Evaluation of attractant flavours for use in oral vaccine baits for badgers (<Emphasis Type="Italic">Meles meles</Emphasis>)

European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis infection (tuberculosis) in Ireland and the UK and are implicated in the transmission of infection to livestock. Vaccination of badgers with the human BCG vaccine (Bacille Calmette Guerin) is considered as an important strategy to reduce the burden of disease in this species, and a pragmatic approach is likely to involve oral vaccination. In this study, we evaluated nine different flavours for use as attractants in a prototype oral vaccine bait for European badgers (M. meles): aniseed, apple, cocoa powder, carob powder, curry, fish, garlic, peanut and strawberry. The bait matrix was composed of a natural lipid formulation, developed as a vehicle for oral vaccination against tuberculosis in wildlife. A ‘food for work’ paradigm was employed during the trials to ensure the animals were actively seeking the baits. The trials showed carob and cocoa powders were equally attractive and more attractive than any of the other candidates. Carob and cocoa show potential as bait attractants for badgers and might form part of a novel vaccine delivery system.     

Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.     

Quantitatively measuring in situ flows using a self-contained underwater velocimetry apparatus (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. Field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field-compatible measurement techniques. To avoid these challenges, scientists typically use controlled laboratory environments to study animal-fluid interactions. However, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. Therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment. We designed a self-contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. This apparatus, a self-contained underwater velocimetry apparatus (SCUVA), can be operated by a single scuba diver in depths up to 40 m. Due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. These considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of SCUVA relative to the flow of interest. The following protocol is intended to address these common field challenges and to maximize measurement success.     

Re-structuring of marine communities exposed to environmental change: a global study on the interactive effects of species and functional richness

Species richness is the most commonly used but controversial biodiversity metric in studies on aspects of community stability such as structural composition or productivity. The apparent ambiguity of theoretical and experimental findings may in part be due to experimental shortcomings and/or heterogeneity of scales and methods in earlier studies. This has led to an urgent call for improved and more realistic experiments. In a series of experiments replicated at a global scale we translocated several hundred marine hard bottom communities to new environments simulating a rapid but moderate environmental change. Subsequently, we measured their rate of compositional change (re-structuring) which in the great majority of cases represented a compositional convergence towards local communities. Re-structuring is driven by mortality of community components (original species) and establishment of new species in the changed environmental context. The rate of this re-structuring was then related to various system properties. We show that availability of free substratum relates negatively while taxon richness relates positively to structural persistence (i.e., no or slow re-structuring). Thus, when faced with environmental change, taxon-rich communities retain their original composition longer than taxon-poor communities. The effect of taxon richness, however, interacts with another aspect of diversity, functional richness. Indeed, taxon richness relates positively to persistence in functionally depauperate communities, but not in functionally diverse communities. The interaction between taxonomic and functional diversity with regard to the behaviour of communities exposed to environmental stress may help understand some of the seemingly contrasting findings of past research.     

Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis

Dimethylarginine dimethylaminohydrolase (DDAH) regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N(omega)-methyl-l-arginine (NMMA), and asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA). Pharmacological regulation of nitric oxide synthesis is an important goal, but the catalytic mechanism of DDAH remains largely unexplored. A DDAH from Pseudomonas aeruginosa was cloned, and asymmetrically methylated arginine analogues were shown to be the preferred substrates, with ADMA displaying a slightly higher k(cat)/K(M) value than NMMA. DDAH is similar to members of a larger superfamily of guanidino-modifying enzymes, some of which have been shown to use an S-alkylthiouronium intermediate during catalysis. No covalent intermediates were found to accumulate during steady-state turnover reactions of DDAH with NMMA or ADMA. However, identification of a new substrate with an activated leaving group, S-methyl-l-thiocitrulline (SMTC), enabled acid trapping and ESI-MS characterization of a transient covalent adduct with a mass of 158 +/- 10 Da that accumulates during steady-state turnover. Subsequent trapping, proteolysis, peptide mapping and fragmentation by mass spectrometry, and site-directed mutagenesis demonstrated that this covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation. The use of covalent catalysis clearly links DDAH to this superfamily of enzymes and suggests that an S-alkylthiouronium intermediate may be a conserved feature in their mechanisms.     

srf-3, a mutant of Caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates

srf-3 is a mutant of C. elegans that is resistant to infection by Microbacterium nematophilum and to binding of the biofilm produced by Yersinia pseudotuberculosis and Yersinia pestis. Recently, SRF-3 was characterized as a nucleotide sugar transporter of the Golgi apparatus occurring exclusively in hypodermal seam cells, pharyngeal cells, and spermatheca. Based on the above observations, we hypothesized that srf-3 may have altered glyconjugates that may enable the mutant nematode to grow unaffected in the presence of the above pathogenic bacteria. Following analyses of N- and O-linked glycoconjugates of srf-3 and wild type nematodes using a combination of enzymatic degradation, permethylation, and mass spectrometry, we found in srf-3 a 65% reduction of acidic O-linked glycoconjugates containing glucuronic acid and galactose as well as a reduction of N-linked glycoconjugates containing galactose and fucose. These results are consistent with the specificity of SRF-3 for UDP-galactose and strongly suggest that the above glycoconjugates play an important role in allowing adhesion of M. nematophilum or Y. pseudotuberculosis biofilm to wild type C. elegans. Furthermore, because seam cells as well as pharyngeal cells secrete their glycoconjugates to the cuticle and surrounding surfaces, the results also demonstrate the critical role of these cells and their secreted glycoproteins in nematode-bacteria interactions and offer a mechanistic basis for strategies to block such recognition processes.     

The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A approximately 29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.     

Heparin-mediated conformational changes in fibronectin expose vascular endothelial growth factor binding sites

Regulation of angiogenesis involves interactions between vascular endothelial growth factor (VEGF) and components of the extracellular matrix, including fibronectin and heparan sulfate. In the present study, we identified two classes of VEGF binding sites on fibronectin. One was constitutively available whereas the availability of the other was modulated by the conformational state of fibronectin. Atomic force microscopy studies revealed that heparin and hydrophilic substrates promoted the extended conformation of fibronectin, leading to increased VEGF binding. The ability of heparin to enhance VEGF binding to fibronectin was dependent on the chemical composition and chain length of heparin, since long (>22 saccharides) heparin chains with sulfation on the 6-O and N positions of glucosamine units were required for full activity. Treatment of the complex endothelial extracellular matrix with heparin also increased VEGF binding, suggesting that heparin/heparan sulfate might regulate VEGF interactions within the extracellular matrix by controlling the structure and organization of fibronectin matrices.