The Good, the Bad, and the Ugly of ROS: New Insights on Aging and Aging-Related Diseases from Eukaryotic and Prokaryotic Model Organisms

Aging is associated with the accumulation of cellular damage over the course of a lifetime. This process is promoted in large part by reactive oxygen species (ROS) generated via cellular metabolic and respiratory pathways. Pharmacological, nonpharmacological, and genetic interventions have been used to target cellular and mitochondrial networks in an effort to decipher aging and age-related disorders. While ROS historically have been viewed as a detrimental byproduct of normal metabolism and associated with several pathologies, recent research has revealed a more complex and beneficial role of ROS in regulating metabolism, development, and lifespan. In this review, we summarize the recent advances in ROS research, focusing on both the beneficial and harmful roles of ROS, many of which are conserved across species from bacteria to humans, in various aspects of cellular physiology. These studies provide a new context for our understanding of the parts ROS play in health and disease. Moreover, we highlight the utility of bacterial models to elucidate the molecular pathways by which ROS mediate aging and aging-related diseases

Protein Posttranslational Modifications: Roles in Aging and Age-Related Disease

Aging is characterized by the progressive decline of biochemical and physiological function in an individual. Consequently, aging is a major risk factor for diseases like cancer, obesity, and type 2 diabetes. The cellular and molecular mechanisms of aging are not well understood, nor is the relationship between aging and the onset of diseases. One of the hallmarks of aging is a decrease in cellular proteome homeostasis, allowing abnormal proteins to accumulate. This phenomenon is observed in both eukaryotes and prokaryotes, suggesting that the underlying molecular processes are evolutionarily conserved. Similar protein aggregation occurs in the pathogenesis of diseases like Alzheimer’s and Parkinson’s. Further, protein posttranslational modifications (PTMs), either spontaneous or physiological/pathological, are emerging as important markers of aging and aging-related diseases, though clear causality has not yet been firmly established. This review presents an overview of the interplay of PTMs in aging-associated molecular processes in eukaryotic aging models. Understanding PTM roles in aging could facilitate targeted therapies or interventions for age-related diseases. In addition, the study of PTMs in prokaryotes is highlighted, revealing the potential of simple prokaryotic models to uncover complex aging-associated molecular processes in the emerging field of microbiogerontology

Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes

BACKGROUND: Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. Well-to-well variation between cell-line based in vitro assays is practically unavoidable, and variation between individual animals is the cause of large deviations in the observed capacity for infection when animal models are used. One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains. RESULTS: A fluorescent marker system, allowing visualization and identification of single L. monocytogenes cells as well as colonies in a non-destructive manner, was developed. Five different fluorescent labels are available, and allowed simultaneous visual discrimination between three differently labelled strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations. The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible. CONCLUSION: The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between differently labelled bacteria after internalization in these cells

Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3d individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion- aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of "soft" intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.Comment: PLoS Computational Biology (2013

Dissecting the Genetic Components of Adaptation of Escherichia coli to the Mouse Gut

While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments

Genetic information transfer promotes cooperation in bacteria

Many bacterial species are social, producing costly secreted “public good” molecules that enhance the growth of neighboring cells. The genes coding for these cooperative traits are often propagated via mobile genetic elements and can be virulence factors from a biomedical perspective. Here, we present an experimental framework that links genetic information exchange and the selection of cooperative traits. Using simulations and experiments based on a synthetic bacterial system to control public good secretion and plasmid conjugation, we demonstrate that horizontal gene transfer can favor cooperation. In a well-mixed environment, horizontal transfer brings a direct infectious advantage to any gene, regardless of its cooperation properties. However, in a structured population transfer selects specifically for cooperation by increasing the assortment among cooperative alleles. Conjugation allows cooperative alleles to overcome rarity thresholds and invade bacterial populations structured purely by stochastic dilution effects. Our results provide an explanation for the prevalence of cooperative genes on mobile elements, and suggest a previously unidentified benefit of horizontal gene transfer for bacteria

A synthetic growth switch based on controlled expression of RNA polymerase

International audienceThe ability to control growth is essential for fundamental studies of bacterial physiology and biotechnological applications. We have engineered an Escherichia coli strain in which the transcription of a key component of the gene expression machinery, RNA polymerase, is under the control of an inducible promoter. By changing the inducer concentration in the medium, we can adjust the RNA polymerase concentration and thereby switch bacterial growth between zero and the maximal growth rate supported by the medium. We show that our synthetic growth switch functions in a medium-independent and reversible way, and we provide evidence that the switching phenotype arises from the ultrasensitive response of the growth rate to the concentration of RNA polymerase. We present an application of the growth switch in which both the wild-type E. coli strain and our modified strain are endowed with the capacity to produce glycerol when growing on glucose. Cells in which growth has been switched off continue to be metabolically active and harness the energy gain to produce glycerol at a twofold higher yield than in cells with natural control of RNA polymerase expression. Remarkably, without any further optimization, the improved yield is close to the theoretical maximum computed from a flux balance model of E. coli metabolism. The proposed synthetic growth switch is a promising tool for gaining a better understanding of bacterial physiology and for applications in synthetic biology and biotechnology

Emergence of Variability in Isogenic Escherichia coli Populations Infected by a Filamentous Virus

The spread of epidemics not only depends on the average number of parasites produced per host, but also on the existence of highly infectious individuals. It is widely accepted that infectiousness depends on genetic and environmental determinants. However, even in clonal populations of host and viruses growing in homogeneous conditions, high variability can exist. Here we show that Escherichia coli cells commonly display high differentials in viral burst size, and address the kinetics of emergence of such variability with the non-lytic filamentous virus M13. By single-cell imaging of a virally-encoded fluorescent reporter, we monitor the viral charge distribution in infected bacterial populations at different time following infection. A mathematical model assuming autocatalytic virus replication and inheritance of bacterial growth rates quantitatively reproduces the experimental distributions, demonstrating that deterministic amplification of small host inhomogeneities is a mechanism sufficient to explain large and highly skewed distributions. This mechanism of amplification is general and may occur whenever a parasite has an initial phase of exponential growth within its host. Moreover, it naturally reproduces the shift towards higher virulence when the host is experimenting poor conditions, as observed commonly in host-parasite systems

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants