a nano-scale level are providing new insights into disease mechanisms. Here, we apply Atomic Force Microscopy (AFM) to quantitatively measure the change in biomechanical properties associated with ageing Caenorhabditis elegans in addition to capturing high-resolution topographical images of cuticle senescence. We show that distinct dietary restriction regimes and genetic pathways that increase lifespan lead to radically different healthspan outcomes. Hence, our data support the view that prolonged lifespan does not always coincide with extended healthspan. Importantly, we identify the insulin signalling pathway in C. elegans and interventions altering bacterial physiology as increasing both lifespan and healthspan. Overall, AFM provides a highly sensitive technique to measure organismal biomechanical fitness and delivers an approach to screen for health-improving conditions, an essential step towards healthy ageing., The development of reliable measures of health in ageing organisms is a need in ageing research. Using atomic force microscopy, here, the authors assess whole body stiffness of worms and show that it reflects organismal fitness.

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA., Aging, 12 (24), ISSN:1945-4589

[Image: see text] Caenorhabditis elegans has become a key model organism within biology. In particular, the transparent gut, rapid growing time, and ability to create a defined gut microbiota make it an ideal candidate organism for understanding and engineering the host microbiota. Here we present the development of an experimental model that can be used to characterize whole-cell bacterial biosensors in vivo. A dual-plasmid sensor system responding to isopropyl β-d-1-thiogalactopyranoside was developed and fully characterized in vitro. Subsequently, we show that the sensor was capable of detecting and reporting on changes in the intestinal environment of C. elegans after introducing an exogenous inducer into the environment. The protocols presented here may be used to aid the rational design of engineered bacterial circuits, primarily for diagnostic applications. In addition, the model system may serve to reduce the use of current animal models and aid in the exploration of complex questions within general nematode and host–microbe biology.

Redox signalling is an important modulator of diverse biological pathways and processes, and operates through specific post-translational modification of redox-sensitive thiols on cysteine residues 1–4. Critically, redox signalling is distinct from irreversible oxidative damage and functions as a reversible ‘redox switch’ to regulate target proteins. H2O2 acts as the major effector of redox signalling, both directly and through intracellular thiol redox relays 5,6. Dysregulation of redox homeostasis has long been implicated in the pathophysiology of many age-related diseases, as well as in the ageing process itself, however the underlying mechanisms remain largely unclear 7,8. To study redox signalling by H2O2in vivo and explore its involvement in metabolic health and longevity, we used the fruit fly Drosophila as a model organism, with its tractable lifespan and strong evolutionary conservation with mammals 9. Here we report that inducing an endogenous redox-shift, by manipulating levels of the H2O2-degrading enzyme catalase, improves health and robustly extends lifespan in flies, independently of oxidative stress resistance and dietary restriction. We find that the catalase redox-shifted flies are acutely sensitive to starvation stress, which relies on autophagy as a vital survival mechanism. Importantly, we show that autophagy is essential for the lifespan extension of the catalase flies. Furthermore, using redox-inactive knock-in mutants of Atg4a, a major effector of autophagy, we show that the lifespan extension in response to catalase requires a key redox-regulatory cysteine residue, Cys102 in Atg4a. These findings demonstrate that redox regulation of autophagy can extend lifespan, confirming the importance of redox signalling in ageing and as a potential pro-longevity target.

Caenorhabditis elegans has become a key model organism within biology. In particular, the transparent gut, rapid growing time, and ability to create a defined gut microbiota make it an ideal candidate organism for understanding and engineering the host microbiota. Here we present the development of an experimental model that can be used to characterize whole-cell bacterial biosensors in vivo. A dual-plasmid sensor system responding to isopropyl β-d-1-thiogalactopyranoside was developed and fully characterized in vitro. Subsequently, we show that the sensor was capable of detecting and reporting on changes in the intestinal environment of C. elegans after introducing an exogenous inducer into the environment. The protocols presented here may be used to aid the rational design of engineered bacterial circuits, primarily for diagnostic applications. In addition, the model system may serve to reduce the use of current animal models and aid in the exploration of complex questions within general nematode and host-microbe biology.

