Site-specific chlorophyll reference conditions for lakes in Northern and Western Europe

The Water Framework Directive (WFD) requires EU Member States to assess the “ecological status” of surface waters. As a component of ecological status, many European countries are developing a classification scheme for chlorophyll concentrations as a measure of phytoplankton biomass. The chlorophyll classification must be based on the degree of divergence of a water body from an appropriate baseline or ‘reference condition’. This article describes the development of a series of regression models for predicting reference chlorophyll concentrations on a site-specific basis. For model development, a large dataset of European lakes considered to be in reference condition, 466 lakes in total, was assembled. Data were included from 12 European countries, but lakes from Northern and Western Europe dominated and made up 92% of all reference lakes. Data have been collated on chlorophyll concentration, altitude, mean depth, alkalinity, humic type, surface area and geographical region. Regression models were developed for estimating site-specific reference chlorophyll concentrations from significant predictor ‘typology’ variables. Reference chlorophyll concentrations were found to vary along a number of environmental gradients. Concentrations increased with colour and alkalinity and decreased with lake depth and altitude. Forward selection was used to identify independent explanatory variables in regression models for predicting site-specific reference chlorophyll concentrations. Depth was selected as an explanatory variable in all models. Alkalinity was included in models for low colour and humic lakes and altitude was included in models for low colour and very humic lakes. Uncertainty in the models was quite high and arises from errors in the data used to develop the models (including natural temporal and spatial variability in data) and also from additional explanatory variables not considered in the models, particularly nutrient concentrations, retention time and grazing. Despite these uncertainties, site-specific reference conditions are still recommended in preference to type-specific reference conditions, as they use the individual characteristics of a site known to influence phytoplankton biomass, rather than adopt standards set to generally represent a large population of lakes of a particular type. For this reason, site-specific reference conditions should result in reduced error in ecological status classifications, particularly for lakes close to typology boundaries.     

Budburst and leaf area expansion measured with a novel mobile camera system and simple color thresholding

Plant phenology relates strongly to primary productivity and the energy that enters into ecological food webs, and thus is vital in understanding ecosystem function and the effects of climate and climate change. The manual collection of phenological data is labor-intensive and not easily scalable, thus the ability to quantify leaf flush and other parameters at many locations requires innovative new methodologies such as the use of visible light digital cameras. Improved imaging performance was obtained by using a cabled, mobile camera system that allowed a repeated image census of branches of Rhododendron occidentale in the understory along a 30 m transect during leaf flush. Automatic division of acquired images into areas of interest (leaves) and background for calculating leaf area was accomplished by thresholding images in different color spaces. Transformation of the color space into the hue, saturation, and luminance (HSL) color space before thresholding resulted in a mean RMS error of 21.2 cm² compared to hand-counts of leaf area. Thresholding in the native red, green, and blue (RGB) color space to isolate leaves resulted in a larger error, as did using algebraic combinations of the color components or color ratios. Relating physiological function to images, as for sap flow for branches of R. occidentale, indicates that the greening and calculated leaf area of a species as detected by imagers requires additional meteorological sensor data for interpretation.     

LIPID CLASS,CAROTENOID, AND TOXIN DYNAMICS OF KARENIA BREVIS (DINOPHYCEAE) DURING DIEL VERTICAL MIGRATION1

The internal lipid, carotenoid, and toxin concentrations of Karenia brevis (C. C. Davis) Gert Hansen and Moestrup are influenced by its ability to use ambient light and nutrients for growth and reproduction. This study investigated changes in K. brevis toxicity, lipid class, and carotenoid concentrations in low‐light, nitrate‐replete (250 μmol quanta · m^?2 · s^?1, 80 μM NO₃); high‐light, nitrate‐replete (960 μmol quanta · m^?2 · s^?1, 80 μM NO₃); and high‐light, nitrate‐reduced (960 μmol quanta · m^?2 · s^?1, <5 μM NO₃) mesocosms. Reverse‐phase HPLC quantified the epoxidation state (EPS) of the xanthophyll‐cycle pigments diadinoxanthin and diatoxanthin, and a Chromarod Iatroscan thin layer chromatography/flame ionization detection (TLC/FID) system quantified changes in lipid class concentrations. EPS did not exceed 0.20 in the low‐light mesocosm, but increased to 0.65 in the high‐light mesocosms. Triacylglycerol and monogalactosyldiacylglycerol (MGDG) were the largest lipid classes consisting of 9.3% to 48.7% and 37.3% to 69.7% of total lipid, respectively. Both lipid classes also experienced the greatest concentration changes in high‐light experiments. K. brevis increased EPS and toxin concentrations while decreasing its lipid concentrations under high light. K. brevis may mobilize its toxins into the surrounding environment by reducing lipid concentrations, such as sterols, limiting competition, or toxins are released because lipids are decreased in high light, reducing any protective mechanism against their own toxins.     

