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Abstract While previous studies identified common genetic variants associated with longevity in centenarians, the role of the rare loss-of-function (LOF) mutation burden remains largely unexplored. Here, we investigated the burden of rare LOF mutations in Ashkenazi Jewish individuals from the Longevity Genes Project and LonGenity study cohorts using whole-exome sequencing data. We found that centenarians had a significantly lower burden (11-22%) of LOF mutations compared to controls. Similar effects were also observed in their offspring. Gene-level burden analysis identified 35 genes with depleted LOF mutations in centenarians, with 14 of these validated in the UK Biobank. Mendelian randomization and multi-omic analyses on these genes identified RGP1, PCNX2, and ANO9 as longevity genes with consistent causal effects on multiple aging-related traits and altered expression during aging. Our findings suggest that a protective genetic background, characterized by a reduced burden of damaging variants, contributes to exceptional longevity, likely acting in concert with specific protective variants to promote healthy aging.

Abstract DNA methylation (DNAm) is one of the most reliable biomarkers of aging across mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, DNAm gain is less characterized. Studies have demonstrated that CpGs which gain methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. However, whole-genome examination of all PRC2 targets as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, we show that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the “PRC2-AgeIndex,” defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells which can distinguish the effect of different anti-aging interventions.

Abstract Organismal aging is inherently connected to the aging of its constituent cells and systems. Reducing the biological age of the organism may be assisted by reducing the age of its cells - an approach exemplified by partial cell reprogramming through the expression of Yamanaka factors or exposure to chemical cocktails. It is crucial to protect cell type identity during partial reprogramming, as cells need to retain or rapidly regain their functions following the treatment. Another critical issue is the ability to quantify biological age as reprogrammed older cells acquire younger states. We discuss recent advances in reprogramming-induced rejuvenation and offer a critical review of this procedure and its relationship to the fundamental nature of aging. We further comparatively analyze partial reprogramming, full reprogramming and transdifferentiation approaches, assess safety concerns and emphasize the importance of distinguishing rejuvenation from dedifferentiation. Finally, we highlight translational opportunities that the reprogramming-induced rejuvenation approach offers.

Summary: Iron serves as a cofactor for enzymes involved in several steps of protein translation, but the control of translation during iron limitation is not understood at the molecular level. Here, we report a genome-wide analysis of protein translation in response to iron deficiency in yeast using ribosome profiling. We show that iron depletion affects global protein synthesis and leads to translational repression of multiple genes involved in iron-related processes. Furthermore, we demonstrate that the RNA-binding proteins Cth1 and Cth2 play a central role in this translational regulation by repressing the activity of the iron-dependent Rli1 ribosome recycling factor and inhibiting mitochondrial translation and heme biosynthesis. Additionally, we found that iron deficiency represses MRS3 mRNA translation through increased expression of antisense long non-coding RNA. Together, our results reveal complex gene expression and protein synthesis remodeling in response to low iron, demonstrating how this important metal affects protein translation at multiple levels.

Abstract Skeletal muscle aging is characterized by the loss of muscle mass, strength and function, mainly attributed to the atrophy of glycolytic fibers. Underlying mechanisms driving the skeletal muscle functional impairment are yet to be elucidated. To unbiasedly uncover its molecular mechanisms, we recurred to gene expression and metabolite profiling in a glycolytic muscle, Extensor digitorum longus (EDL), from young and aged C57BL/6JRj mice. Employing multi-omics approaches we found that the main age-related changes are connected to mitochondria, exhibiting a downregulation in mitochondrial processes. Consistent is the altered mitochondrial morphology. We further compared our mouse EDL aging signature with human data from the GTEx database, reinforcing the idea that our model may recapitulate muscle loss in humans. We are able to show that age-related mitochondrial downregulation is likely to be detrimental, as gene expression signatures from commonly used lifespan extending interventions displayed the opposite direction compared to our EDL aging signature.

