Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium Pseudomonas syringae pv. glycinea

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.     

New baseline environmental assessment of mosquito ecology in northern Haiti during increased urbanization

The catastrophic 2010 earthquake in Port‐au‐Prince, Haiti, led to the large‐scale displacement of over 2.3 million people, resulting in rapid and unplanned urbanization in northern Haiti. This study evaluated the impact of this unplanned urbanization on mosquito ecology and vector‐borne diseases by assessing land use and change patterns. Land‐use classification and change detection were carried out on remotely sensed images of the area for 2010 and 2013. Change detection identified areas that went from agricultural, forest, or bare‐land pre‐earthquake to newly developed and urbanized areas post‐earthquake. Areas to be sampled for mosquito larvae were subsequently identified. Mosquito collections comprised five genera and ten species, with the most abundant species being Culex quinquefasciatus 35% (304/876), Aedes albopictus 27% (238/876), and Aedes aegypti 20% (174/876). All three species were more prevalent in urbanized and newly urbanized areas. Anopheles albimanus, the predominate malaria vector, accounted for less than 1% (8/876) of the collection. A set of spectral indices derived from the recently launched Landsat 8 satellite was used as covariates in a species distribution model. The indices were used to produce probability surfaces maps depicting the likelihood of presence of the three most abundant species within 30 m pixels. Our findings suggest that the rapid urbanization following the 2010 earthquake has increased the amount of area with suitable habitats for urban mosquitoes, likely influencing mosquito ecology and posing a major risk of introducing and establishing emerging vector‐borne diseases.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

Flavone 5-O-glucosides from Daphne sericea

The aerial parts of Daphne sericea yielded two new flavonoids, luteolin 7-methyl ether 5-β-d-glucoside and luteolin 7,3′-dimethyl ether 5-β-d-glucoside, as well as luteolin 7-methyl ether, isovitexin, apigenin and its 7-β-d-glucoside.     

Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics.We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics.Utilizing HRM, we profiled acetaminophen (APAP)¹-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD).Our findings imply that DIA should be the preferred method for quantitative protein profiling.Quantitative mass spectrometry is a powerful and widely used approach to identify differentially abundant proteins, e.g. for proteome profiling and biomarker discovery (1). Several tens of thousands of peptides and thousands of proteins can be routinely identified from a single sample injection in shotgun proteomics (2). Shotgun proteomics, however, is limited by low analytical reproducibility. This is due to the complexity of the samples that results in under sampling (supplemental Fig. 1) and to the fact that the acquisition of MS2 spectra is often triggered outside of the elution peak apex. As a result, only 17% of the detectable peptides are typically fragmented, and less than 60% of those are identified. This translates in reliable identification of only 10% of the detectable peptides (3). The overlap of peptide identification across technical replicates is typically 35–60% (4), which results in inconsistent peptide quantification. Alternatively to shotgun proteomics, selected reaction monitoring (SRM) enables quantification of up to 200–300 peptides at very high reproducibility, accuracy, and precision (5–8).Data-independent acquisition (DIA), a novel acquisition type, overcomes the semistochastic nature of shotgun proteomics (9–18). Spectra are acquired according to a predefined schema instead of dependent on the data. Targeted analysis of DIA data was introduced with SWATH-MS (19). For the originally published SWATH-MS, the mass spectrometer cycles through 32 predefined, contiguous, 25 Thomson wide precursor windows, and records high-resolution fragment ion spectra (19). This results in a comprehensive measurement of all detectable precursors of the selected mass range. The main novelty of SWATH-MS was in the analysis of the collected DIA data. Predefined fragment ions are extracted using precompiled spectrum libraries, which results in SRM-like data. Such targeted analyses are now enabled by several publicly available computational tools, in particular Spectronaut², Skyline (20), and OpenSWATH (21). The accuracy of peptide identification is evaluated based on the mProphet method (22).We introduce a novel SWATH-MS-type DIA workflow termed hyper reaction monitoring (HRM) (reviewed in (23)) implemented on a Thermo Scientific Q Exactive platform. It consists of comprehensive DIA acquisition and targeted data analysis with retention-time-normalized spectral libraries (24). Its high accuracy of peptide identification and quantification is due to three aspects. First, we developed a novel, improved DIA method. Second, we reimplemented the mProphet (22) approach in the software Spectronaut (www.spectronaut.org). Third, we developed large, optimized, and retention-time-normalized (iRT) spectral libraries.We compared HRM and state-of-the-art shotgun proteomics in terms of ability to discover differentially abundant proteins. For this purpose, we used a “profiling standard sample set” with 12 non-human proteins spiked at known absolute concentrations into a stable human cell line protein extract. This resulted in quasi complete data sets for HRM and the detection of a larger number of differentially abundant proteins as compared with shotgun proteomics. We utilized HRM to identify changes in the proteome in primary three-dimensional human liver microtissues after APAP exposure (25–27). These primary hepatocytes exhibit active drug metabolism. With a starting material of only 12,000 cells per sample, the abundance of 2,830 proteins was quantified over an APAP concentration range. Six novel NAPQI-cysteine proteins adducts that might be relevant for the toxicity of APAP were found and quantified mainly on mitochondrion-related proteins.     

Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.     

Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae)

We used a molecular phylogenetic approach to investigate species delimitations and diversification in the mussel drills of the Ocinebrina edwardsii complex by means of a combination of nuclear (internal transcribed spacer 2, ITS2) and mitochondrial [cytochrome oxidase subunit I (COI) and 16S] sequences. Our sample included 243 specimens ascribed to seven currently accepted species from 51 sites. Five of the samples were from either the type locality of a nominal species or a close nearby locality (O. edwardsii from Corsica, O. carmelae and O. piantonii from the Kerkennah Islands, O. hispidula from the Gulf of Gabès and O. leukos from the Canary Islands), one from the inferred original locality (O. ingloria from Venice Lagoon), and specimens assigned in the recent literature to O. nicolai. We used a combination of distance‐ and tree‐based species delimitation methods to identify Molecular Operational Taxonomic Units (MOTUs) to compare with the a priori species identifications. The consensus tree obtained by BEAST on the COI alignment allows the recognition of several distinct clades supported by the three species delimitation methods employed. The eight‐MOTUs scenario, shared by the Automatic Barcode Gap Discovery (ABGD) and Generalized Mixed Yule‐Coalescent (GMYC) methods, comprises the following major clades: clade A contains the south Tunisian species Ocinebrina piantonii Cecalupo, Buzzurro & Mariani from which the sympatric taxon O. carmelae Cecalupo, Buzzurro & Mariani (new synonym) cannot be separated; clades B and C bring together all populations from the Aegean Sea and some from the Ionian Sea, respectively; clade D groups, on the one hand, the south Tunisian samples morphologically assigned to O. hispidula Pallary and, on the other, Atlantic and Alboran Sea samples (including the Canarian taxon O. leukos Houart); clade E includes a sample from the type locality of O. edwardsii and several samples from the Tyrrhenian Sea; clades F and G correspond to a few samples from the Venice Lagoon and the Tyrrhenian Sea, respectively; clade H groups the bulk of samples from the Adriatic Sea, including samples from the Venice Lagoon morphologically identified as Ocinebrina ingloria (Crosse), and some from the Ionian Sea. No final conclusions could be reached to reconcile the currently recognized morphological taxa with the clades suggested by the COI data. The geographical structure proposed by the mitochondrial markers is similar to that found in other marine invertebrates and partially corresponds to the species defined by shell characters. We propose here a framework for the revision of the Ocinebrina edwardsii species complex, suggesting a geographical pattern for the diversification of this group in the studied area. © 2013 The Linnean Society of London     

Directed seed dispersal towards areas with low conspecific tree density by a scatter‐hoarding rodent