Caenorhabditis elegans has become a key model organism within biology. In particular, the transparent gut, rapid growing time and ability to create a defined gut microbiota make it an ideal candidate organism for understanding and engineering the host microbiota. Here we present the development of an experimental model which can be used to characterise whole-cell bacterial biosensors in vivo. A dual-plasmid sensor system responding to isopropyl β-D-1-thiogalactopyranoside was developed and fully characterised in vitro. Subsequently, we show the sensor was capable of detecting and reporting on changes in the intestinal environment of C. elegans after introducing exogenous inducer into the environment. The protocols presented here may be used for aiding the rational design of engineered bacterial circuits, primarily for diagnostic applications. In addition, the model system may serve to reduce the use of current animal models and aid in the exploration of complex questions within general nematode and host-microbe biology.

Summary Loss of proteostasis is a fundamental process driving aging. Proteostasis is affected by the accuracy of translation, yet the physiological consequence of having fewer protein synthesis errors during multi-cellular organismal aging is poorly understood. Our phylogenetic analysis of RPS23, a key protein in the ribosomal decoding center, uncovered a lysine residue almost universally conserved across all domains of life, which is replaced by an arginine in a small number of hyperthermophilic archaea. When introduced into eukaryotic RPS23 homologs, this mutation leads to accurate translation, as well as heat shock resistance and longer life, in yeast, worms, and flies. Furthermore, we show that anti-aging drugs such as rapamycin, Torin1, and trametinib reduce translation errors, and that rapamycin extends further organismal longevity in RPS23 hyperaccuracy mutants. This implies a unified mode of action for diverse pharmacological anti-aging therapies. These findings pave the way for identifying novel translation accuracy interventions to improve aging., Graphical abstract, Highlights • Evolutionarily selected arginine in RPS23 is present only in hyperthermophilic archaea • RPS23 K60R mutation in flies leads to improved accuracy of protein synthesis with age • Yeast, worm, and fly RPS23 K60R mutants are longer-lived, healthier, and heat resistant • Anti-aging drugs, rapamycin, torin1, and trametinib, increase translation accuracy, Martinez-Miguel et al. demonstrate that improving translation fidelity by mutating a single amino acid in the decoding center of the ribosome suffices to improve health and longevity in yeast, worms, and flies. This work provides a direct link between fewer errors in translation and longevity.

Caenorhabditis elegans is an excellent model for high‐throughput experimental approaches but lacks an automated means to pinpoint time of death during survival assays over a short time frame, that is, easy to implement, highly scalable, robust, and versatile. Here, we describe an automated, label‐free, high‐throughput method using death‐associated fluorescence to monitor nematode population survival (dubbed LFASS for label‐free automated survival scoring), which we apply to severe stress and infection resistance assays. We demonstrate its use to define correlations between age, longevity, and severe stress resistance, and its applicability to parasitic nematodes. The use of LFASS to assess the effects of aging on susceptibility to severe stress revealed an unexpected increase in stress resistance with advancing age, which was largely autophagy‐dependent. Correlation analysis further revealed that while severe thermal stress resistance positively correlates with lifespan, severe oxidative stress resistance does not. This supports the view that temperature‐sensitive protein‐handling processes more than redox homeostasis underpin aging in C. elegans. That the ages of peak resistance to infection, severe oxidative stress, heat shock, and milder stressors differ markedly suggests that stress resistance and health span do not show a simple correspondence in C. elegans.

Caenorhabditis elegans is an excellent model for high-throughput experimental approaches, but lacks a robust, versatile and automated means to pinpoint time of death. Here we describe an automated, label-free, high-throughput method using death-associated fluorescence to monitor nematode survival, which we apply to stress and infection resistance assays. We demonstrate its use to define correlations between age, longevity and stress-resistance, and reveal an autophagy-dependent increase in acute stress resistance in early adulthood.

The gut microbiota is a key player regulating human physiology and can adjust its structure and metabolic activity in response to host health. A recent Nature paper by Forslund et al. (2015) reveals that microbiota adaptation to metformin treatment are responsible for the drug's therapeutic effects against type 2 diabetes.

