Metabolic Evolution of a Deep-Branching Hyperthermophilic Chemoautotrophic Bacterium

Aquifex aeolicus is a deep-branching hyperthermophilic chemoautotrophic bacterium restricted to hydrothermal vents and hot springs. These characteristics make it an excellent model system for studying the early evolution of metabolism. Here we present the whole-genome metabolic network of this organism and examine in detail the driving forces that have shaped it. We make extensive use of phylometabolic analysis, a method we recently introduced that generates trees of metabolic phenotypes by integrating phylogenetic and metabolic constraints. We reconstruct the evolution of a range of metabolic sub-systems, including the reductive citric acid (rTCA) cycle, as well as the biosynthesis and functional roles of several amino acids and cofactors. We show that A. aeolicus uses the reconstructed ancestral pathways within many of these sub-systems, and highlight how the evolutionary interconnections between sub-systems facilitated several key innovations. Our analyses further highlight three general classes of driving forces in metabolic evolution. One is the duplication and divergence of genes for enzymes as these progress from lower to higher substrate specificity, improving the kinetics of certain sub-systems. A second is the kinetic optimization of established pathways through fusion of enzymes, or their organization into larger complexes. The third is the minimization of the ATP unit cost to synthesize biomass, improving thermodynamic efficiency. Quantifying the distribution of these classes of innovations across metabolic sub-systems and across the tree of life will allow us to assess how a tradeoff between maximizing growth rate and growth efficiency has shaped the long-term metabolic evolution of the biosphere.Comment: 25 pages, 5 figures, 5 tables, 2 supplementary file

Incremental cost effectiveness of proton pump inhibitors for the prevention of non-steroidal anti-inflammatory drug ulcers: a pharmacoeconomic analysis linked to a case-control study

Introduction We estimated the cost effectiveness of concomitant proton pump inhibitors (PPIs) in relation to the occurrence of non-steroidal anti-inflammatory drug (NSAID) ulcer complications. Methods This study was linked to a nested case-control study. Patients with NSAID ulcer complications were compared with matched controls. Only direct medical costs were reported. For the calculation of the incremental cost effectiveness ratio we extrapolated the data to 1,000 patients using concomitant PPIs and 1,000 patients not using PPIs for 1 year. Sensitivity analysis was performed by 'worst case' and 'best case' scenarios in which the 95% confidence interval (Cl) of the odds ratio (OR) and the 95% Cl of the cost estimate of a NSAID ulcer complication were varied. Costs of PPIs was varied separately. Results In all, 104 incident cases and 284 matched controls were identified from a cohort of 51,903 NSAID users with 10,402 NSAID exposition years. Use of PPIs was associated with an adjusted OR of 0.33 (95% Cl 0.17 to 0.67; p = 0.002) for NSAID ulcer complications. In the extrapolation the estimated number of NSAID ulcer complications was 13.8 for non-PPI users and 3.6 for PPI users. The incremental total costs were (sic) 50,094 higher for concomitant PPIs use. The incremental cost effectiveness ratio was (sic) 4,907 per NSAID ulcer complication prevented when using the least costly PPIs. Conclusions Concomitant use of PPIs for the prevention of NSAID ulcer complications costs (sic) 4,907 per NSAID ulcer complication prevented when using the least costly PPIs. The price of PPIs highly influenced the robustness of the results

The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion

<p>Abstract</p> <p>Background</p> <p>The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.</p> <p>Results</p> <p>The 386 carbohydrate was not essential for folding of <it>wt </it>gp120. However, its removal improved folding of a gp120 variant lacking the 385–418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.</p> <p>Conclusion</p> <p>The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.</p

