Characterization of growth and metabolism of the haloalkaliphile Natronomonas pharaonis

Natronomonas pharaonis is an archaeon adapted to two extreme conditions: high salt concentration and alkaline pH. It has become one of the model organisms for the study of extremophilic life. Here, we present a genome-scale, manually curated metabolic reconstruction for the microorganism. The reconstruction itself represents a knowledge base of the haloalkaliphile's metabolism and, as such, would greatly assist further investigations on archaeal pathways. In addition, we experimentally determined several parameters relevant to growth, including a characterization of the biomass composition and a quantification of carbon and oxygen consumption. Using the metabolic reconstruction and the experimental data, we formulated a constraints-based model which we used to analyze the behavior of the archaeon when grown on a single carbon source. Results of the analysis include the finding that Natronomonas pharaonis, when grown aerobically on acetate, uses a carbon to oxygen consumption ratio that is theoretically near-optimal with respect to growth and energy production. This supports the hypothesis that, under simple conditions, the microorganism optimizes its metabolism with respect to the two objectives. We also found that the archaeon has a very low carbon efficiency of only about 35%. This inefficiency is probably due to a very low P/O ratio as well as to the other difficulties posed by its extreme environment

Hydroxybenzothiazoles as New Nonsteroidal Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1)

17β-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17β-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17β-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17β-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17β-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC50-values in the nanomolar range for the transformation of E1 to E2 by 17β-HSD1, reasonable selectivity against 17β-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17β-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17β-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17β-HSD1 inhibitors for the development of potential therapeutics

Fertile Prototaxites taiti: a basal ascomycete with inoperculate, polysporous asci lacking croziers

The affinities of Prototaxites have been debated ever since its fossils, some attaining tree-trunk proportions, were discovered in Canadian Lower Devonian rocks in 1859. Putative assignations include conifers, red and brown algae, liverworts and fungi (some lichenised). Detailed anatomical investigation led to the reconstruction of the type species, P. logani, as a giant sporophore (basidioma) of an agaricomycete (= holobasidiomycete), but evidence for its reproduction remained elusive. Tissues associated with P. taiti in the Rhynie chert plus charcoalified fragments from southern Britain are investigated here to describe the reproductive characters and hence affinities of Prototaxites. Thin sections and peels (Pragian Rhynie chert, Aberdeenshire) were examined using light and confocal microscopy; Přídolí and Lochkovian charcoalified samples (Welsh Borderland) were liberated from the rock and examined with scanning electron microscopy. Prototaxites taiti possessed a superficial hymenium comprising an epihymenial layer, delicate septate paraphyses, inoperculate polysporic asci lacking croziers and a subhymenial layer composed predominantly of thin-walled hyphae and occasional larger hyphae. Prototaxites taiti combines features of extant Taphrinomycotina (Neolectomycetes lacking croziers) and Pezizomycotina (epihymenial layer secreted by paraphyses) but is not an ancestor of the latter. Brief consideration is given to its nutrition and potential position in the phylogeny of the Ascomycota

Insights in 17β-HSD1 Enzyme Kinetics and Ligand Binding by Dynamic Motion Investigation

BACKGROUND: Bisubstrate enzymes, such as 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), exist in solution as an ensemble of conformations. 17beta-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17beta-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. CONCLUSIONS/SIGNIFICANCE: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the betaF alphaG'-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17beta-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17beta-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17beta-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis

Species Used for Drug Testing Reveal Different Inhibition Susceptibility for 17beta-Hydroxysteroid Dehydrogenase Type 1

Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17β-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17β-HSD types 1, 2, 4, 5 and 7 but also against 17β-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17β-HSDs analyzed were observed. Especially, the rodent 17β-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17β-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution

Tracking the evolutionary history of Cortinarius species in section Calochroi, with transoceanic disjunct distributions

