The genomic landscape of compensatory evolution.

Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, as deleterious mutations are common in natural populations, they generate a strong selection pressure to mitigate their detrimental effects through compensatory genetic changes. This process can potentially influence directions of adaptive evolution by enabling evolutionary routes that are otherwise inaccessible. Therefore, the extent to which compensatory mutations shape genomic evolution is of central importance. Here, we studied the capacity of the baker's yeast genome to compensate the complete loss of genes during evolution, and explored the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid baker's yeast genotypes, all of which initially displayed slow growth owing to the deletion of a single gene. Compensatory evolution following gene loss was rapid and pervasive: 68% of the genotypes reached near wild-type fitness through accumulation of adaptive mutations elsewhere in the genome. As compensatory mutations have associated fitness costs, genotypes with especially low fitnesses were more likely to be subjects of compensatory evolution. Genomic analysis revealed that as compensatory mutations were generally specific to the functional defect incurred, convergent evolution at the molecular level was extremely rare. Moreover, the majority of the gene expression changes due to gene deletion remained unrestored. Accordingly, compensatory evolution promoted genomic divergence of parallel evolving populations. However, these different evolutionary outcomes are not phenotypically equivalent, as they generated diverse growth phenotypes across environments. Taken together, these results indicate that gene loss initiates adaptive genomic changes that rapidly restores fitness, but this process has substantial pleiotropic effects on cellular physiology and evolvability upon environmental change. Our work also implies that gene content variation across species could be partly due to the action of compensatory evolution rather than the passive loss of genes

Molecular Modeling and Simulation: Force Field Development, Evaporation Processes and Thermophysical Properties of Mixtures

To gain physical insight into the behavior of fluids on a microscopic level as well as to broaden the data base for thermophysical properties especially for mixtures, molecular modeling and simulation is utilized in this work. Various methods and applications are discussed, including a procedure for the development of new force field models. The evaporation of liquid nitrogen into a supercritical hydrogen atmosphere is presented as an example for large scale molecular dynamics simulation. System-size dependence and scaling behavior are discussed in the context of Kirkwood-Buff integration. Further, results for thermophysical mixture properties are presented, i.e. the Henry’s law constant of aqueous systems and diffusion coefficients of a ternary mixture

Abstract WP431: Functionally Distinct Neuronal Nitric Oxide Synthase Expressed in the Brain Microvascular Endothelial Cells Mediates Anoxic-injury to Blood-Brain Barrier

Objective: Mice with genetic deletion of endothelial (eNOS; protective) and neuronal (nNOS; detrimental) nitric oxide synthase isoforms exhibit dramatically opposite consequences of ischemic brain injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify nNOS and characterize its functional role in primary brain microvascular endothelial cells (MECs). Methods and Results: MECs from humans (hMECs), rats (rMECs), and mice (mMECs) along with cultured primary rat cortical neurons were used. In addition, rat brain microvessels were freshly isolated. Transendothelial electrical resistance (TEER) measurements of monolayers of hMECs cultured in transwells were used to quantitate in vitro blood-brain barrier (BBB) integrity. Immunocytochemistry identified von Willebrand factor, eNOS, and nNOS in MECs but stained negative for glial (GFAP) and neuronal (Neu1) markers. PCR studies confirmed the expression of eNOS and nNOS mRNA in MECs and microvessels. We utilized electron spin resonance spectrometry to measure reactive oxygen species (ROS) (1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; CMH) and NO (colloid Fe(DETC)2). Inhibition of nNOS (N-ω-Propyl-L-arginine and ARL-17477) reduced ROS but increased NO levels in MECs and rat brain microvessels. In contrast, eNOS inhibitor (L-N5-(1-Iminoethyl)ornithine) increased ROS but reduced NO levels. Inhibition of nNOS in neurons, similarly increased ROS and decreased NO levels. siRNA targeting rat nNOS in rMECs was able to knockdown nNOS mRNA as well as ROS levels. BBB studies of hMECs treated with NOS inhibitors followed by oxygen-glucose deprivation (OGD) revealed that nNOS inhibition increased TEER at baseline and promoted TEER recovery following OGD. In contrast, eNOS inhibition had no effect on TEER at baseline but weakly albeit transiently helps in the post-OGD recovery of BBB function. Conclusions: Thus, we identified a constitutively active nNOS in MECs that is functionally distinct from the nNOS isoform expressed in neurons and eNOS. In addition, nNOS inhibition enhances the BBB integrity and affords protection against anoxic-injury induced impairment of BBB function. </jats:p

Genomic analyses of evolutionary compensation.

<p>(A) Distribution of different mutational events (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s007" target="_blank">Table S2</a>). The inlet shows the color coding and the average value of total mutational events per genotype. (B) The originally deleted gene and the gene with identified <i>de novo</i> mutation participated more often in the same protein complex, were more often assigned to the same functional category and showed significantly more similar genetic interaction and expression profile than expected by random shuffling of the knock-out gene–mutated gene network. Dashed line represents no enrichment; */**/*** indicates <i>p</i>-value<0.05/0.01/0.001, respectively. The x axis is logarithmically scaled. (C) Δ<i>rpl6b</i> evolved lines showed duplication of the chromosomal region (or the complete chromosome) carrying a duplicate with redundant function (<i>RPL6A</i>). The gene positions are marked by arrows below the corresponding chromosome, copy numbers are shown by color codes. (D) Dosage compensation of Δrpl6b by increased copy number of RPL6A (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s010" target="_blank">Table S5</a>). Copy number of <i>RPL6A</i> was increased by transforming the <i>RPL6A</i> bearing plasmid of the MoBY ORF Library. As the vector carries a selectable marker and a yeast centromere, the plasmid is present in one to three copies per cell. As a control, strains were transformed with the empty centromeric plasmid. Relative fitness was measured as colony sizes on agar plates, values were normalized to the wild-type control with a single genomic copy of RPL6A. All strains were grown on synthetic complete medium without uracil to select for the plasmids. Error bars show standard error.</p