MsrB1 is a thiol-dependent enzyme that reduces protein methionine-R-sulfoxide and regulates inflammatory response in macrophages. Therefore, MsrB1 could be a promising therapeutic target for the control of inflammation. To identify MsrB1 inhibitors, we construct a redox protein-based fluorescence biosensor composed of MsrB1, a circularly permutated fluorescent protein, and the thioredoxin1 in a single polypeptide chain. This protein-based biosensor, named RIYsense, efficiently measures protein methionine sulfoxide reduction by ratiometric fluorescence increase. We used it for high-throughput screening of potential MsrB1 inhibitors among 6868 compounds. A total of 192 compounds were selected based on their ability to reduce relative fluorescence intensity by more than 50% compared to the control. Then, we used molecular docking simulations of the compound on MsrB1, affinity assays, and MsrB1 activity measurement to identify compounds with reliable and strong inhibitory effects. Two compounds were selected as MsrB1 inhibitors: 4-[5-(4-ethylphenyl)-3-(4-hydroxyphenyl)-3,4-dihydropyrazol-2-yl]benzenesulfonamide and 6-chloro-10-(4-ethylphenyl)pyrimido[4,5-b]quinoline-2,4-dione. They are heterocyclic, polyaromatic compounds with a substituted phenyl moiety interacting with the MsrB1 active site, as revealed by docking simulation. These compounds were found to decrease the expression of anti-inflammatory cytokines such as IL-10 and IL-1rn, leading to auricular skin swelling and increased thickness in an ear edema model, effectively mimicking the effects observed in MsrB1 knockout mice. In summary, using a novel redox protein-based fluorescence biosensor, we identified potential MsrB1 inhibitors that can regulate the inflammatory response, particularly by influencing the expression of anti-inflammatory cytokines. These compounds are promising tools for understanding MsrB1’s role during inflammation and eventually controlling inflammation in therapeutic approaches.

Osteoarthritis is caused by the gradual accumulation of oxidative stress in cartilage. Here, the authors show that dysregulation of the selenium metabolic pathway underlies a shift in redox homeostasis in chondrocytes, leading to chronic osteoarthritic changes in joints.

The exceptionally long-lived naked mole-rat is characterized by the lack of increased mortality with aging. Here the authors perform epigenetic studies to show that naked mole-rats epigenetically age despite their non-increasing mortality rate.

Ying et al. conduct a multi-instrument Mendelian randomization study looking at the link between aging and COVID-19 risk. They observe an association between genetic variation implicated in longevity and decreased risk of COVID-19 infection and hospitalization, with Notch signaling and immune system development loci found to be important in aging-related COVID-19 risk.

A comprehensive transcriptomic survey of the pig could enable mechanistic understanding of tissue specialization and accelerate its use as a biomedical model. Here the authors characterize four distinct transcript types in 31 adult pig tissues to dissect their distinct structural and transcriptional features and uncover transcriptomic variability related to tissue physiology.

Aneuploidy (abnormal chromosome number) can enable rapid adaptation to stress conditions, but it also entails fitness costs from gene imbalance. Here, the authors experimentally evolve yeast while forcing maintenance of aneuploidy to identify the mechanisms that promote tolerance of aneuploidy.

Contemporary studies on aging and longevity have largely overlooked the role that adaptation plays in lifespan variation across species. Emerging evidence indicates that the genetic signals of extended lifespan may be maintained by natural selection, suggesting that longevity could be a product of organismal adaptation. The mechanisms of adaptation in long-lived animals are believed to account for the modification of physiological function. Here, we first review recent progress in comparative biology of long-lived animals, together with the emergence of adaptive genetic factors that control longevity and disease resistance. We then propose that hitchhiking of adaptive genetic changes is the basis for lifespan changes and suggest ways to test this evolutionary model. As individual adaptive or adaptation-linked mutations/substitutions generate specific forms of longevity effects, the cumulative beneficial effect is largely nonrandom and is indirectly favored by natural selection. We consider this concept in light of other proposed theories of aging and integrate these disparate ideas into an adaptive evolutionary model, highlighting strategies in decoding genetic factors of lifespan control.

Abstract Epigenetic clocks comprise a set of CpG sites whose DNA methylation levels measure subject age. These clocks are acknowledged as a highly accurate molecular correlate of chronological age in humans and other vertebrates. Also, extensive research is aimed at their potential to quantify biological aging rates and test longevity or rejuvenating interventions. Here, we discuss key challenges to understand clock mechanisms and biomarker utility. This requires dissecting the drivers and regulators of age-related changes in single-cell, tissue- and disease-specific models, as well as exploring other epigenomic marks, longitudinal and diverse population studies, and non-human models. We also highlight important ethical issues in forensic age determination and predicting the trajectory of biological aging in an individual.