Metabolic evolution and the self-organization of ecosystems

Metabolism mediates the flow of matter and energy through the biosphere. We examined how metabolic evolution shapes ecosystems by reconstructing it in the globally abundant oceanic phytoplankter Prochlorococcus To understand what drove observed evolutionary patterns, we interpreted them in the context of its population dynamics, growth rate, and light adaptation, and the size and macromolecular and elemental composition of cells. This multilevel view suggests that, over the course of evolution, there was a steady increase in Prochlorococcus' metabolic rate and excretion of organic carbon. We derived a mathematical framework that suggests these adaptations lower the minimal subsistence nutrient concentration of cells, which results in a drawdown of nutrients in oceanic surface waters. This, in turn, increases total ecosystem biomass and promotes the coevolution of all cells in the ecosystem. Additional reconstructions suggest that Prochlorococcus and the dominant cooccurring heterotrophic bacterium SAR11 form a coevolved mutualism that maximizes their collective metabolic rate by recycling organic carbon through complementary excretion and uptake pathways. Moreover, the metabolic codependencies of Prochlorococcus and SAR11 are highly similar to those of chloroplasts and mitochondria within plant cells. These observations lead us to propose a general theory relating metabolic evolution to the self-amplification and self-organization of the biosphere. We discuss the implications of this framework for the evolution of Earth's biogeochemical cycles and the rise of atmospheric oxygen.Simons Foundation (Grant SCOPE 329108)Gordon and Betty Moore Foundation (Grant 3778)Gordon and Betty Moore Foundation (Grant 495.01

The compositional and evolutionary logic of metabolism

Metabolism displays striking and robust regularities in the forms of modularity and hierarchy, whose composition may be compactly described. This renders metabolic architecture comprehensible as a system, and suggests the order in which layers of that system emerged. Metabolism also serves as the foundation in other hierarchies, at least up to cellular integration including bioenergetics and molecular replication, and trophic ecology. The recapitulation of patterns first seen in metabolism, in these higher levels, suggests metabolism as a source of causation or constraint on many forms of organization in the biosphere. We identify as modules widely reused subsets of chemicals, reactions, or functions, each with a conserved internal structure. At the small molecule substrate level, module boundaries are generally associated with the most complex reaction mechanisms and the most conserved enzymes. Cofactors form a structurally and functionally distinctive control layer over the small-molecule substrate. Complex cofactors are often used at module boundaries of the substrate level, while simpler ones participate in widely used reactions. Cofactor functions thus act as "keys" that incorporate classes of organic reactions within biochemistry. The same modules that organize the compositional diversity of metabolism are argued to have governed long-term evolution. Early evolution of core metabolism, especially carbon-fixation, appears to have required few innovations among a small number of conserved modules, to produce adaptations to simple biogeochemical changes of environment. We demonstrate these features of metabolism at several levels of hierarchy, beginning with the small-molecule substrate and network architecture, continuing with cofactors and key conserved reactions, and culminating in the aggregation of multiple diverse physical and biochemical processes in cells.Comment: 56 pages, 28 figure

Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of Monocyte Migration in Immune-Mediated Inflammatory Disease

Background: Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man. Methods/Principal Findings: We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (99m-Tc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4x10(-3) (0.95-5.1x10(-3)) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion. Conclusions/Significance: The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention

Single cell genomes of Prochlorococcus, Synechococcus, and sympatric microbes from diverse marine environments

Prochlorococcus and Synechococcus are the dominant primary producers in marine ecosystems and perform a significant fraction of ocean carbon fixation. These cyanobacteria interact with a diverse microbial community that coexists with them. Comparative genomics of cultivated isolates has helped address questions regarding patterns of evolution and diversity among microbes, but the fraction that can be cultivated is miniscule compared to the diversity in the wild. To further probe the diversity of these groups and extend the utility of reference sequence databases, we report a data set of single cell genomes for 489 Prochlorococcus, 50 Synechococcus, 9 extracellular virus particles, and 190 additional microorganisms from a diverse range of bacterial, archaeal, and viral groups. Many of these uncultivated single cell genomes are derived from samples obtained on GEOTRACES cruises and at well-studied oceanographic stations, each with extensive suites of physical, chemical, and biological measurements. The genomic data reported here greatly increases the number of available Prochlorococcus genomes and will facilitate studies on evolutionary biology, microbial ecology, and biological oceanography

