Glycoproteins are species-specific markers and major IgE reactants in grass pollens

Grass pollen allergic patients are concomitantly exposed and sensitized to pollens from multiple Pooideae (i.e. common grass) species. As such, they are currently desensitized by allergen‐specific immunotherapy using extracts made from mixes of pollens from Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis. Herein, we demonstrate that species‐specific glycoprotein patterns are documented by 1D and 2D electrophoresis and Western blotting analysis, which can be used as an identity test for such pollens. Most allergens are glycoproteins bearing complex N‐glycans encompassing β1,2 xylose and α1,3 fucose glycoepitopes. Glycoepitope destruction using periodate oxidation has no impact on seric IgE reactivity in 75% atopic patients (n = 24). The latter have thus no significant IgE responses to carbohydrate‐containing epitopes. In contrast, periodate treatment strongly impairs IgE recognition of glycoallergens in 25% of patients tested, demonstrating the presence of carbohydrate‐specific IgE in those patients. While the clinical impact of carbohydrate‐specific IgE is still a matter of controversy, the presence of these IgE in the serum of many allergic patients illustrates the need for cross‐reacting carbohydrate epitope‐free recombinant allergens to develop relevant diagnostic tests. These data also support the pertinence of mixing multiple grass pollens to desensitize atopic patients, with the aim to broaden the repertoire of glycoepitopes in the vaccine, thus mimicking natural exposure conditions.     

Nonproteolytic properties of murine alternatively spliced tissue factor: implications for integrin-mediated signaling in murine models

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.     

Key outcomes from stakeholder workshops at a symposium to inform the development of an Australian national plan for rare diseases

ABSTRACT: BACKGROUND: Calls have been made for governments to adopt a cohesive approach to rare diseases through the development of national plans. At present, Australia does not have a national plan for rare diseases. To progress such a plan an inaugural Australian Rare Diseases Symposium was held in Western Australia in April 2011. This paper describes the key issues identified by symposium attendees for the development of a national plan, compares these to the content of EUROPLAN and national plans elsewhere and discusses how the outcomes might be integrated for national planning. METHODS: The symposium was comprised of a series of plenary sessions followed by workshops. The topics covered were; 1) Development of national plans for rare diseases; 2) Patient empowerment; 3) Patient care, support and management; 4) Research and translation; 5) Networks, partnerships and collaboration. All stakeholders within the rare diseases community were invited to participate, including: people affected by rare diseases such as patients, carers, and families; clinicians and allied health practitioners; social and disability services; researchers; patient support groups; industry (e.g. pharmaceutical, biotechnology and medical device companies); regulators and policy-makers. RESULTS: All of these stakeholder groups were represented at the symposium. Workshop participants indicated the need for a national plan, a national peak body, a standard definition of 'rare diseases', education campaigns, lobbying of government, research infrastructure, streamlined whole-of-lifetime service provision, case co-ordination, early diagnosis, support for health professionals and dedicated funding. CONCLUSIONS: These findings are consistent with frameworks and initiatives being undertaken internationally (such as EUROPLAN), and with national plans in other countries. This implies that the development of an Australian national plan could plausibly draw on frameworks for plan development that have been proposed for use in other jurisdictions. The translation of the symposium outcomes to government policy (i.e. a national plan) requires the consideration of several factors such as the under-representation of some stakeholder groups (e.g. clinicians) and the current lack of evidence required to translate some of the symposium outcomes to policy options. The acquisition of evidence provides a necessary first step in a comprehensive planning approach.     

High-resolution melting technology: a new tool for studying the Wolbachia endosymbiont diversity in the field

