Inducibility of chemical defenses in Norway spruce bark is correlated with unsuccessful mass attacks by the spruce bark beetle

Secondary attraction to aggregation pheromones plays a central role in the host colonization behavior of the European spruce bark beetle Ips typographus. However, it is largely unknown how the beetles pioneering an attack locate suitable host trees, and eventually accept or reject them. To find possible biomarkers for host choice by I. typographus, we analyzed the chemistry of 58 Norway spruce (Picea abies) trees that were subsequently either (1) successfully attacked and killed, (2) unsuccessfully attacked, or (3) left unattacked. The trees were sampled before the main beetle flight in a natural Norway spruce-dominated forest. No pheromones were used to attract beetles to the experimental trees. To test the trees' defense potential, each tree was treated in a local area with the defense hormone methyl jasmonate (MeJ), and treated and untreated bark were analyzed for 66 different compounds, including terpenes, phenolics and alkaloids. The chemistry of MeJ-treated bark correlated strongly with the success of I. typographus attack, revealing major chemical differences between killed trees and unsuccessfully attacked trees. Surviving trees produced significantly higher amounts of most of the 39 analyzed mono-, sesqui-, and diterpenes and of 4 of 20 phenolics. Alkaloids showed no clear pattern. Differences in untreated bark were less pronounced, where only 1,8-cineole and (-)-limonene were significantly higher in unsuccessfully attacked trees. Our results show that the potential of individual P. abies trees for inducing defense compounds upon I. typographus attack may partly determine tree resistance to this bark beetle by inhibiting its mass attack.     

Quantifying trait selection driving community assembly: a test in herbaceous plant communities under contrasted land use regimes

Plant traits are particularly important in determining plant community structure. However, how can one identify which traits are the most important in driving community assembly? Here we propose a method 1) to quantify the direction and strength of trait selection during community assembly and 2) to obtain parsimonious lists of traits that can predict species relative abundances in plant communities. We tested our method using floristic data from 32 plots experiencing different treatments (fertilisation and grazing) in southern France. Twelve functional traits were measured on 68 species. We determined the direction and strength of selection on these 12 traits using a metric derived from a maximum entropy model (i.e. lambda). We then determined our parsimonious list of traits using a backward selection of traits based on these lambda values (for all treatments and in each treatment separately). We finally compared our method to two other methods: one based on iterative RLQ and the other based on an entropy‐based forward selection of traits. We found major differences in the direction and strength of selection across the 12 traits and treatments. From the 12 traits, plant vegetative and reproductive heights, leaf dry matter content leaf nitrogen content, specific leaf area, and leaf phosphorus content were particularly important for predicting species relative abundances when considering all treatments together. Our method yielded results similar to those produced by the entropy‐based approach but differed from those produced by the iterative RLQ, whose selected traits could not significantly predict species relative abundances. Together these results suggest that the assembly of these communities is primarily driven by a small number of key functional traits. We argue that our method provides an objective way of determining a parsimonious list of traits that together accurately predict community structure and which, despite its complementarities with entropy‐based method, offers significant advantages.     

The J-UNIO protocol for automated protein structure determination by NMR in solution

The J-UNIO (JCSG protocol using the software UNIO) procedure for automated protein structure determination by NMR in solution is introduced. In the present implementation, J-UNIO makes use of APSY-NMR spectroscopy, 3D heteronuclear-resolved [(1)H,(1)H]-NOESY experiments, and the software UNIO. Applications with proteins from the JCSG target list with sizes up to 150 residues showed that the procedure is highly robust and efficient. In all instances the correct polypeptide fold was obtained in the first round of automated data analysis and structure calculation. After interactive validation of the data obtained from the automated routine, the quality of the final structures was comparable to results from interactive structure determination. Special advantages are that the NMR data have been recorded with 6-10 days of instrument time per protein, that there is only a single step of chemical shift adjustments to relate the backbone signals in the APSY-NMR spectra with the corresponding backbone signals in the NOESY spectra, and that the NOE-based amino acid side chain chemical shift assignments are automatically focused on those residues that are heavily weighted in the structure calculation. The individual working steps of J-UNIO are illustrated with the structure determination of the protein YP_926445.1 from Shewanella amazonensis, and the results obtained with 17 JCSG targets are critically evaluated.     

The role of Drosophila ninaG oxidoreductase in visual pigment chromophore biogenesis

We previously reported (Sarfare, S., Ahmad, S. T., Joyce, M. V., Boggess, B., and O'Tousa, J. E. (2005) J. Biol. Chem. 280, 11895-11901) that the Drosophila ninaG gene encodes an oxidoreductase involved in the biosynthesis of the (3S)-3-hydroxyretinal serving as chromophore for Rh1 rhodopsin and that ninaG mutant flies expressing Rh4 as the major opsin accumulate large amounts of a different retinoid. Here, we show that this unknown retinoid is 11-cis-3-hydroxyretinol. Reversed phase high performance liquid chromatography coupled with a photodiode array UV-visible absorbance detector and mass spectrometer revealed a major product eluting at a retention time, t(r), of 3.5 min with a lambda(max) of approximately 324 nm and with a base peak in the mass spectrum at m/z 285. These observations are identical with those of the 3-hydroxyretinol standard. The base peak in the electrospray ionization mass spectrum arises from the loss of a water molecule from the protonated molecule at m/z 303 because of fragmentation in the ion source. These results suggest that 11-cis-3-hydroxyretinol is an intermediate required for chromophore biogenesis in Drosophila. We further show that ninaG mutants fed on retinal as the sole source of vitamin A are able to synthesize 3-hydroxyretinoids. Thus, the NinaG oxidoreductase is not responsible for the initial hydroxylation of the retinal ring but rather acts in a subsequent step in chromophore production. These data are used to review chromophore biosynthesis and propose that NinaG acts in the conversion of (3R)-3-hydroxyretinol to the 3S enantiomer.     

High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison

We report the development of a heteroduplex-based mutation detection method using multicapillary automated sequencers, known as conformation-sensitive capillary electrophoresis (CSCE). Our optimized CSCE protocol detected 93 of 95 known base substitution sequence variants. Since the optimization of the method, we have analyzed 215 Mb of DNA and identified 3397 unique variants. An analysis of this data set indicates that the sensitivity of CSCE is above 95% in the central 56% of the average PCR product. To fully exploit the mutation detection capacity of this method, we have developed software, canplot, which automatically compares normal and test results to prioritize samples that are most likely to contain variants. Using multiple fluorescent dyes, CSCE has the capacity to screen over 2.2 Mb on one ABI3730 each day. Therefore this technique is suitable for projects where a rapid and sensitive DNA mutation detection system is required.     

Fabricating optical fiber imaging sensors using ink jet printing technology: a pH sensor proof-of-concept

We demonstrate the feasibility of using Drop-on-Demand microjet printing technology for fabricating imaging sensors by reproducibly printing an array of photo-polymerizable sensing elements, containing a pH sensitive indicator, on the surface of an optical fiber image guide. The reproducibility of the microjet printing process is excellent for microdot (i.e. micrometer-sized polymer) sensor diameter (92.2+/-2.2 microm), height (35.0+/-1.0 microm), and roundness (0.00072+/-0.00023). pH sensors were evaluated in terms of pH sensing ability (< or =2% sensor variation), response time, and hysteresis using a custom fluorescence imaging system. In addition, the microjet technique has distinct advantages over other fabrication methods, which are discussed in detail.     

Nuclear Magnetic Resonance Structure Shows that the Severe Acute Respiratory Syndrome Coronavirus-Unique Domain Contains a Macrodomain Fold

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for “middle of the SARS-unique domain”) in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.