Comparative Analysis of Yeast Metabolic Network Models Highlights Progress,Opportunities for Metabolic Reconstruction

We have compared 12 genome-scale models of the Saccharomyces cerevisiae metabolic network published since 2003 to evaluate progress in reconstruction of the yeast metabolic network. We compared the genomic coverage, overlap of annotated metabolites, predictive ability for single gene essentiality with a selection of model parameters, and biomass production predictions in simulated nutrient-limited conditions. We have also compared pairwise gene knockout essentiality predictions for 10 of these models. We found that varying approaches to model scope and annotation reflected the involvement of multiple research groups in model development; that single-gene essentiality predictions were affected by simulated medium, objective function, and the reference list of essential genes; and that predictive ability for single-gene essentiality did not correlate well with predictive ability for our reference list of synthetic lethal gene interactions (R = 0.159). We conclude that the reconstruction of the yeast metabolic network is indeed gradually improving through the iterative process of model development, and there remains great opportunity for advancing our understanding of biology through continued efforts to reconstruct the full biochemical reaction network that constitutes yeast metabolism. Additionally, we suggest that there is opportunity for refining the process of deriving a metabolic model from a metabolic network reconstruction to facilitate mechanistic investigation and discovery. This comparative study lays the groundwork for developing improved tools and formalized methods to quantitatively assess metabolic network reconstructions independently of any particular model application, which will facilitate ongoing efforts to advance our understanding of the relationship between genotype and cellular phenotype.     

Induction and evolution of cytomegalovirus-specific CD4+ T cell clonotypes in rhesus macaques

CMV infection induces robust CD4+ T cell responses in immunocompetent hosts that orchestrate immune control of viral replication, dissemination, and disease. In this study, we characterized the clonotypic composition of CD4+ T cell populations specific for rhesus CMV (RhCMV) in chronically infected adult rhesus macaques (RM) and in juvenile RM undergoing primary RhCMV infection and subsequent secondary challenge with RhCMV. In adult RM with established chronic infection, RhCMV-specific CD4+ T cell populations exhibited stable, pauciclonal structures with skewed hierarchies dominated by two or three clonotypes. During primary infection, in contrast, the initial RhCMV-specific CD4+ T cell populations were highly polyclonal and progressive evolution to the chronic pattern manifest in adults occurred over the ensuing 2-3 years. Clear patterns of clonal succession were observed during this maturation process, such that clonotypes present in the acute phase were largely replaced over time. However, rechallenge with RhCMV expanded virus-specific CD4+ T cell clonotypes identified solely during acute infection. These findings indicate that, during persistent viral infection, substantial selection pressures and ongoing clonotype recruitment shape the specific CD4+ T cell repertoire and that rapidly exhausted or superseded clonotypes often remain within the memory T cell pool.     

Chromoplast-Specific Proteins in Capsicum annuum

Chromoplasts are a common differentiation state of plastids in which the photosynthetic apparatus is absent and carotenoids accumulate to high levels. As a first step toward the isolation of chromoplast-specific genes, we have examined plastids of the bell pepper, Capsicum annuum L., for the presence of chromoplast-specific proteins. Intact chromoplasts were isolated from mature fruits of C. annuum var Emerald Giant, Golden Cal Wonder, and DNAP VS-12 by differential centrifugation followed by isopycnic sedimentation in gradients of silica sols. The plastids were then fractionated into soluble and membrane components and the proteins analyzed by one- and two-dimensional gel electrophoresis using isoelectric focusing, sodium dodecyl sulfate, and sodium dodecyl sulfate-urea gels. Two polypeptides with M_r of 35,000 and 58,000 accumulate to high levels in membrane fractions of chromoplasts of var Emerald Giant. These polypeptides are either not detectable or barely detectable in chloroplasts from immature fruits. Both polypeptides have been purified to near homogeneity. Yellow chromoplasts from var Golden Cal Wonder and red chromoplasts from var DNAP VS-12 contained the 35-kilodalton polypeptide, but not the 58-kilodalton species.     

Ring species as bridges between microevolution and speciation

A demonstration of how small changes can lead to species-level differences is provided by ring species, in which two reproductively isolated forms are connected by a chain of intermediate populations. We review proposed cases of ring species and the insights they provide into speciation. Ring species have been viewed both as illustrations of the history of divergence of two species from their common ancestor and as demonstrations that speciation can occur in spite of gene flow between the diverging forms. Theoretical models predict that speciation with gene flow can occur when there is divergent ecological selection, and geographical differentiation increases the likelihood of speciation. Thus ring species are ideal systems for research into the role of both ecological and geographical differentiation in speciation, but few examples have been studied in detail. The Greenish warbler is a ring species in which two northward expansions around the Tibetan plateau have been accompanied by parallel evolution in morphology, ecology, and song length and complexity. However, songs have diverged in structure, resulting in a lack of recognition where the reproductively isolated forms come into contact in Siberia. Our analysis suggests that these differences could have arisen even with gene flow, and that parallel rather than divergent ecological changes have led to divergence in sexually selected traits and subsequent speciation.     