Folates act as co-factors for transfer of one-carbon units for nucleotide production, methylation and other biosynthetic reactions. Comprehensive profiling of multiple folates can be achieved using liquid chromatography tandem mass spectrometry, enabling determination of their relative abundance that may provide an indication of metabolic differences between cell types. For example, cell lines exposed to methotrexate showed a dose-dependent elevation of dihydrofolate, consistent with inhibition of dihydrofolate reductase. We analysed the folate profile of E. coli sub-types as well as cell lines and embryonic tissue from both human and mouse. The folate profile of bacteria differed markedly from those of all the mammalian samples, most notably in the greater abundance of formyl tetrahydrofolate. The overall profiles of mouse and human fibroblasts and mid-gestation mouse embryos were broadly similar, with specific differences. The major folate species in these cell types was 5-methyl tetrahydrofolate, in contrast to lymphoblastoid cell lines in which the predominant form was tetrahydrofolate. Analysis of embryonic human brain revealed a shift in folate profile with increasing developmental stage, with a decline in relative abundance of dihydrofolate and increase in 5-methyl tetrahydrofolate. These cell type-specific and developmental changes in folate profile may indicate differential requirements for the various outputs of folate metabolism. Electronic supplementary material The online version of this article (doi:10.1007/s11010-013-1613-y) contains supplementary material, which is available to authorized users.

Summary Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease., Graphical Abstract, Highlights • Drug-microbe-host high-throughput screens reveal new mechanisms for cancer drugs • Microbes integrate nutritional and drug cues regulating treatment efficacy in the host • Ribonucleotide co-metabolism of cancer pro-drugs exists between host and microbe • Imbalanced bacterial deoxynucleotides synergize 5-FU-induced autophagic cell death, A three-way high-throughput screen involving host-microbe-drug interactions reveals that the beneficial impact of some drugs can be due to effects of drug-dependent alterations by gut microbe composition rather than direct action of the therapeutic itself.

Aging (senescence) includes causal mechanisms (etiologies) of late-life disease, which remain poorly understood. According to the recently proposed hyperfunction theory, based on the older theory of antagonistic pleiotropy, senescent pathologies can arise from futile, post-reproductive run-on of processes that in early life promote fitness. Here we apply this idea to investigate the etiology of senescent pathologies in the reproductive system of Caenorhabditis elegans hermaphrodites, particularly distal gonad degeneration and disintegration. Hermaphrodite germ cells frequently undergo “physiological” (non-damage-induced) apoptosis (PA) to provision growing oocytes. Run-on of such PA is a potential cause of age-related gonad degeneration. We document the continuation of germline apoptosis in later life, and report that genetically blocking or increasing PA retards or accelerates degeneration, respectively. In wild-type males, which lack germ line apoptosis, gonad disintegration does not occur. However, mutational induction of PA in males does not lead to gonad disintegration. These results suggest that as germ-cell proliferation rate declines markedly in aging hermaphrodites (but not males), run-on of PA becomes a pathogenic mechanism that promotes gonad degeneration. This illustrates how hyperfunction, or non-adaptive run-on in later life of a process that promotes fitness in early life, can promote atrophic senescent pathology in C. elegans.

Highlights ► Here we test whether iron-catalyzed oxidative damage contributes to organismal ageing. ► We develop new methodologies to measure free iron in vivo in C. elegans. ► Moderate iron supplementation can increase oxidative damage without reducing lifespan. ► Iron chelation or increasing ferritin levels increase resistance to oxidative stress but do not increase lifespan. ► Our findings argue against the oxidative damage theory of ageing., Iron-catalyzed generation of free radicals leads to molecular damage in vivo, and has been proposed to contribute to organismal ageing. Here we investigate the role of free iron in ageing in the nematode Caenorhabditis elegans. Media supplementation with Fe(III) increased free iron levels in vivo, as detected by continuous-wave electron paramagnetic resonance spectroscopy and elevated expression of the iron-sensitive reporter transgene pftn-1::gfp. Increased free iron levels caused elevated levels of protein oxidation and hypersensitivity to tert-butyl hydroperoxide (t-BOOH) given 9 mM Fe(III) or greater, but 15 mM Fe(III) or greater was required to reduce lifespan. Treatment with either an iron chelator (deferoxamine) or over-expression of ftn-1, encoding the iron sequestering protein ferritin, increased resistance to t-BOOH and, in the latter case, reduced protein oxidation, but did not increase lifespan. Expression of ftn-1 is greatly increased in long-lived daf-2 insulin/IGF-1 receptor mutants. In this context, deletion of ftn-1 decreased t-BOOH resistance, but enhanced both daf-2 mutant longevity and constitutive dauer larva formation, suggesting an effect of ferritin on signaling. These results show that high levels of iron can increase molecular damage and reduce lifespan, but overall suggest that iron levels within the normal physiological range do not promote ageing in C. elegans.