The Emergence and Early Evolution of Biological Carbon-Fixation

The fixation of into living matter sustains all life on Earth, and embeds the biosphere within geochemistry. The six known chemical pathways used by extant organisms for this function are recognized to have overlaps, but their evolution is incompletely understood. Here we reconstruct the complete early evolutionary history of biological carbon-fixation, relating all modern pathways to a single ancestral form. We find that innovations in carbon-fixation were the foundation for most major early divergences in the tree of life. These findings are based on a novel method that fully integrates metabolic and phylogenetic constraints. Comparing gene-profiles across the metabolic cores of deep-branching organisms and requiring that they are capable of synthesizing all their biomass components leads to the surprising conclusion that the most common form for deep-branching autotrophic carbon-fixation combines two disconnected sub-networks, each supplying carbon to distinct biomass components. One of these is a linear folate-based pathway of reduction previously only recognized as a fixation route in the complete Wood-Ljungdahl pathway, but which more generally may exclude the final step of synthesizing acetyl-CoA. Using metabolic constraints we then reconstruct a “phylometabolic” tree with a high degree of parsimony that traces the evolution of complete carbon-fixation pathways, and has a clear structure down to the root. This tree requires few instances of lateral gene transfer or convergence, and instead suggests a simple evolutionary dynamic in which all divergences have primary environmental causes. Energy optimization and oxygen toxicity are the two strongest forces of selection. The root of this tree combines the reductive citric acid cycle and the Wood-Ljungdahl pathway into a single connected network. This linked network lacks the selective optimization of modern fixation pathways but its redundancy leads to a more robust topology, making it more plausible than any modern pathway as a primitive universal ancestral form

Gas-Phase Terahertz Spectroscopy and the Study of Complex Interstellar Chemistry

Terahertz spectroscopy holds great promise in the advancement of the field of astrochemistry. The sensitive observation of interstellar THz radiation is expected to lower detection limits and allow the study of larger and more complex species than is currently possible at millimeter wavelengths, which will place further constraints on chemical models and permit a direct comparison to the organic compounds seen in carbonaceous chondrites. With the successful recent launch of the Herschel Space Telescope, which will give high-fidelity access to interstellar THz radiation for the first time, and the completion of the Atacama Large Millimeter Array (ALMA) by 2013, the THz astronomy era is upon us. Unfortunately, laboratory THz spectroscopy presents significant challenges and will be soon be lagging behind the newly available observational platforms. Technologies to extend the capabilities of high-resolution spectroscopic systems into the THz domain are actively being pursued on many fronts, but affordable systems that are broadly tunable, sensitive and achieve the necessary resolution are not yet available. The work in this thesis should therefore be seen as part of the effort in the transition from centimeter-/millimeter-wave to THz spectroscopy that is currently taking place in the astrochemistry community. As part of this thesis, observational searches for the complex organics hydroxyacetone (CH₃COCH₂OH), 2-cyanoethanol (OHCH₂CH₂CN) and methoxyacetonitrile (CH₃OCH₂CN) were attempted at millimeter wavelengths. The unsuccessful nature of these searches highlight the current limits of studying interstellar chemistry using pure rotational spectroscopy. The characterization of the laboratory spectra of these molecules is nonetheless important as it will aid in the assignment and description of the rotational substructure and band shapes of their THz torsional spectra, features that may allow their interstellar detection; and this thesis presents methods by which such complex spectra may be rapidly and efficiently collected and fit using automated spectrometers and modern software tools. The description of the spectrum of hydroxyacetone is furthermore of interest due to the presence of the very low barrier to internal rotation in this molecule. Many interstellar compounds, both known and potential future targets, have functional groups capable of internal rotation in their structure; and so the effort in understanding the complex effects of the low barrier rotor in this case will benefit the general effort to further understand internal rotation. In searching for new interstellar molecules, both at millimeter wavelengths and at higher THz frequencies, characterization of the complete spectra of known interstellar molecules is of great importance to allow substraction of their contribution to observational spectra. In this thesis, the ground-state rotational spectrum of methanol, the most important "interstellar weed", is catalogued and described in detail through most of the THz region that will be accessible with Herschel and ALMA. Lastly, as part of the effort to increase the sensitivity of THz spectrometers, the use of Fabry-Perot cavities at these frequencies is explored. Such resonant cavities hold the potential to significantly increase the possible path lengths in spectroscopic system and to allow novel and sensitive detection techniques. Optimal configurations and the limits on achievable path lengths and Q-factors of such cavities are discussed, as are the possible extensions of Fourier Transform MicroWave (FT-MW) techniques to THz frequencies.</p