Contrasting dynamics of a mutator allele in asexual populations of differing size

Mutators have been shown to hitchhike in asexual populations when the anticipated beneficial mutation supply rate of the mutator subpopulation, NU(b) (for subpopulation of size N and beneficial mutation rate U(b)) exceeds that of the wild-type subpopulation. Here, we examine the effect of total population size on mutator dynamics in asexual experimental populations of Saccharomyces cerevisiae. Although mutators quickly hitchhike to fixation in smaller populations, mutator fixation requires more and more time as population size increases; this observed delay in mutator hitchhiking is consistent with the expected effect of clonal interference. Interestingly, despite their higher beneficial mutation supply rate, mutators are supplanted by the wild type in very large populations. We postulate that this striking reversal in mutator dynamics is caused by an interaction between clonal interference, the fitness cost of the mutator allele, and infrequent large-effect beneficial mutations in our experimental populations. Our work thus identifies a potential set of circumstances under which mutator hitchhiking can be inhibited in natural asexual populations, despite recent theoretical predictions that such populations should have a net tendency to evolve ever-higher genomic mutation rates.     

Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology

We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two ∼750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5–10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5′ overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5′ overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.     

Compartmentalization of ROS-mediated signal transduction

Localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion of the same components. It has been suggested that ROS-generating complexes follow the principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model of redox signaling compartmentalization. Nevertheless, evidence of local ROS production and the restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH oxidase isoforms, their adapter proteins, and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant systems which prevent uncontrolled ROS diffusion from sites of generation to the adjacent subcellular compartments; the current data on the specific localization of NADPH oxidase activity and its influence on intracellular processes; and the recent evidence of the ROS diffusion restriction.     

Predicting the functional impact of protein mutations: application to cancer genomics

As large-scale re-sequencing of genomes reveals many protein mutations, especially in human cancer tissues, prediction of their likely functional impact becomes important practical goal. Here, we introduce a new functional impact score (FIS) for amino acid residue changes using evolutionary conservation patterns. The information in these patterns is derived from aligned families and sub-families of sequence homologs within and between species using combinatorial entropy formalism. The score performs well on a large set of human protein mutations in separating disease-associated variants (∼19 200), assumed to be strongly functional, from common polymorphisms (∼35 600), assumed to be weakly functional (area under the receiver operating characteristic curve of ∼0.86). In cancer, using recurrence, multiplicity and annotation for ∼10 000 mutations in the COSMIC database, the method does well in assigning higher scores to more likely functional mutations (‘drivers’). To guide experimental prioritization, we report a list of about 1000 top human cancer genes frequently mutated in one or more cancer types ranked by likely functional impact; and, an additional 1000 candidate cancer genes with rare but likely functional mutations. In addition, we estimate that at least 5% of cancer-relevant mutations involve switch of function, rather than simply loss or gain of function.     

Prediction of High Incidence of Dengue in the Philippines

Background

Accurate prediction of dengue incidence levels weeks in advance of an outbreak may reduce the morbidity and mortality associated with this neglected disease. Therefore, models were developed to predict high and low dengue incidence in order to provide timely forewarnings in the Philippines.

Methods

Model inputs were chosen based on studies indicating variables that may impact dengue incidence. The method first uses Fuzzy Association Rule Mining techniques to extract association rules from these historical epidemiological, environmental, and socio-economic data, as well as climate data indicating future weather patterns. Selection criteria were used to choose a subset of these rules for a classifier, thereby generating a Prediction Model. The models predicted high or low incidence of dengue in a Philippines province four weeks in advance. The threshold between high and low was determined relative to historical incidence data.

Principal Findings

Model accuracy is described by Positive Predictive Value (PPV), Negative Predictive Value (NPV), Sensitivity, and Specificity computed on test data not previously used to develop the model. Selecting a model using the F_0.5 measure, which gives PPV more importance than Sensitivity, gave these results: PPV = 0.780, NPV = 0.938, Sensitivity = 0.547, Specificity = 0.978. Using the F₃ measure, which gives Sensitivity more importance than PPV, the selected model had PPV = 0.778, NPV = 0.948, Sensitivity = 0.627, Specificity = 0.974. The decision as to which model has greater utility depends on how the predictions will be used in a particular situation.

Conclusions

This method builds prediction models for future dengue incidence in the Philippines and is capable of being modified for use in different situations; for diseases other than dengue; and for regions beyond the Philippines. The Philippines dengue prediction models predicted high or low incidence of dengue four weeks in advance of an outbreak with high accuracy, as measured by PPV, NPV, Sensitivity, and Specificity.     

Hydrogen peroxide probes directed to different cellular compartments

Background

Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.

Principal Findings

Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events.

Conclusions

We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.     

Targeted Metabolomics Reveals a Male Pheromone and Sex-Specific Ascaroside Biosynthesis in Caenorhabditis elegans

In the model organism Caenorhabditis elegans, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC-MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.     

Rearrangement of the morphological structure and degradation of the extracellular matrix in amphibian embryos after short-term disruption of cell contacts

The changes in cell structure of explants of presumptive early gastrula mesoderm of Xenopus laevis and dynamics of extracellular matrix formation has been studied for 6 h after their isolation. After 6 h, control explants of any dorsoventral level turned into dense spheric homogeneous cell clusters. Explants of dorsal and lateral sectors, treated with medium without Ca2+ and Mg2+ for 30 s, bent and separated into several layers, and in ventral sector explants, cavities, surrounded by polarized cells, arose. In control explants, the amount of extracellular matrix on the surface and in intercellular spaces increased, and in the experimental explants, extracellular matrix disappeared and was not observed for several hours. Previously described differential transformations of the treated explants (Georgiev, Belousov, 1986) are supposed to be determined by extracellular matrix degradation and the degradation-associated complications of cell structure of the explant.