The superoxide free radical (O2•−) has been viewed as a likely major contributor to aging. If this is correct, then superoxide dismutase (SOD), which removes O2•−, should contribute to longevity assurance. In Caenorhabditis elegans, overexpression (OE) of the major cytosolic Cu/Zn-SOD, sod-1, increases life span. But is this increase caused by enhanced antioxidant defense? sod-1 OE did not reduce measures of lipid oxidation or glycation and actually increased levels of protein oxidation. The effect of sod-1 OE on life span was dependent on the DAF-16/FoxO transcription factor (TF) and, partially, on the heat shock TF HSF-1. Similarly, overexpression of sod-2 (major mitochondrial Mn-SOD) resulted in life-span extension that was daf-16 dependent. sod-1 OE increased steady-state hydrogen peroxide (H2O2) levels in vivo. However, co-overexpression of catalase did not suppress the life-span extension, arguing against H2O2 as a cause of longevity. sod-1 OE increased hsp-4 expression, suggesting increased endoplasmic reticulum (ER) stress. Moreover, longevity was partially suppressed by inactivation of ire-1 and xbp-1, mediators of the ER stress response. This suggests that high levels of SOD-1 protein may challenge protein-folding homeostasis, triggering a daf-16- and hsf-1-dependent stress response that extends life span. These findings imply that SOD overexpression increases C. elegans life span, not by removal of O2•−, but instead by activating longevity-promoting transcription factors., Highlights ► sod-1 over-expression increases levels of cellular ROS and of molecular damage. ► sod-1 over-expression effects on lifespan are dependent on DAF-16 (FoxO) and HSF-1. ► Increased lifespan is partially suppressed by inactivation of ER stress mediators. ► sod-2 (MnSOD) over-expression effects on lifespan are dependent on DAF-16.

Summary Hydrogen peroxide (H2O2) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H2O2 in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H2O2 levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H2O2 to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H2O2 that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H2O2 with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H2O2 correlates with aging, it may not be causative., Highlights ► A mitochondria-targeted mass spectrometry probe measures mitochondrial H2O2 in vivo ► Overall mitochondrial H2O2 increases with age but can be independent of life span ► Increased physical activity leads to a decrease in mitochondrial H2O2 ► Hypotheses dependent on overall mitochondrial ROS can now be assessed in vivo

According to the mitochondrial theory of aging, mitochondrial dysfunction increases intracellular reactive oxidative species production, leading to the oxidation of macromolecules and ultimately to cell death. In this study, we investigated the role of the mitochondrial methionine sulfoxide reductase B2 in the protection against oxidative stress. We report, for the first time, that overexpression of methionine sulfoxide reductase B2 in mitochondria of acute T-lymphoblastic leukemia MOLT-4 cell line, in which methionine sulfoxide reductase A is missing, markedly protects against hydrogen peroxide-induced oxidative stress by scavenging reactive oxygen species. The addition of hydrogen peroxide provoked a time-gradual increase of intracellular reactive oxygen species, leading to a loss in mitochondrial membrane potential and to protein carbonyl accumulation, whereas in methionine sulfoxide reductase B2-overexpressing cells, intracellular reactive oxygen species and protein oxidation remained low with the mitochondrial membrane potential highly maintained. Moreover, in these cells, delayed apoptosis was shown by a decrease in the cleavage of the apoptotic marker poly(ADP-ribose) polymerase-1 and by the lower percentage of Annexin-V-positive cells in the late and early apoptotic stages. We also provide evidence for the protective mechanism of methionine sulfoxide reductase B2 against protein oxidative damages. Our results emphasize that upon oxidative stress, the overexpression of methionine sulfoxide reductase B2 leads to the preservation of mitochondrial integrity by decreasing the intracellular reactive oxygen species build-up through its scavenging role, hence contributing to cell survival and protein maintenance.