Abstract Background High-throughput sequencing often provides a foundation for experimental analyses in the life sciences. For many such methods, an intermediate layer of bioinformatics data analysis is the genomic signal track constructed by short read mapping to a particular genome assembly. There are many software tools to visualize genomic tracks in a web browser or with a stand-alone graphical user interface. However, there are only few command-line applications suitable for automated usage or production of publication-ready visualizations. Results Here we present svist4get, a command-line tool for customizable generation of publication-quality figures based on data from genomic signal tracks. Similarly to generic genome browser software, svist4get visualizes signal tracks at a given genomic location and is able to aggregate data from several tracks on a single plot along with the transcriptome annotation. The resulting plots can be saved as the vector or high-resolution bitmap images. We demonstrate practical use cases of svist4get for Ribo-Seq and RNA-Seq data. Conclusions svist4get is implemented in Python 3 and runs on Linux. The command-line interface of svist4get allows for easy integration into bioinformatics pipelines in a console environment. Extra customization is possible through configuration files and Python API. For convenience, svist4get is provided as pypi package. The source code is available at https://bitbucket.org/artegorov/svist4get/

The human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included all seven known Sec machinery proteins and two critical selenoproteins, GPX4 and TXNRD1. Additionally, these nine genes were further positively associated with PRDX6 and negatively with SCD, linking the latter two genes to the essential role of selenium. We analyzed the essentiality scores of gene knockouts in this cluster across one thousand cancer cell lines and found that Sec metabolism genes are strongly selective for a subset of primary tissues, suggesting that certain cancer cell lineages are particularly dependent on selenium. A separate outstanding cluster included selenophosphate synthetase SEPHS1, which was linked to a group of transcription factors, whereas the remaining selenoproteins were linked neither to these clusters nor among themselves. The data suggest that key components of Sec machinery have already been identified and that their primary role is to support the functions of GPX4 and TXNRD1, with further functional links to PRDX6 and SCD.

Abstract Advanced age is associated with accumulation of damage and other deleterious changes and a consequential systemic decline of function. This decline affects all organs and systems in an organism, leading to their inadaptability to the environment, and therefore is thought to be inevitable for humans and most animal species. However, in vitro and in vivo application of reprogramming strategies, which convert somatic cells to induced pluripotent stem cells, has demonstrated that the aged cells can be rejuvenated. Moreover, the data and theoretical considerations suggest that reversing the biological age of somatic cells (from old to young) and de‐differentiating somatic cells into stem cells represent two distinct processes that take place during rejuvenation, and thus they may be differently targeted. We advance a stemness‐function model to explain these data and discuss a possibility of rejuvenation from the perspective of damage accumulation. In turn, this suggests approaches to achieve rejuvenation of cells in vitro and in vivo.

Summary: Long-lived rodents have become an attractive model for the studies on aging. To understand evolutionary paths to long life, we prepare chromosome-level genome assemblies of the two longest-lived rodents, Canadian beaver (Castor canadensis) and naked mole rat (NMR, Heterocephalus glaber), which were scaffolded with in vitro proximity ligation and chromosome conformation capture data and complemented with long-read sequencing. Our comparative genomic analyses reveal that amino acid substitutions at “disease-causing” sites are widespread in the rodent genomes and that identical substitutions in long-lived rodents are associated with common adaptive phenotypes, e.g., enhanced resistance to DNA damage and cellular stress. By employing a newly developed substitution model and likelihood ratio test, we find that energy and fatty acid metabolism pathways are enriched for signals of positive selection in both long-lived rodents. Thus, the high-quality genome resource of long-lived rodents can assist in the discovery of genetic factors that control longevity and adaptive evolution.

Summary: The human gut microbiome is a complex ecosystem that both affects and is affected by its host status. Previous metagenomic analyses of gut microflora revealed associations between specific microbes and host age. Nonetheless there was no reliable way to tell a host's age based on the gut community composition. Here we developed a method of predicting hosts' age based on microflora taxonomic profiles using a cross-study dataset and deep learning. Our best model has an architecture of a deep neural network that achieves the mean absolute error of 5.91 years when tested on external data. We further advance a procedure for inferring the role of particular microbes during human aging and defining them as potential aging biomarkers. The described intestinal clock represents a unique quantitative model of gut microflora aging and provides a starting point for building host aging and gut community succession into a single narrative.

