Current work reports the use of single-stranded RNA toeholds of different lengths to promote the reassociation of various RNA-DNA hybrids, which results in activation of multiple split functionalities inside human cells. The process of reassociation is analyzed and followed with a novel computational multistrand secondary structure prediction algorithm and various experiments. All of our previously designed RNA/DNA nanoparticles employed single-stranded DNA toeholds to initiate reassociation. The use of RNA toeholds is advantageous because of the simpler design rules, the shorter toeholds, and the smaller size of the resulting nanoparticles (by up to 120 nucleotides per particle) compared to the same hybrid nanoparticles with single-stranded DNA toeholds. Moreover, the cotranscriptional assemblies result in higher yields for hybrid nanoparticles with ssRNA toeholds.

Flavin-Containing Monooxygenases are conserved xenobiotic-detoxifying enzymes. Recent studies have revealed endogenous functions of FMOs in regulating longevity in Caenorhabditis elegans and in regulating aspects of metabolism in mice. To explore the cellular mechanisms of FMO’s endogenous function, here we demonstrate that all five functional mammalian FMOs may play similar endogenous roles to improve resistance to a wide range of toxic stresses in both kidney and liver cells. We further find that stress-activated c-Jun N-terminal kinase activity is enhanced in FMO-overexpressing cells, which may lead to increased survival under stress. Furthermore, FMO expression modulates cellular metabolic activity as measured by mitochondrial respiration, glycolysis, and metabolomics analyses. FMO expression augments mitochondrial respiration and significantly changes central carbon metabolism, including amino acid and energy metabolism pathways. Together, our findings demonstrate an important endogenous role for the FMO family in regulation of cellular stress resistance and major cellular metabolic activities including central carbon metabolism.

Flavin containing monooxygenases (FMOs) are promiscuous enzymes known for metabolizing a wide range of exogenous compounds. In C. elegans, fmo-2 expression increases lifespan and healthspan downstream of multiple longevity-promoting pathways through an unknown mechanism. Here, we report that, beyond its classification as a xenobiotic enzyme, fmo-2 expression leads to rewiring of endogenous metabolism principally through changes in one carbon metabolism (OCM). These changes are likely relevant, as we find that genetically modifying OCM enzyme expression leads to alterations in longevity that interact with fmo-2 expression. Using computer modeling, we identify decreased methylation as the major OCM flux modified by FMO-2 that is sufficient to recapitulate its longevity benefits. We further find that tryptophan is decreased in multiple mammalian FMO overexpression models and is a validated substrate for FMO-2. Our resulting model connects a single enzyme to two previously unconnected key metabolic pathways and provides a framework for the metabolic interconnectivity of longevity-promoting pathways such as dietary restriction. FMOs are well-conserved enzymes that are also induced by lifespan-extending interventions in mice, supporting a conserved and important role in promoting health and longevity through metabolic remodeling.

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An organism’s ability to perceive and respond to changes in its environment is crucial for its health and survival. Our approach to identify molecular mechanisms of aging is to focus on common mechanisms downstream of multiple pathways. This approach led to our discovery of a gene, flavin-containing monooxygenase (fmo)-2, that is both necessary and sufficient to increase lifespan and healthspan downstream of several longevity interventions, including dietary restriction and hypoxia. Surprisingly, we also find that in both hypoxia and dietary restriction models, fmo-2 is induced by cell non-autonomous signaling pathways, consistent with the worms’ perceiving the stress (e.g. low oxygen, lack of food) and changing physiology as a result. Our current work focuses on 1) the signaling networks that regulate stress perception and integrate multiple signals to change physiology, and 2) the mechanism of FMO-2-mediated longevity. Our new data suggest that these cell non autonomous networks pathways utilize both overlapping and distinct signaling mechanisms to converge on upregulation of the same gene. They also suggest that these pathways can be manipulated by small molecule drugs to increase lifespan by “tricking” the organism into activating stress response networks. We further find that FMO enzyme expression has a drastic effect on endogenous metabolism, primarily through tryptophan and one carbon metabolism. Ultimately, we aim to leverage our results in a translational framework to identify key signals, genes, and mechanisms where organisms respond to the perception of environmental stress to improve health and slow aging.