Aging has been associated with zinc deficiency, leading to chronic inflammation and subsequent oxidative stress, especially in the immune system. The increased oxidative stress provokes the accumulation of oxidized proteins, raising the problem of the efficacy of intracellular protein maintenance systems responsible for the elimination of oxidatively modified proteins. Our objective was to analyse the effect of zinc supplementation in the elderly on protein maintenance in peripheral blood lymphocytes. The status of the proteasome, which is in charge of oxidized protein degradation and the repair enzymes peptide methionine sulfoxide reductases, which can reverse methionine oxidation in proteins, were analysed on peripheral blood lymphocytes collected from 20 elderly subjects (age range between 59 and 85 years old) before and after zinc supplementation (10mg of zinc per day for 48+/-2 days). A decrease of oxidized protein content in zinc supplemented subjects was observed and was associated with an increase of expression levels and/or activities of proteasome and methionine sulfoxide reductases. Our results indicate that zinc treatment could enhance the anti-oxidative defences of peripheral blood lymphocytes by increasing the activities of protein maintenance systems responsible for the elimination of oxidatively modified proteins.

Improvements in healthcare and nutrition have generated remarkable increases in life expectancy worldwide. This is one of the greatest achievements of the modern world yet it also presents a grave challenge: as more people survive into later life, more also experience the diseases of old age, including type 2 diabetes (T2D), cardiovascular disease (CVD) and cancer. Developing new ways to improve health in the elderly is therefore a top priority for biomedical research. Although our understanding of the molecular basis of these morbidities has advanced rapidly, effective novel treatments are still lacking. Alternative drug development strategies are now being explored, such as the repurposing of existing drugs used to treat other diseases. This can save a considerable amount of time and money since the pharmacokinetics, pharmacodynamics and safety profiles of these drugs are already established, effectively enabling preclinical studies to be bypassed. Metformin is one such drug currently being investigated for novel applications. The present review provides a thorough and detailed account of our current understanding of the molecular pharmacology and signalling mechanisms underlying biguanide–protein interactions. It also focuses on the key role of the microbiota in regulating age-associated morbidities and a potential role for metformin to modulate its function. Research in this area holds the key to solving many of the mysteries of our current understanding of drug action and concerted effects to provide sustained and long-life health.

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Methionine is among the amino acids the most susceptible to oxidation by almost all forms of ROS, resulting in both S and R diasteroisomeric forms of methionine sulfoxide. These modifications can be repaired specifically by the peptide methionine sulfoxide reductase A and B enzymes (MsrA and MsrB), respectively. MsrA has been detected in several organisms going from prokaryotes to eukaryotes. MsrA is tightly implicated in protection against oxidative stress and in protein maintenance, which is critical in the aging process. Several studies have shown that overexpression of MsrA led to an increased resistance against oxidative stress, while MsrA null mutants are more sensitive toward oxidative stress. Since oxidative damage is a key factor in aging, overexpression of MsrA in some organisms led to an increased life span whereas deletion of the gene led to the opposite. MsrA could also be involved, by regulating the function and/or expression of target proteins, in ROS-mediated signal transduction. In fact, changes in gene expression, including certain oxidative stress-response genes, have been observed when MsrA is overexpressed. This review elaborates on the current knowledge in the implication of the Msr system in protection against oxidative stress and aging.

A GENiE (EU COST action, www.worm-genie.eu) workshop was held at the Babraham Institute in Cambridge, UK on April 19 and 20, 2017, to discuss global challenges around Caenorhabditis elegans metabol...