Structural and Biophysical Characterization of the Proteins Interacting with the Herpes Simplex Virus 1 Origin of Replication

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA_15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (K_d ∼ 6 nm) followed by a second binding event (K_d ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA_15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.The initiation of DNA replication for most double-stranded DNA (dsDNA)⁶ viral genomes begins with the recognition of the origin by specific origin-binding proteins. The herpes simplex virus type 1 (HSV-1) genome encodes seven proteins required for origin-dependent DNA replication. These are the DNA polymerase (UL30) and its accessory protein (UL42), a heterotrimeric helicase-primase complex (UL5, UL8, and UL52), the single-stranded DNA-binding protein (ICP8 or UL29), and the origin-binding protein (UL9) (reviewed in Ref. 1). HSV-1 contains three functional origins, oriL and two copies of oriS. OriS, which is about 80 bp in length, consists of three UL9 recognition sites, in Boxes I, II, and III, which are arranged in two overlapping palindromes (2). Box I and Box III are part of an evolutionarily conserved palindrome that forms a stable hairpin in single-stranded DNA, which may be important in the origin rearrangement (3) during initiation of replication. Box I and II are separated by an AT-rich spacer sequence, which varies in length and nucleotide composition between the different members of the α-herpesvirus subfamily (2, 4–6).UL9 is a homodimer in solution, and EM studies, with UL9 bound to oriS, indicate the existence of a dimer or pair of dimers assembled on oriS (7). Several reports indicate that UL9 can physically interact not only with ICP8 (8) but also with other members of the HSV-1 replication complex, including UL8 (9) and UL42 (10). Thus UL9 functions as a docking protein to recruit these essential replication proteins to the viral origins. ICP8 stimulates the helicase activity of UL9 (11, 12) and binds to its C-terminal 27-aa residues (13). In the presence of ICP8, UL9 will open dsDNA containing Box I, leading to a conformational change in the origin, thus facilitating unwinding (14–16). As stated above, the changes in DNA conformation in the complete oriS may be more complex (3). Recently, it has been suggested that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by UL9. oriS* contains a hairpin formed by complementary base pairing between Box I and Box III in oriS (17). UL9, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-bp double-stranded minimal oriS fragment to oriS* and form a UL9-oriS* complex. The formation of a UL9-oriS* complex requires ATP hydrolysis (18). Therefore, the UL9-oriS* complex may serve as an assembly site for the herpesvirus replisome. Macao et al. (3) proposed a model in which full-length UL9 would be required to adopt a different conformation when binding to oriS or oriS*. The implication is that UL9 partially unwinds and introduces a hairpin into the origin of replication and that the formation of oriS* is aided, in some way, by ICP8 and requires ATP hydrolysis. Macao et al. (3) suggest that the length of the single-stranded tail of the probe DNA determines the stoichiometry of the UL9-DNA complex. oriS may bind two molecules of UL9, whereas oriS* may only bind one because the hairpin formation prevents the second interaction.Photo-cross-linking studies have shown that, although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I, suggesting that the C terminus of UL9 undergoes a conformational change upon binding to Box I (19). The results reported here are consistent with this observation. To date there is no three-dimensional structural information available on the full-length UL9 or either of the functionally characterized (helicase and DNA binding) domains. The ability to adopt different conformations and a tendency to proteolytic degradation may be responsible for this. It has been shown that UL9 binds with very high specificity to the Box I through its DNA-binding domain, consisting of the C-terminal 317 aa (UL9ct) (20, 21). Although the importance of the binding between UL9ct and oriS for the viral life cycle is well established, the mechanism behind this interaction still remains unclear. Even though UL9ct exists as a monomer in solution, uncertainty remains as to whether one or two molecules bind to a single Box I recognition sequence. Some reports have suggested that one UL9ct molecule binds to a single copy of the sequence (22–24), whereas others have proposed that UL9ct forms a dimer when bound to DNA (25, 26). This apparent difference may well result from the different protein concentrations used in different assays/experiments, which in turn highlights the difficulty of translating in vitro equilibrium experiments into cellular nonequilibrium situations.A few years ago, the crystal structure of a 60-residue C-terminal deletion mutant of ICP8 (ICP8ΔC) was determined to 3 Å resolution (Protein Data Bank code 1URJ (27)). The structure of ICP8ΔC consists of a large N-terminal domain (aa 9–1038) and a smaller entirely helical C-terminal domain (aa 1049–1120) connected to the N-terminal domain by a disordered linker (aa 1038–1049) spanning around 18 Å in the crystal structure. ICP8 preferentially binds ssDNA over dsDNA in a nonsequence-specific and cooperative manner (28). ICP8 is a zinc metalloprotein containing one zinc atom per molecule, which is coordinated by three cysteines (Cys-499, Cys-502, and Cys-510) and a histidine (His-512) (27).In this study, we show that the 60-amino acid C-terminal deletion of ICP8 (ICP8ΔC) binds strongly to UL9ct. We present three low resolution structures in solution using small angle x-ray scattering as follows: that of the UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer dsDNA (dsDNA_15-mer) containing the Box I sequence. Using these data and a variety of biophysical techniques, we demonstrate that the stoichiometries of the UL9ct-dsDNA_15-mer and UL9ct-ICP8ΔC-dsDNA_15-mer complexes are 2:1 and 2:1:1, respectively, at the micromolar protein concentrations used in this study. Using EM we visualize the assembly of the ICP8ΔC-UL9ct complex on oriS and estimate the size of the complex.     

