From sea to land and beyond : new insights into the evolution of euthyneuran Gastropoda (Mollusca)
- Background The Euthyneura are considered to be the most successful and diverse group of Gastropoda. Phylogenetically, they are riven with controversy. Previous morphology-based phylogenetic studies have been greatly hampered by rampant parallelism in morphological characters or by incomplete taxon sampling. Based on sequences of nuclear 18S rRNA and 28S rRNA as well as mitochondrial 16S rRNA and COI DNA from 56 taxa, we reconstructed the phylogeny of Euthyneura utilising Maximum Likelihood and Bayesian inference methods. The evolution of colonization of freshwater and terrestrial habitats by pulmonate Euthyneura, considered crucial in the evolution of this group of Gastropoda, is reconstructed with Bayesian approaches. Results We found several well supported clades within Euthyneura, however, we could not confirm the traditional classification, since Pulmonata are paraphyletic and Opistobranchia are either polyphyletic or paraphyletic with several clades clearly distinguishable. Sacoglossa appear separately from the rest of the Opisthobranchia as sister taxon to basal Pulmonata. Within Pulmonata, Basommatophora are paraphyletic and Hygrophila and Eupulmonata form monophyletic clades. Pyramidelloidea are placed within Euthyneura rendering the Euthyneura paraphyletic. Conclusion Based on the current phylogeny, it can be proposed for the first time that invasion of freshwater by Pulmonata is a unique evolutionary event and has taken place directly from the marine environment via an aquatic pathway. The origin of colonisation of terrestrial habitats is seeded in marginal zones and has probably occurred via estuaries or semi-terrestrial habitats such as mangroves.
Molecular similarity for machine learning in drug development : poster presentation
- Poster presentation In pharmaceutical research and drug development, machine learning methods play an important role in virtual screening and ADME/Tox prediction. For the application of such methods, a formal measure of similarity between molecules is essential. Such a measure, in turn, depends on the underlying molecular representation. Input samples have traditionally been modeled as vectors. Consequently, molecules are represented to machine learning algorithms in a vectorized form using molecular descriptors. While this approach is straightforward, it has its shortcomings. Amongst others, the interpretation of the learned model can be difficult, e.g. when using fingerprints or hashing. Structured representations of the input constitute an alternative to vector based representations, a trend in machine learning over the last years. For molecules, there is a rich choice of such representations. Popular examples include the molecular graph, molecular shape and the electrostatic field. We have developed a molecular similarity measure defined directly on the (annotated) molecular graph, a long-standing established topological model for molecules. It is based on the concepts of optimal atom assignments and iterative graph similarity. In the latter, two atoms are considered similar if their neighbors are similar. This recursive definition leads to a non-linear system of equations. We show how to iteratively solve these equations and give bounds on the computational complexity of the procedure. Advantages of our similarity measure include interpretability (atoms of two molecules are assigned to each other, each pair with a score expressing local similarity; this can be visualized to show similar regions of two molecules and the degree of their similarity) and the possibility to introduce knowledge about the target where available. We retrospectively tested our similarity measure using support vector machines for virtual screening on several pharmaceutical and toxicological datasets, with encouraging results. Prospective studies are under way.
Genome-wide analysis of growth phase-dependent translational and transcriptional regulation in halophilic archaea : research article
Stephan C. Schuster
- Background Differential expression of genes can be regulated on many different levels. Most global studies of gene regulation concentrate on transcript level regulation, and very few global analyses of differential translational efficiencies exist. The studies have revealed that in Saccharomyces cerevisiae, Arabidopsis thaliana, and human cell lines translational regulation plays a significant role. Additional species have not been investigated yet. Particularly, until now no global study of translational control with any prokaryotic species was available. Results A global analysis of translational control was performed with two haloarchaeal model species, Halobacterium salinarum and Haloferax volcanii. To identify differentially regulated genes, exponentially growing and stationary phase cells were compared. More than 20% of H. salinarum transcripts are translated with non-average efficiencies. By far the largest group is comprised of genes that are translated with above-average efficiency specifically in exponential phase, including genes for many ribosomal proteins, RNA polymerase subunits, enzymes, and chemotaxis proteins. Translation of 1% of all genes is specifically repressed in either of the two growth phases. For comparison, DNA microarrays were also used to identify differential transcriptional regulation in H. salinarum, and 17% of all genes were found to have non-average transcript levels in exponential versus stationary phase. In H. volcanii, 12% of all genes are translated with non-average efficiencies. The overlap with H. salinarum is negligible. In contrast to H. salinarum, 4.6% of genes have non-average translational efficiency in both growth phases, and thus they might be regulated by other stimuli than growth phase. Conclusions For the first time in any prokaryotic species it was shown that a significant fraction of genes is under differential translational control. Groups of genes with different regulatory patterns were discovered. However, neither the fractions nor the identity of regulated genes are conserved between H. salinarum and H. volcanii, indicating that prokaryotes as well as eukaryotes use differential translational control for the regulation of gene expression, but that the identity of regulated genes is not conserved For 70 H. salinarum genes potentiation of regulation was observed, but for the majority of regulated genes either transcriptional or translational regulation is employed.