Death of the nematode Caenorhabditis elegans involves a conserved necrotic cell death cascade which generates endogenous blue anthranilate fluorescence, allowing death to be visualized., For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters—not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals—e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death., Author Summary In the nematode Caenorhabditis elegans, intestinal lysosome-related organelles (or “gut granules”) contain a bright blue fluorescent substance of unknown identity. This has similar spectral properties to lipofuscin, a product of oxidative damage known to accumulate with age in postmitotic mammalian cells. Blue fluorescence seems to increase in aging worm populations, and lipofuscin has been proposed to be the source. To analyze this further, we measure fluorescence levels after exposure to oxidative stress and during aging in individually tracked worms. Surprisingly, neither of these conditions increases fluorescence levels; instead blue fluorescence increases in a striking and rapid burst at death. Such death fluorescence (DF) also appears in young worms when killed, irrespective of age or cause of death. We chemically identify DF as anthranilic acid glucosyl esters derived from tryptophan, and not lipofuscin. In addition, we show that DF generation in the intestine is dependent upon the necrotic cell death cascade, previously characterized as a driver of neurodegeneration. We find that necrosis spreads in a rapid wave along the intestine by calcium influx via innexin ion channels, accompanied by cytosolic acidosis. Inhibition of necrosis pathway components can delay stress-induced death, supporting its role as a driver of organismal death. This necrotic cascade provides a model system to study neurodegeneration and organismal death.

Many animal species live in close association with commensal and symbiotic microbes (microbiota). Recent studies have revealed that the status of gastrointestinal tract microbiota can influence nutrition-related syndromes such as obesity and type-2 diabetes, and perhaps aging. These morbidities have a profound impact in terms of individual suffering, and are an increasing economic burden to modern societies. Several theories have been proposed for the influence of microbiota on host metabolism, but these largely remain to be proven. In this article we discuss how microbiota may be manipulated (via pharmacology, diet, or gene manipulation) in order to alter metabolism, immunity, health and aging in the host. The nematode Caenorhabditis elegans in combination with one microbial species is an excellent, defined model system to investigate the mechanisms of host–microbiota interactions, particularly given the combined power of worm and microbial genetics. We also discuss the multifaceted nature of the worm–microbe relationship, which likely encompasses predation, commensalism, pathogenicity and necromeny.

SummaryThe biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy—and also to its side effects, which include folate deficiency and gastrointestinal upset.PaperClip

Overexpression of sirtuins (NAD+-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila melanogaster1,2,3. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background4. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression1 abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression2 also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported3, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent3. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.

During the last decade, biogerontologists have labored to understand the biological basis of the aging process by studying the genes and signaling pathways that regulate it. But the last year has seen a breakthrough in a different direction: toward treatments that might slow aging by mimicking the effects of dietary restriction.

Cell culture and in vitro models are the basis for much biological research, especially in human immunology. Ex vivo studies of T cell physiology employ conditions attempting to mimic the in vivo situation as closely as possible. Despite improvements in controlling the cellular milieu in vitro, most of what is known about T cell behavior in vitro is derived from experiments on T cells exposed to much higher oxygen levels than are normal in vivo. In this study, we report a reduced proliferative response and increased apoptosis susceptibility after T cell activation at 2% oxygen compared to in air. To explain this observation, we tested the hypothesis of an impaired efficacy of intracellular protective mechanisms including antioxidant levels, oxidized protein repair (methionine sulfoxide reductases), and degradation (proteasome) activities. Indeed, after activation, there was a significant accumulation of intracellular oxidized proteins at more physiological oxygen levels concomitant with a reduced GSH:GSSG ratio. Proteasome and methionine sulfoxide reductase activities were also reduced. These data may explain the increased apoptotic rate observed at more physiological oxygen levels. Altogether, this study highlights the importance of controlling oxygen levels in culture when investigating oxygen-dependent phenomena such as oxidative stress.

Methionine sulfoxide reductase A overexpressing WI-38 SV40 human fibroblasts have been previously shown to exhibit higher resistance to oxidative stress by decreasing intracellular reactive oxygen species content and oxidative damage to proteins [C.R. Picot, I. Petropoulos, M. Perichon, M. Moreau, C. Nizard, B. Friguet, Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress, Free Radic Biol Med 39 (2005) 1332-1341]. In order to get further insight into the molecular mechanisms underlying this resistance to oxidative stress, proteins that are differentially expressed in methionine sulfoxide reductase A overexpressing cells were identified by 2D gel and Western blot quantitative analyses. Five proteins were shown to be differentially expressed and were identified by mass spectrometry, some of them were related to either cellular protection against oxidative stress, apoptosis or premature ageing.