Volatile Organic Compounds in Uremia

Background

Although “uremic fetor” has long been felt to be diagnostic of renal failure, the compounds exhaled in uremia remain largely unknown so far. The present work investigates whether breath analysis by ion mobility spectrometry can be used for the identification of volatile organic compounds retained in uremia.

Methods

Breath analysis was performed in 28 adults with an eGFR ≥60 ml/min per 1.73 m², 26 adults with chronic renal failure corresponding to an eGFR of 10–59 ml/min per 1.73 m², and 28 adults with end-stage renal disease (ESRD) before and after a hemodialysis session. Breath analysis was performed by ion mobility spectrometryafter gas-chromatographic preseparation. Identification of the compounds of interest was performed by thermal desorption gas chromatography/mass spectrometry.

Results

Breath analyses revealed significant differences in the spectra of patients with and without renal failure. Thirteen compounds were chosen for further evaluation. Some compounds including hydroxyacetone, 3-hydroxy-2-butanone and ammonia accumulated with decreasing renal function and were eliminated by dialysis. The concentrations of these compounds allowed a significant differentiation between healthy, chronic renal failure with an eGFR of 10–59 ml/min, and ESRD (p<0.05 each). Other compounds including 4-heptanal, 4-heptanone, and 2-heptanone preferentially or exclusively occurred in patients undergoing hemodialysis.

Conclusion

Impairment of renal function induces a characteristic fingerprint of volatile compounds in the breath. The technique of ion mobility spectrometry can be used for the identification of lipophilic uremic retention molecules.     

Sipunculan larvae and "cosmopolitan" species

Sipuncula is a relatively small taxon with roughly 150 recognized species. Many species are geographically widespread or "cosmopolitan." The pelagosphera larvae of some species are estimated to spend several months in the plankton. However, recent molecular evidence suggests that many of the "cosmopolitan" species actually represent species-complexes, some not even monophyletic. Herein, we present data on three sipunculan species with different developmental modes that occur both in the Sea of Japan and in the Northeast Pacific. The development of the three species-Phascolosoma agassizii, Thysanocardia nigra, and Themiste pyroides-is exceptionally well studied in both regions of the Pacific. Significant differences have been observed between the two regions with respect to egg size, developmental mode, and developmental timing. In general, eggs are larger and development slower in the Northeast Pacific when compared with the Sea of Japan. These differences have been explained as a result of phenotypic plasticity exhibited under different environmental conditions, in particular temperature, but we show that the populations of all three species are also remarkably distinct genetically and that gene flow between the two regions is extremely unlikely. In Thysanocardia nigra, we even found two very distinct genetic lineages within the same location in the Northeast Pacific. The amount of genetic divergence between populations from the Sea of Japan and those from the Northeast Pacific is not correlated with developmental mode. Themiste pyroides, the species with the most abbreviated development, actually has the least degree of genetic divergence between the regions. Analyses of molecular variance show that the majority of the observed variation in all three species is between the regions. We conclude that all three "cosmopolitan" species actually represent complexes of cryptic or pseudo-cryptic species. These examples demonstrate that a solid taxonomic framework based on molecular and morphological evidence is a prerequisite for evaluating relationships between dispersal capabilities, species' ranges, and the connectivity of populations.     

Crystal structure of a ring-cleaving cyclohexane-1,2-dione hydrolase, a novel member of the thiamine diphosphate enzyme family