AbstractHIF-1-mediated adaptation to changes in oxygen availability is a critical aspect of healthy physiology. HIF is regulated by a conserved mechanism whereby EGLN/PHD family members hydroxylate HIF in an oxygen-dependent manner, targeting it for ubiquitination by Von-Hippel-Lindau (VHL) family members, leading to its proteasomal degradation. The activity of the only C. elegans PHD family member, EGL-9, is also regulated by a hydrogen sulfide sensing cysteine-synthetase-like protein, CYSL-1, which is, in turn, regulated by RHY-1/acyltransferase. Over the last decade multiple seminal studies have established a role for the hypoxic response in regulating longevity, with mutations in vhl-1 substantially extending C. elegans lifespan through a HIF-1-dependent mechanism. However, studies on other components of the hypoxic signaling pathway that similarly stabilize HIF-1 have shown more mixed results, suggesting that mutations in egl-9 and rhy-1 frequently fail to extend lifespan. Here, we show that egl-9 and rhy-1 mutants suppress the long-lived phenotype of vhl-1 mutants. We also show that RNAi of rhy-1 extends lifespan of wild-type worms while decreasing lifespan of vhl-1 mutant worms. We further identify VHL-1-independent gene expression changes mediated by EGL-9 and RHY-1 and find that a subset of these genes contributes to longevity regulation. The resulting data suggest that changes in HIF-1 activity derived by interactions with EGL-9 likely contribute greatly to its role in regulation of longevity.

The rapid progress of the past three decades has led the geroscience field near a point where human interventions in aging are plausible. Advances across scientific areas, such as high throughput “-omics” approaches, have led to an exponentially increasing quantity of data available for biogerontologists. To best translate the lifespan and healthspan extending interventions discovered by basic scientists into preventative medicine, it is imperative that the current data are comprehensively utilized to generate testable hypotheses about translational interventions. Building a translational pipeline for geroscience will require both systematic efforts to identify interventions that extend healthspan across taxa and diagnostics that can identify patients who may benefit from interventions prior to the onset of an age-related morbidity. Databases and computational tools that organize and analyze both the wealth of information available on basic biogerontology research and clinical data on aging populations will be critical in developing such a pipeline. Here, we review the current landscape of databases and computational resources available for translational aging research. We discuss key platforms and tools available for aging research, with a focus on how each tool can be used in concert with hypothesis driven experiments to move closer to human interventions in aging.

An organism’s ability to respond to stress is crucial for long-term survival. These stress responses are coordinated by distinct but overlapping pathways, many of which also regulate longevity across taxa. Our previous work identified a cell non-autonomous signaling pathway led by the hypoxia-inducible factor and resulting in induction of flavin-containing monooxygenase-2 (fmo-2) to promote health and longevity. Our current work identifies a distinct cell non-autonomous pathway downstream of dietary restriction (DR) that also relies on fmo-2 induction to promote health and longevity. We now find that these cell non-autonomous pathways can be mimicked by small molecule interventions that increase longevity by inducing fmo-2. Based on the commonalities of these pathways, we hypothesized that fmo-2, a classically annotated xenobiotic enzyme, might play a key endogenous role in responding to metabolic stress. Our resulting data, using metabolic profiling and further epistatic analysis, both support this hypothesis and link fmo-2’s mechanism to modifications in one-carbon metabolism (OCM), a key intermediate pathway consisting of the folate and methionine cycles. Using mathematical modeling and a labeled metabolomics approach, we were able to further identify the likely mechanism of fmo-2-mediated metabolic effects and connect them to both OCM and downstream components. We propose that fmo-2 is induced cell non-autonomously to modify systemic metabolism and longevity, and that fmo-2 is a key member of a conserved metabolic stress response.