Waves of genomic hitchhikers shed light on the evolution of gamebirds (Aves: Galliformes) : research article
Jan Ole Kriegs
- Background The phylogenetic tree of Galliformes (gamebirds, including megapodes, currassows, guinea fowl, New and Old World quails, chicken, pheasants, grouse, and turkeys) has been considerably remodeled over the last decades as new data and analytical methods became available. Analyzing presence/absence patterns of retroposed elements avoids the problems of homoplastic characters inherent in other methodologies. In gamebirds, chicken repeats 1 (CR1) are the most prevalent retroposed elements, but little is known about the activity of their various subtypes over time. Ascertaining the fixation patterns of CR1 elements would help unravel the phylogeny of gamebirds and other poorly resolved avian clades. Results We analyzed 1,978 nested CR1 elements and developed a multidimensional approach taking advantage of their transposition in transposition character (TinT) to characterize the fixation patterns of all 22 known chicken CR1 subtypes. The presence/absence patterns of those elements that were active at different periods of gamebird evolution provided evidence for a clade (Cracidae + (Numididae + (Odontophoridae + Phasianidae))) not including Megapodiidae; and for Rollulus as the sister taxon of the other analyzed Phasianidae. Genomic trace sequences of the turkey genome further demonstrated that the endangered African Congo Peafowl (Afropavo congensis) is the sister taxon of the Asian Peafowl (Pavo), rejecting other predominantly morphology-based groupings, and that phasianids are monophyletic, including the sister taxa Tetraoninae and Meleagridinae. Conclusions The TinT information concerning relative fixation times of CR1 subtypes enabled us to efficiently investigate gamebird phylogeny and to reconstruct an unambiguous tree topology. This method should provide a useful tool for investigations in other taxonomic groups as well.
The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles
Jürgen M. Steiner
- Background Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most informations on protein translocation are obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. Results To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. Conclusion The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a primitive translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has subsequently been lost in the transit sequence, but can be found at the C-terminal position of the translocating pore. Thereby, the current hypothesis of Omp85 being the prokaryotic contribution to the ancestral Toc translocon can be supported.
Snake bite envenoming in Bangladesh and the challenge of biodiversity
Kernel learning for ligand-based virtual screening:discovery of a new PPARgamma agonist
- Poster presentation at 5th German Conference on Cheminformatics: 23. CIC-Workshop Goslar, Germany. 8-10 November 2009 We demonstrate the theoretical and practical application of modern kernel-based machine learning methods to ligand-based virtual screening by successful prospective screening for novel agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma) . PPARgamma is a nuclear receptor involved in lipid and glucose metabolism, and related to type-2 diabetes and dyslipidemia. Applied methods included a graph kernel designed for molecular similarity analysis , kernel principle component analysis , multiple kernel learning , and, Gaussian process regression . In the machine learning approach to ligand-based virtual screening, one uses the similarity principle  to identify potentially active compounds based on their similarity to known reference ligands. Kernel-based machine learning  uses the "kernel trick", a systematic approach to the derivation of non-linear versions of linear algorithms like separating hyperplanes and regression. Prerequisites for kernel learning are similarity measures with the mathematical property of positive semidefiniteness (kernels). The iterative similarity optimal assignment graph kernel (ISOAK)  is defined directly on the annotated structure graph, and was designed specifically for the comparison of small molecules. In our virtual screening study, its use improved results, e.g., in principle component analysis-based visualization and Gaussian process regression. Following a thorough retrospective validation using a data set of 176 published PPARgamma agonists , we screened a vendor library for novel agonists. Subsequent testing of 15 compounds in a cell-based transactivation assay  yielded four active compounds. The most interesting hit, a natural product derivative with cyclobutane scaffold, is a full selective PPARgamma agonist (EC50 = 10 ± 0.2 microM, inactive on PPARalpha and PPARbeta/delta at 10 microM). We demonstrate how the interplay of several modern kernel-based machine learning approaches can successfully improve ligand-based virtual screening results.