Assessing what is needed to resolve a molecular phylogeny: simulations and empirical data from emydid turtles

Background  

Section Calochroi is one of the most species-rich lineages in the genus Cortinarius (Agaricales, Basidiomycota) and is widely distributed across boreo-nemoral areas, with some extensions into meridional zones. Previous phylogenetic studies of Calochroi (incl. section Fulvi) have been geographically restricted; therefore, phylogenetic and biogeographic relationships within this lineage at a global scale have been largely unknown. In this study, we obtained DNA sequences from a nearly complete taxon sampling of known species from Europe, Central America and North America. We inferred intra- and interspecific phylogenetic relationships as well as major morphological evolutionary trends within section Calochroi based on 576 ITS sequences, 230 ITS + 5.8S + D1/D2 sequences, and a combined dataset of ITS + 5.8S + D1/D2 and RPB1 sequences of a representative subsampling of 58 species.     

Chondrostereum purpureum and Fusarium tumidum independently reduce regrowth in gorse (Ulex europaeus)

An experiment was conducted in two gorse populations (Ulex europaeus) in which Chondrostereum purpureum was applied each month as mycelial-agar cultures to the wounds of decapitated stems of previously untreated gorse plants to determine its potential as a mycoherbicide. Summer-autumn (Feb-May) or late winter-early spring (Aug-Sept) applications were effective in both populations, halving stem stump survival (from an average of 56 to 29%). Another experiment in the same gorse populations evaluated the combined effects of C. purpureum applied in May to decapitated stems, and Fusarium tumidum applied as spores in an invert emulsion to regenerative shoots 5-6 months later. There was no evidence of synergism between the two fungi; each pathogen independently reduced the density of regenerative shoots on the decapitated stems by 39-63% averaged over the 12 months following their respective applications. It is concluded that both pathogens have potential as mycoherbicides for gorse regenerating after stem decapitation.     

<Emphasis Type="Italic">Plectosphaerella cucumerina</Emphasis> as a bioherbicide for <Emphasis Type="Italic">Cirsium arvense</Emphasis>: proof of concept

Plectosphaerella cucumerina (Lindf.) W. Gams was evaluated as a bioherbicide for Cirsium arvense L. (Scop.) using a Canadian and a New Zealand isolate. Both isolates defoliated C. arvense when applied at 10¹³ conidia ha^?1 in water volumes ranging from 250 to 6400 l ha^?1 with a rapid decline in effect with declining conidial dose. Repeat application and the addition of the adjuvant Pulse^® penetrant to the conidial suspension increased the disease severity in C. arvense. Maximum disease occurred at 20 °C with a 48 h post-application dew period. The experiments demonstrate that P. cucumerina can defoliate C. arvense under the environmental conditions of temperate pastures where the weed is problematic. The results also show that modifications to formulation and strategic application may reduce the 48 h dew period requirement and risk to non-target species respectively, supporting the conclusion that the fungus has potential as a bioherbicide for C. arvense.     

Structure of the C-terminal domain of Neisseria heparin binding antigen (NHBA), one of the main antigens of a novel vaccine against Neisseria meningitidis

Neisseria heparin binding antigen (NHBA), also known as GNA2132 (genome-derived Neisseria antigen 2132), is a surface-exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology. It is one the three main antigens of a multicomponent vaccine against serogroup B meningitis (4CMenB), which has just completed phase III clinical trials in infants. In contrast to the other two main vaccine components, little is known about the origin of the immunogenicity of this antigen, and about its ability to induce a strong cross-bactericidal response in animals and humans. To characterize NHBA in terms of its structural/immunogenic properties, we have analyzed its sequence and identified a C-terminal region that is highly conserved in all strains. We demonstrate experimentally that this region is independently folded, and solved its three-dimensional structure by nuclear magnetic resonance. Notably, we need detergents to observe a single species in solution. The NHBA domain fold consists of an 8-strand β-barrel that closely resembles the C-terminal domains of N. meningitidis factor H-binding protein and transferrin-binding protein B. This common fold together with more subtle structural similarities suggest a common ancestor for these important antigens and a role of the β-barrel fold in inducing immunogenicity against N. meningitidis. Our data represent the first step toward understanding the relationship between structural, functional, and immunological properties of this important vaccine component.