Undescribed toxin in pseudomembranous colitis.

A girl aged 12 developed pseudomembranous colitis after a short course of oral penicillin. She had no history of adverse reaction to penicillin before or after the illness. No pathogenic bacteria, mycoplasmas, or viruses were found in her faeces, but they did contain a toxin. Toxin was also found in four of five other patients with pseudomembranous colitis but not in six specimens obtained from patients with diarrhoea caused by other disorders. Further studies may show that pseudomembranous colitis is caused by a bacterial toxin.     

Linking leaf veins to growth and mortality rates: an example from a subtropical tree community

A fundamental goal in ecology is to link variation in species function to performance, but functional trait–performance investigations have had mixed success. This indicates that less commonly measured functional traits may more clearly elucidate trait–performance relationships. Despite the potential importance of leaf vein traits, which are expected to be related to resource delivery rates and photosynthetic capacity, there are few studies, which examine associations between these traits and demographic performance in communities. Here, we examined the associations between species traits including leaf venation traits and demographic rates (Relative Growth Rate, RGR and mortality) as well as the spatial distributions of traits along soil environment for 54 co‐occurring species in a subtropical forest. Size‐related changes in demographic rates were estimated using a hierarchical Bayesian approach. Next, Kendall's rank correlations were quantified between traits and estimated demographic rates at a given size and between traits and species‐average soil environment. Species with denser venation, smaller areoles, less succulent, or thinner leaves showed higher RGR for a wide range of size classes. Species with leaves of denser veins, larger area, cheaper construction costs or thinner, or low‐density wood were associated with high mortality rates only in small size classes. Lastly, contrary to our expectations, acquisitive traits were not related to resource‐rich edaphic conditions. This study shows that leaf vein traits are weakly, but significantly related to tree demographic performance together with other species traits. Because leaf traits associated with an acquisitive strategy such as denser venation, less succulence, and thinner leaves showed higher growth rate, but similar leaf traits were not associated with mortality, different pathways may shape species growth and survival. This study suggests that we are still not measuring some of key traits related to resource‐use strategies, which dictate the demography and distributions of species.     

Assessment of Rival Males through the Use of Multiple Sensory Cues in the Fruitfly Drosophila pseudoobscura

Environments vary stochastically, and animals need to behave in ways that best fit the conditions in which they find themselves. The social environment is particularly variable, and responding appropriately to it can be vital for an animal’s success. However, cues of social environment are not always reliable, and animals may need to balance accuracy against the risk of failing to respond if local conditions or interfering signals prevent them detecting a cue. Recent work has shown that many male Drosophila fruit flies respond to the presence of rival males, and that these responses increase their success in acquiring mates and fathering offspring. In Drosophila melanogaster males detect rivals using auditory, tactile and olfactory cues. However, males fail to respond to rivals if any two of these senses are not functioning: a single cue is not enough to produce a response. Here we examined cue use in the detection of rival males in a distantly related Drosophila species, D. pseudoobscura, where auditory, olfactory, tactile and visual cues were manipulated to assess the importance of each sensory cue singly and in combination. In contrast to D. melanogaster, male D. pseudoobscura require intact olfactory and tactile cues to respond to rivals. Visual cues were not important for detecting rival D. pseudoobscura, while results on auditory cues appeared puzzling. This difference in cue use in two species in the same genus suggests that cue use is evolutionarily labile, and may evolve in response to ecological or life history differences between species.     

The chondrocyte-intrinsic circadian clock is disrupted in human osteoarthritis

Peripheral clocks are essential for driving cell differentiation. In osteoarthritis, loss of the normal differentiated chondrocyte (cartilage cell) phenotype is causative of disease. We investigated whether clock gene expression differed in osteoarthritic compared to “healthy” chondrocytes and used RNAi to determine whether the differences observed could affect chondrocyte phenotype. Following serum shock, PER2 expression was significantly higher, whereas BMAL1 expression was significantly lower, in osteoarthritic chondrocytes. Knockdown of BMAL1 in “healthy” chondrocytes was associated with higher cell proliferation and MMP13 expression, features characteristic of the osteoarthritic chondrocyte phenotype. Chondrocyte-intrinsic clock disruption may be a critical early step in osteoarthritis development.     

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.