The thiamine diphosphate (ThDP) dependent flavoenzyme cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) catalyses a key step of a novel anaerobic degradation pathway for alicyclic alcohols by converting cyclohexane-1,2-dione (CDO) to 6-oxohexanoate and further to adipate using NAD(+) as electron acceptor. To gain insights into the molecular basis of these reactions CDH from denitrifying anaerobe Azoarcus sp. strain 22Lin was structurally characterized at 1.26 ? resolution. Notably, the active site funnel is rearranged in an unprecedented manner providing the structural basis for the specific binding and cleavage of an alicyclic compound. Crucial features include a decreased and displaced funnel entrance, a semi-circularly shaped loop segment preceding the C-terminal arm and the attachment of the C-terminal arm to other subunits of the CDH tetramer. Its structural scaffold and the ThDP activation is related to that observed for other members of the ThDP enzyme family. The selective binding of the competitive inhibitor 2-methyl-2,4-pentane-diol (MPD) to the open funnel of CDH reveals an asymmetry of the two active sites found also in the dimer of several other ThDP dependent enzymes. The substrate binding site is characterized by polar and non-polar moieties reflected in the structures of MPD and CDO and by three prominent histidine residues (His28, His31 and His76) that most probably play a crucial role in substrate activation. The NAD(+) dependent oxidation of 6-oxohexanoate remains enigmatic as the redox-active cofactor FAD seems not to participate in catalysis, and no obvious NAD(+) binding site is found. Based on the structural data both reactions are discussed.     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.     

miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.     

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli

The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H(+)-dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.     

Functional implications of plasma membrane condensation for T cell activation

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.     

Investigating the mechanisms of photosynthetic proteins using continuum electrostatics

Computational methods based on continuum electrostatics are widely used in theoretical biochemistry to analyze the function of proteins. Continuum electrostatic methods in combination with quantum chemical and molecular mechanical methods can help to analyze even very complex biochemical systems. In this article, applications of these methods to proteins involved in photosynthesis are reviewed. After giving a short introduction to the basic concepts of the continuum electrostatic model based on the Poisson-Boltzmann equation, we describe the application of this approach to the docking of electron transfer proteins, to the comparison of isofunctional proteins, to the tuning of absorption spectra, to the analysis of the coupling of electron and proton transfer, to the analysis of the effect of membrane potentials on the energetics of membrane proteins, and to the kinetics of charge transfer reactions. Simulations as those reviewed in this article help to analyze molecular mechanisms on the basis of the structure of the protein, guide new experiments, and provide a better and deeper understanding of protein functions.     

Effects of reproductive compensation, gamete discounting and reproductive assurance on mating-system diversity in hermaphrodites

Hermaphroditism allows considerable scope for contributing genes to subsequent generations through various mixtures of selfed and outcrossed offspring. The fitness consequences of different family compositions determine the evolutionarily stable mating strategy and depend on the interplay of genetic features, the nature of mating, and factors that govern offspring development. This theoretical article considers the relative contributions of these influences and their interacting effects on mating-system evolution, given a fixed genetic load within a population. Strong inbreeding depression after offspring gain independence selects for exclusive outcrossing, regardless of the intensity of predispersal inbreeding depression, unless insufficient mating limits offspring production. The extent to which selfing evolves under weak postdispersal inbreeding depression depends on predispersal inbreeding depression and the opportunity for resource limitation of offspring production. Mixed selfing and outcrossing is an evolutionarily stable strategy (ESS) if selfed zygotes survive poorly, but selfed offspring survive well, and maternal individuals produce enough "extra" eggs that deaths of unviable outcrossed embryos do not impact offspring production (reproductive compensation). Mixed mating can also be an ESS, despite weak lifetime inbreeding depression, if self-mating reduces the number of male gametes available for outcrossing (male-gamete discounting). Reproductive compensation and male-gamete discounting act largely independently on mating-system evolution. ESS mating systems always involve either complete fertilization or fertilization of enough eggs to induce resource competition among embryos, so although reproductive assurance is adaptive with insufficient mating, it is never an ESS. Our results illustrate the theoretical importance of different constraints on offspring production (availability of male gametes, egg production, and maternal resources) for both the course and outcome of mating-system evolution, whereas unequal competition between selfed and outcrossed embryos has limited effect. These results also underscore the significance of heterogeneity in the nature and intensity of inbreeding depression during the life cycle for the evolution of hermaphrodite mating systems.