Scatter‐hoarding animals spread out cached seeds to reduce density‐dependent theft of their food reserves. This behaviour could lead to directed dispersal into areas with lower densities of conspecific trees, where seed and seedling survival are higher, and could profoundly affect the spatial structure of plant communities. We tested this hypothesis with Central American agoutis and Astrocaryum standleyanum palm seeds on Barro Colorado Island, Panama. We radio‐tracked seeds as they were cached and re‐cached by agoutis, calculated the density of adult Astrocaryum trees surrounding each cache, and tested whether the observed number of trees around seed caches declined more than expected under random dispersal. Seedling establishment success was negatively dependent on seed density, and agoutis carried seeds towards locations with lower conspecific tree densities, thus facilitating the escape of seeds from natural enemies. This behaviour may be a widespread mechanism leading to highly effective seed dispersal by scatter‐hoarding animals.     

Identification and QTL mapping of whitefly resistance components in Solanum galapagense

Solanum galapagense is closely related to the cultivated tomato and can show a very good resistance towards whitefly. A segregating population resulting from a cross between the cultivated tomato and a whitefly resistant S. galapagense was created and used for mapping whitefly resistance and related traits, which made it possible to study the genetic basis of the resistance. Quantitative trait loci (QTL) for adult survival co-localized with type IV trichome characteristics (presence, density, gland longevity and gland size). A major QTL (Wf-1) was found for adult survival and trichome characters on Chromosome 2. This QTL explained 54.1 % of the variation in adult survival and 81.5 % of the occurrence of type IV trichomes. A minor QTL (Wf-2) for adult survival and trichome characters was identified on Chromosome 9. The major QTL was confirmed in F3 populations. Comprehensive metabolomics, based on GCMS profiling, revealed that 16 metabolites segregating in the F2 mapping population were associated with Wf-1 and/or Wf-2. Analysis of the 10 most resistant and susceptible F2 genotypes by LCMS showed that several acyl sugars were present in significantly higher concentration in the whitefly resistant genotypes, suggesting a role for these components in the resistance as well. Our results show that whitefly resistance in S. galapagense seems to inherit relatively simple compared to whitefly resistance from other sources and this offers great prospects for resistance breeding as well as elucidating the underlying molecular mechanism(s) of the resistance.     

Effects of combined ozone and nitrogen deposition on the in situ properties of eleven key plant species of a subalpine pasture

Tropospheric O₃ and deposition of reactive N threaten the composition and function of natural and semi-natural vegetation even in remote regions. However, little is known about effects of these pollutants individually or in combination on plant species in alpine habitats. We analyzed 11 frequent plant species of a subalpine Geo-Montani-Nardetum pasture exposed at 2,000 m a.s.l. in the Swiss Alps during 3 years using a factorial free-air exposure system with three concentrations of O₃ and five rates of N application. The aim was to detect subtle effects on leaf chlorophyll and N concentrations, leaf weight, specific leaf area (SLA), and δ¹⁸O and δ¹³C as proxies for gas exchange. We expected that the species’ responsiveness to O₃ and N would be related to their functional traits and that N-induced changes in these traits would modify the species’ response to O₃ via increased growth and higher leaf conductance (g _s). Most species reacted to N supply with the accumulation of N and chlorophyll, but with no change in SLA, g _s, and growth, except Carex sempervirens which showed increased water use efficiency and leaf weight. Elevated O₃ reduced g _s in most species, but this was not related to a reduction in leaf weight, which was recorded in half of the species. Contrary to our expectation, the magnitude of the response to both O₃ and N was not related to species-specific traits such as SLA or g _s. No pronounced O₃ × N interactions were observed. In conclusion, since for most species neither N nor gas exchange limited growth, their short-term response to O₃ and N and to their combination was small. O₃ × N interactive effects are expected to be more pronounced in habitats where species are more responsive to N due to favorable growth conditions in terms of nutrient availability and temperature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.