Oxidative stress induces CHIP-mediated ubiquitination and roteasomal degradation of soluble guanylyl cyclase : oral presentation
Peter M. Schmidt
Harald HHW Schmidt
- Oxidative stress attenuates the NO-cGMP pathway, e.g. in the vascular system, through scavenging of free NO radicals by superoxide O2•-, by inactivation of soluble guanylyl cyclase (sGC) via oxidation of its central Fe2+ ion, and by down-regulation of sGC protein levels. While the former pathways are well established, the molecular mechanisms underlying the latter are still obscure. Using oxidative sGC inhibitor ODQ we demonstrate rapid down-regulation of sGC protein in mammalian cells. Co-incubation with proteasomal inhibitor MG132 results in accumulation of ubiquitinated sGC whereas sGC activator BAY 58–2667 prevents ubiquitination. ODQ-induced down-regulation of sGC is mediated through selective ubiquitination of its b subunit, and BAY 58–2667 abrogates this effect. Ubiquitination of sGC-b is dramatically enhanced by E3 ligase CHIP. Our data indicate that oxidative stress promotes ubiquitination of sGC b subunit through E3 ligase CHIP, and that sGC activator 58–2667 reverts this effect, most likely through stabilization of the heme-free b subunit. Thus the deleterious effects of oxidative stress can be counter-balanced by an activator of a key enzyme of vascular homeostasis.
cGMP-dependent protein kinase regulates Rap1 signaling in platelets : poster presentation
- cGMP- and cAMP-dependent protein kinases (cGK and cAK) mediate the inhibitory effects of endothelium-derived messenger molecules nitric oxide and prostacyclin on platelets. To understand the mechanisms involved in platelet inhibition we searched for new substrates of cGK and cAK. We identified Rap1GAP2, the only GTPase-activating protein of Rap1 in platelets. Rap1 is a guanine-nucleotide binding protein that controls integrin activity, platelet adhesion and aggregation. Rap1GAP2 is required to turn over Rap1-GTP to Rap1-GDP resulting in the inactivation of integrins and reduced cellular adhesion. Using phospho-specific antibodies we demonstrate phosphorylation of endogenous Rap1GAP2 on serine 7 by cGK and cAK in intact platelets. Yeast-two-hybrid screening revealed an interaction of the phosphoserine/-threonine binding adapter protein 14-3-3 with Rap1GAP2, and we mapped the 14-3-3 binding site to the N-terminus of Rap1GAP2 close to the cGK/cAK phosphorylation site. We could show that 14-3-3 binding to Rap1GAP2 requires phosphorylation of serine 9. Platelet activation by ADP and thrombin treatment induces Rap1GAP2 serine 9 phosphorylation and enhances the attachment of 14-3-3 to Rap1GAP2. In contrast, phosphorylation of serine 7 by cGK/cAK leads to the detachment of 14-3-3. Furthermore, Rap1GAP2 serine 7 phosphorylation correlates with the inhibition of Rap1-GTP formation by cGMP and cAMP in platelets. Cell adhesion experiments provide additional evidence that Rap1GAP2 is activated by the detachment of 14-3-3. Point mutants of Rap1GAP2 deficient in 14-3-3 binding inhibit Rap1-mediated cell adhesion significantly stronger than a Rap1GAP2 mutant that binds 14-3-3 constitutively. Our findings define a novel regulatory mechanism that might contribute to both platelet activation and endothelial inhibition of platelet adhesion and aggregation.
Cysteine-rich protein 2 is a downstream effector of cGMP-dependent protein kinase I in nociception : poster presentation
- The experience of pain is mediated by a specialized sensory system, the nociceptive system. There is considerable evidence that the cGMP/cGMP kinase I (cGKI) signaling pathway modulates the nociceptive processing within the spinal cord. However, downstream targets of cGKI in this context have not been identified to date. In this study we investigated whether cysteine-rich protein 2 (CRP2) is a downstream effector of cGKI in the spinal cord and is involved in nociceptive processing. Immunohistochemistry of the mouse spinal cord revealed that CRP2 is expressed in superficial laminae of the dorsal horn. CRP2 is colocalized with cGKI and with markers of primary afferent C fibers. Importantly, the majority of CRP2 mRNA-positive dorsal root ganglion (DRG) neurons express cGKI and CRP2 is phosphorylated in a cGMP-dependent manner. To elucidate the functional role of CRP2 in nociception, we investigated the nociceptive behavior of CRP2-deficient (CRP2-/-) mice. Touch perception and acute thermal nociception were unaltered in CRP2-/- mice. However, CRP2-/- mice showed an increased nociceptive behavior in models of persistent pain as compared to wild type mice. Intrathecal administration of cGKI activating cGMP analogs increased the nociceptive behavior in wild type but not in CRP2-/- mice, indicating that the presence of CRP2 was essential for cGMP/cGKI-mediated nociception. These data indicate that CRP2 is a new downstream effector of cGKI-mediated spinal nociceptive processing and point to an inhibitory role of CRP2 in the generation of inflammatory pain.