Evidence that conserved essential genes are enriched for pro-longevity factors.

At the cellular level, many aspects of aging are conserved across species. This has been demonstrated by numerous studies in simple model organisms like Saccharomyces cerevisiae, Caenorhabdits elegans, and Drosophila melanogaster. Because most genetic screens examine loss of function mutations or decreased expression of genes through reverse genetics, essential genes have often been overlooked as potential modulators of the aging process. By taking the approach of increasing the expression level of a subset of conserved essential genes, we found that 21% of these genes resulted in increased replicative lifespan in S. cerevisiae. This is greater than the ~ 3.5% of genes found to affect lifespan upon deletion, suggesting that activation of essential genes may have a relatively disproportionate effect on increasing lifespan. The results of our experiments demonstrate that essential gene overexpression is a rich, relatively unexplored means of increasing eukaryotic lifespan.

WormBot, an open-source robotics platform for survival and behavior analysis in C. elegans.

Caenorhabditis elegans is a popular organism for aging research owing to its highly conserved molecular pathways, short lifespan, small size, and extensive genetic and reverse genetic resources. Here we describe the WormBot, an open-source robotic image capture platform capable of conducting 144 parallel C. elegans survival and behavioral phenotyping experiments. The WormBot uses standard 12-well tissue culture plates suitable for solid agar media and is built from commercially available robotics hardware. The WormBot is controlled by a web-based interface allowing control and monitoring of experiments from any internet connected device. The standard WormBot hardware features the ability to take both time-lapse bright field images and real-time video micrographs, allowing investigators to measure lifespan, as well as heathspan metrics as worms age. The open-source nature of the hardware and software will allow for users to extend the platform and implement new software and hardware features. This extensibility, coupled with the low cost and simplicity of the system, allows the automation of C. elegans survival analysis even in small laboratory settings with modest budgets.

Inter-organ regulation of haem homeostasis.

Haem is an iron-containing cofactor required for life. Many cellular processes rely on haem and failure to maintain iron homeostasis results in numerous pathological conditions. A study now identifies a Caenorhabditis elegans inter-organ signalling pathway in which secreted intestinal HRG-7 and neuronally secreted BMP signals coordinate animal haem homeostasis.

Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans isp-1(qm150) Rieske iron-sulfur protein.

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.

Correction: Why Is Aging Conserved and What Can We Do about it?

[This corrects the article DOI: 10.1371/journal.pbio.1002131.].

Why is aging conserved and what can we do about it?

The field of aging research has progressed rapidly over the past few decades. Genetic modulators of aging rate that are conserved over a broad evolutionary distance have now been identified. Several physiological and environmental interventions have also been shown to influence the rate of aging in organisms ranging from yeast to mammals. Here we briefly review these conserved pathways and interventions and highlight some key unsolved challenges that remain. Although the molecular mechanisms by which these modifiers of aging act are only partially understood, interventions to slow aging are nearing clinical application, and it is likely that we will begin to reap the benefits of aging research prior to solving all of the mysteries that the biology of aging has to offer.

Rapid construction of empirical RNA fitness landscapes.

Evolution is an adaptive walk through a hypothetical fitness landscape, which depicts the relationship between genotypes and the fitness of each corresponding phenotype. We constructed an empirical fitness landscape for a catalytic RNA by combining next-generation sequencing, computational analysis, and "serial depletion," an in vitro selection protocol. By determining the reaction rate constant for every point mutant of a catalytic RNA, we demonstrated that abundance in serially depleted pools correlates with biochemical activity (correlation coefficient r = 0.67, standard score Z = 7.4). Therefore, enumeration of each genotype by deep sequencing yielded a fitness landscape containing ~10(7) unique sequences, without requiring measurement of the phenotypic fitness for each sequence. High-throughput mapping between genotype and phenotype may apply to artificial selections, host-pathogen interactions, and other biomedically relevant evolutionary phenomena.

SEWAL: an open-source platform for next-generation sequence analysis and visualization.

Next-generation DNA sequencing platforms provide exciting new possibilities for in vitro genetic analysis of functional nucleic acids. However, the size of the resulting data sets presents computational and analytical challenges. We present an open-source software package that employs a locality-sensitive hashing algorithm to enumerate all unique sequences in an entire Illumina sequencing run (∼ 10(8) sequences). The algorithm results in quasilinear time processing of entire Illumina lanes (∼ 10(7) sequences) on a desktop computer in minutes. To facilitate visual analysis of sequencing data, the software produces three-dimensional scatter plots similar in concept to Sewall Wright and John Maynard Smith's adaptive or fitness landscape. The software also contains functions that are particularly useful for doped selections such as mutation frequency analysis, information content calculation, multivariate statistical functions (including principal component analysis), sequence distance metrics, sequence searches and sequence comparisons across multiple Illumina data sets. Source code, executable files and links to sample data sets are available at http://www.sourceforge.net/projects/sewal.

Structure-guided engineering of the regioselectivity of RNA ligase ribozymes.

Ribozyme-catalyzed RNA synthesis is central to the RNA world hypothesis. No natural RNA polymerase ribozymes have been discovered. However, ribozymes that catalyze the requisite chemistry, generating a new phosphodiester through attack of a terminal hydroxyl of an RNA on the alpha-phosphate of a triphosphate-activated oligonucleotide, have been isolated by in vitro selection. These experiments often yield ribozymes that generate 2'-5' phosphodiesters rather than conventional 3'-5' linkages. We have determined crystal structures of the duplex formed by the template segment of a representative 2'-5' RNA ligase ribozyme, the class II ligase, and its ligation product. The structures reveal a product-template duplex with a G x A pair at the ligation junction. This sheared pair is flanked on one side by a minor groove-broadening wedge comprised of two unpaired nucleotides. The reported structure of an independently isolated 3'-5' ligase ribozyme, the L1 ligase, shows a product-template duplex that shares the G x A pair with the class II ligase. However, this G x A pair is flanked by G x U wobbles, rather than an unpaired wedge. We demonstrate that these structural differences of the substrate-template duplexes are largely responsible for the divergent regioselectivity of the two ribozymes, independent of their catalytic moieties, by constructing chimeras. The L1 ligase with a class II substrate-template duplex shows a 30-fold increase in 2'-5' bond synthesis, while the class II ligase with an L1 substrate-template duplex produces 3'-5' bonds exclusively. These results demonstrate how local geometry inherent to the substrate-template duplexes controls the regioselectivity of ribozyme-catalyzed RNA ligation reactions.

A major role for zygotic hunchback in patterning the Nasonia embryo.

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.