Adipogenesis is a fundamental process of white adipose tissue function, supporting lipid storage and release, while avoiding its spillover and ectopic accumulation in tissues and organs. During aging adipogenesis is impaired and among other factors, oxidative stress contributes to this process. Adipogenesis requires functional and dynamic mitochondria; however, this organelle itself becomes dysfunctional during aging and accounts for most of reactive oxygen species (ROS) production. Here, we evaluated whether oxidative stress impairs adipogenesis through functional impairment of mitodynamics by utilizing hyperoxia as a continuous source of oxidative stress while maintaining cellular viability. This negatively impacted mitochondrial function, including respiration and dynamics and ultimately blocked adipogenesis. Interestingly, this state was reversible by using the antidiabetic drug, Rosiglitazone, which reduced oxidative stress, restored mitochondrial dynamics and respiration and augmented adipogenesis. Moreover, in vitro results were in agreement with in vivo models of oxidative stress and aging, in which mice depleted of the superoxide dismutase enzyme 1 (SOD1) and old wild-type C57BL/6JRj mice demonstrated the same trend of adipogenic potential. Importantly, in humans the results follow the same pattern, showing a downregulation of adipogenic markers during aging. Since the levels of oxidative stress and peripheral insulin resistance increase with age, while adipogenesis decreases during aging, our model helps to understand a possible way to overcome physiologically low, steady stress conditions and restore adipogenesis, avoiding accumulation of deleterious hypertrophic adipocytes in favor of beneficial hyperplasia.

Summary: An increase in the probability of death has been a defining feature of aging, yet human perinatal mortality starts high and decreases with age. Previous evolutionary models suggested that organismal aging begins after the onset of reproduction. However, we find that mortality and incidence of diseases associated with aging follow a U-shaped curve with the minimum before puberty, whereas quantitative biomarkers of aging, including somatic mutations and DNA methylation, do not, revealing that aging starts early but is masked by early-life mortality. Moreover, our genetic analyses point to the contribution of damaging mutations to early mortality. We propose that mortality patterns are governed, in part, by negative selection against damaging mutations in early life, manifesting after the corresponding genes are first expressed. Deconvolution of mortality patterns suggests that deleterious changes rather than mortality are the defining characteristic of aging and that aging begins in very early life. : Kinzina et al. highlight the importance of adequate definition of aging and address long-standing questions on the onset of aging and causes of early-life mortality. They show that aging is better represented by the accumulation of deleterious changes rather than mortality and thus starts early in life. Keywords: aging, lifespan, mortality, selection, age-related, cancer, incidence, damage, mutations, clock

Naked mole rats (NMRs, Heterocephalus glaber) are long-lived, cancer-resistant rodents. Here, we report the development of an induced pluripotent stem cell (iPSC) line generated from immortalized NMR embryonic fibroblasts transduced with a doxycycline-inducible mouse OSKM polycistronic vector. This iPSC line was shown to express pluripotency-associated markers, form embryoid bodies, differentiate in vitro to the derivatives of three germ layers, and exhibit normal karyotype. The ability of iPSCs to differentiate in vivo was supported by the contribution to interspecific chimera upon injection into mouse blastocysts. This NMR iPSC line may be a useful tool in cancer and aging research.

Summary: Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins. : Yim et al. report that Selenof (15-kDa selenoprotein; Sep15) functions as a gatekeeper of immunoglobulins and, likely, other client proteins en route from the ER to the Golgi apparatus, thereby preventing secretion of dysfunctional proteins and supporting redox quality control. Keywords: Selenof, selenoprotein, oxidoreductase, immunoglobulins, IgM, knockout mouse, endoplasmic reticulum, Sep15, gatekeeper

Whales, dolphins and porpoises are adapted to various aquatic habitats. Here, Zhou et al. show that polymorphisms associated with renal function and the urea cycle have undergone selection in the freshwater Yangtze finless porpoise and provide genomic evidence of incipient speciation.

Summary: Naked mole rats (NMRs) are exceptionally long-lived, cancer-resistant rodents. Identifying the defining characteristics of these traits may shed light on aging and cancer mechanisms. Here, we report the generation of induced pluripotent stem cells (iPSCs) from NMR fibroblasts and their contribution to mouse-NMR chimeric embryos. Efficient reprogramming could be observed under N2B27+2i conditions. The iPSCs displayed a characteristic morphology, expressed pluripotent markers, formed embryoid bodies, and showed typical differentiation patterns. Interestingly, NMR embryonic fibroblasts and the derived iPSCs had propensity for a tetraploid karyotype and were resistant to forming teratomas, but within mouse blastocysts they contributed to both interspecific placenta and fetus. Gene expression patterns of NMR iPSCs were more similar to those of human than mouse iPSCs. Overall, we uncovered unique features of NMR iPSCs and report a mouse-NMR chimeric model. The iPSCs and associated cell culture systems can be used for a variety of biological and biomedical applications. : In this article, Gladyshev and colleagues demonstrate that naked mole rat (NMR) embryonic and adult fibroblasts can be efficiently reprogrammed into induced pluripotent stem cells (iPSCs). NMR iPSCs showed unique features, were resistant to teratoma formation, and contributed to interspecific chimeric embryos. Keywords: naked mole rat, induced pluripotent stem cells, aging, lifespan, interspecific chimera, reprogramming

Abstract Post-translational redox modification of methionine residues often triggers a change in protein function. Emerging evidence points to this reversible protein modification being an important regulatory mechanism under various physiological conditions. Reduction of oxidized methionine residues is catalyzed by methionine sulfoxide reductases (Msrs). Here, we show that one of these enzymes, a selenium-containing MsrB1, is highly expressed in immune-activated macrophages and contributes to shaping cellular and organismal immune responses. In particular, lipopolysaccharide (LPS) induces expression of MsrB1, but not other Msrs. Genetic ablation of MsrB1 did not preclude LPS-induced intracellular signaling in macrophages, but resulted in attenuated induction of anti-inflammatory cytokines, such as interleukin (IL)-10 and the IL-1 receptor antagonist. This anomaly was associated with excessive pro-inflammatory cytokine production as well as an increase in acute tissue inflammation in mice. Together, our findings suggest that MsrB1 controls immune responses by promoting anti-inflammatory cytokine expression in macrophages. MsrB1-dependent reduction of oxidized methionine in proteins may be a heretofore unrecognized regulatory event underlying immunity and inflammatory disease, and a novel target for clinical applications.

Abstract Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects.

Summary: Selenium (Se) is an essential trace element because of its presence in selenoproteins in the form of selenocysteine residue. Both Se deficiency, which compromises selenoprotein functions, and excess Se, which is toxic, have been associated with altered redox homeostasis and adverse health conditions. Surprisingly, we found that, although Se deficiency led to a drastic decline in selenoprotein expression, mice subjected to this dietary regimen for their entire life had normal lifespans. To understand the molecular mechanisms involved, we performed systemic analyses at the level of metabolome, transcriptome, and microRNA profiling. These analyses revealed that Se deficiency reduced amino acid levels, elevated mononucleotides, altered metabolism, and activated signaling pathways linked to longevity-related nutrient sensing. The data show that the metabolic control associated with nutrient sensing coordinately responds to suppressed selenoprotein functions, resulting in normal lifespan under Se deficiency. : Yim et al. report that selenium deficiency in mice is associated with pro-longevity mechanisms because of reduced amino acid levels and altered nutrient signaling. Keywords: selenium, lifespan, selenoproteins, metabolite profiling, transcriptome, metabolic pathway, miRNA, aging, gene expression, nutrition

The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4−/− mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4−/− livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4−/− pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4−/− mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.

Somatic mutations are associated with disease, including cancer. Here, the authors analyse cancer genomic data and show that somatic mutations increase with age and that cancer incidence lags 15 years behind this increase, later in life, mutation and cancer incidence are reduced.

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

Trace elements are essential to all mammals, but their distribution and utilization across species and organs remains unclear. Here, we examined 18 elements in the brain, heart, kidney, and liver of 26 mammalian species and report the elemental composition of these organs, the patterns of utilization across the species, and their correlation with body mass and longevity. Across the organs, we observed distinct distribution patterns for abundant elements, transition metals, and toxic elements. Some elements showed lineage-specific patterns, including reduced selenium utilization in African mole rats, and positive correlation between the number of selenocysteine residues in selenoprotein P and the selenium levels in liver and kidney across mammals. Body mass was linked positively to zinc levels, whereas species lifespan correlated positively with cadmium and negatively with selenium. This study provides insights into the variation of mammalian ionome by organ physiology, lineage specialization, body mass, and longevity.

Subterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis) and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber). Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity.

Selenium (Se) deficiency has been known for many years to be associated with disease, impaired growth and a variety of other metabolic disorders in mammals. Only recently has the major role that Se-containing proteins, designated selenoproteins, play in many aspects of health and development begun to emerge. Se is incorporated into protein by way of the Se-containing amino acid, selenocysteine (Sec). The synthesis of selenoproteins is dependent on Sec tRNA for insertion of Sec, the 21st amino acid in the genetic code, into protein. We have taken advantage of this dependency to modulate the expression of Sec tRNA that in turn modulates the expression of selenoproteins by generating transgenic, conditional knockout, transgenic/standard knockout and transgenic/conditional knockout mouse models, all of which involve the Sec tRNA gene, to elucidate the intracellular roles of this protein class.