<p>Abstract</p> <p>Background</p> <p><it>Cortinarius </it>species in section <it>Calochroi </it>display local, clinal and circumboreal patterns of distribution across the Northern Hemisphere where these ectomycorrhizal fungi occur with host trees throughout their geographical range within a continent, or have disjunct intercontinental distributions, the origins of which are not understood. We inferred evolutionary histories of four species, 1) <it>C</it>. <it>arcuatorum</it>, 2) <it>C. aureofulvus</it>, 3) <it>C</it>. <it>elegantior </it>and 4) <it>C. napus</it>, from populations distributed throughout the Old World, and portions of the New World (Central- and North America) based on genetic variation of 154 haplotype internal transcribed spacer (ITS) sequences from 83 population samples. By describing the population structure of these species across their geographical distribution, we attempt to identify their historical migration and patterns of diversification.</p> <p>Results</p> <p>Models of population structure from nested clade, demographic and coalescent-based analyses revealed genetically differentiated and geographically structured haplotypes in <it>C</it>. <it>arcuatorum </it>and <it>C</it>. <it>elegantior</it>, while <it>C</it>. <it>aureofulvus </it>showed considerably less population structure and <it>C. napus </it>lacked sufficient genetic differentiation to resolve any population structure. Disjunct populations within <it>C</it>. <it>arcuatorum, C. aureofulvus </it>and <it>C</it>. <it>elegantior </it>show little or no morphological differentiation, whereas in <it>C. napus </it>there is a high level of homoplasy and phenotypic plasticity for veil and lamellae colour. The ITS sequences of the type specimens of <it>C. albobrunnoides </it>and <it>C. albobrunnoides </it>var. <it>violaceovelatus </it>were identical to one another and are treated as one species with a wider range of geographic distribution under <it>C. napus</it>.</p> <p>Conclusions</p> <p>Our results indicate that each of the <it>Calochroi </it>species has undergone a relatively independent evolutionary history, hypothesised as follows: 1) a widely distributed ancestral population of <it>C</it>. <it>arcuatorum </it>diverged into distinctive sympatric populations in the New World; 2) two divergent lineages in <it>C</it>. <it>elegantior </it>gave rise to the New World and Old World haplotypes, respectively; and 3) the low levels of genetic divergence within <it>C</it>. <it>aureofulvus </it>and <it>C</it>. <it>napus </it>may be the result of more recent demographic population expansions. The scenario of migration via the Bering Land Bridge provides the most probable explanation for contemporaneous disjunct geographic distributions of these species, but it does not offer an explanation for the low degree of genetic divergence between populations of <it>C. aureofulvus </it>and <it>C. napus</it>. Our findings are mostly consistent with the designation of New World allopatric populations as separate species from the European counterpart species <it>C. arcuatorum </it>and <it>C. elegantior</it>. We propose the synonymy of <it>C. albobrunnoides</it>, <it>C. albobrunnoides </it>var. <it>violaceovelatus </it>and <it>C. subpurpureophyllus </it>var. <it>sulphureovelatus </it>with <it>C. napus</it>. The results also reinforce previous observations that linked <it>C. arcuatorum </it>and <it>C. aureofulvus </it>displaying distributions in parts of North America and Europe. Interpretations of the population structure of these fungi suggest that host tree history has heavily influenced their modern distributions; however, the complex issues related to co-migration of these fungi with their tree hosts remain unclear at this time.</p

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature

Visual Coding in Locust Photoreceptors

Information capture by photoreceptors ultimately limits the quality of visual processing in the brain. Using conventional sharp microelectrodes, we studied how locust photoreceptors encode random (white-noise, WN) and naturalistic (1/f stimuli, NS) light patterns in vivo and how this coding changes with mean illumination and ambient temperature. We also examined the role of their plasma membrane in shaping voltage responses. We found that brightening or warming increase and accelerate voltage responses, but reduce noise, enabling photoreceptors to encode more information. For WN stimuli, this was accompanied by broadening of the linear frequency range. On the contrary, with NS the signaling took place within a constant bandwidth, possibly revealing a ‘preference’ for inputs with 1/f statistics. The faster signaling was caused by acceleration of the elementary phototransduction current - leading to bumps - and their distribution. The membrane linearly translated phototransduction currents into voltage responses without limiting the throughput of these messages. As the bumps reflected fast changes in membrane resistance, the data suggest that their shape is predominantly driven by fast changes in the light-gated conductance. On the other hand, the slower bump latency distribution is likely to represent slower enzymatic intracellular reactions. Furthermore, the Q10s of bump duration and latency distribution depended on light intensity. Altogether, this study suggests that biochemical constraints imposed upon signaling change continuously as locust photoreceptors adapt to environmental light and temperature conditions