Stable infections by maternally transmitted symbionts are frequently found in field populations, especially in arthropods. Many questions remain regarding their contribution to host biology and ecology, and especially on environmental adaptation of their host. Wolbachia is one of the most common endosymbiont of invertebrates. This cytoplasmically inherited endocellular bacterium induces number of reproductive alterations in its arthropod hosts and various fitness effects that allow it to spread in host populations. To better understand the influence of Wolbachia on host phenotypes and consequences of the manipulation of reproduction on the host genetic differentiation, it is crucial to be able to discriminate Wolbachia strains and determine their prevalence, which requires exhaustive screening. In the present report, we proposed the use of a new tool for the population studies, based on the high resolution melting (HRM) analysis, less expensive and faster than the 'classical' methods for large-scale studies. We investigated the effectiveness of HRM to explore and characterize the diversity of Wolbachia strains. Results obtained showed that HRM is a powerful tool to identify strains and detect polymorphism in singly infected hosts. When individuals harboured a mixture of Wolbachia strains (multiple infections), there is a risk of underestimation of the diversity if the proportions of the strains are highly different. However, the same limitations exist for the other techniques commonly used. Overall, this study demonstrated that HRM analysis is a rapid and reliable technique useful for studying, without a priori, Wolbachia strains diversity in field populations.     

A new stalked filter-feeder from the middle Cambrian Burgess Shale, British Columbia, Canada

Burgess Shale-type deposits provide invaluable insights into the early evolution of body plans and the ecological structure of Cambrian communities, but a number of species, continue to defy phylogenetic interpretations. Here we extend this list to include a new soft-bodied animal, Siphusauctum gregarium n. gen. and n. sp., from the Tulip Beds (Campsite Cliff Shale Member, Burgess Shale Formation) of Mount Stephen (Yoho National Park, British Columbia). With 1,133 specimens collected, S. gregarium is clearly the most abundant animal from this locality.This stalked animal (reaching at least 20 cm in length), has a large ovoid calyx connected to a narrow bilayered stem and a small flattened or bulb-like holdfast. The calyx is enclosed by a flexible sheath with six small openings at the base, and a central terminal anus near the top encircled by indistinct openings. A prominent organ, represented by six radially symmetrical segments with comb-like elements, surrounds an internal body cavity with a large stomach, conical median gut and straight intestine. Siphusauctum gregarium was probably an active filter-feeder, with water passing through the calyx openings, capturing food particles with its comb-like elements. It often occurs in large assemblages on single bedding planes suggesting a gregarious lifestyle, with the animal living in high tier clusters. These were probably buried en masse more or less in-situ by rapid mud flow events.Siphusauctum gregarium resembles Dinomischus, another Cambrian enigmatic stalked animal. Principal points of comparison include a long stem with a calyx containing a visceral mass and bract-like elements, and a similar lifestyle albeit occupying different tiering levels. The presence in both animals of a digestive tract with a potential stomach and anus suggest a grade of organization within bilaterians, but relationships with extant phyla are not straightforward. Thus, the broader affinities of S. gregarium remain largely unconstrained.     

Crystal structure of the copper-containing quercetin 2,3-dioxygenase from Aspergillus japonicus

Quercetin 2,3-dioxygenase is a copper-containing enzyme that catalyzes the insertion of molecular oxygen into polyphenolic flavonols. Dioxygenation catalyzed by iron-containing enzymes has been studied extensively, but dioxygenases employing other metal cofactors are poorly understood. We determined the crystal structure of quercetin 2,3-dioxygenase at 1.6 A resolution. The enzyme forms homodimers, which are stabilized by an N-linked heptasaccharide at the dimer interface. The mononuclear type 2 copper center displays two distinct geometries: a distorted tetrahedral coordination, formed by His66, His68, His112, and a water molecule, and a distorted trigonal bipyramidal environment, which additionally comprises Glu73. Manual docking of the substrate quercetin into the active site showed that the different geometries of the copper site might be of catalytic importance.     

Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk

We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment.     

Crystal structure and mutational study of a unique SpoU family archaeal methylase that forms 2'-O-methylcytidine at position 56 of tRNA

The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2′-O-methylation of the cytidine residue in archaeal tRNA, using S-adenosyl-L-methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S-adenosyl-L-methionine at 2.48 Å resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal β-hairpin. aTrm56 forms a dimer. The S-adenosyl-L-methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the λ form as its substrate.     

Impact of transgene inheritance on the mitigation of gene flow between crops and their wild relatives: the example of foxtail millet

Developing genetically modified crop plants that are biologically contained could reduce significantly the potential spread of transgenes to conventional and organic crop plants and to wild or weedy relatives. Among several strategies, the hereditary mode of transmission of transgenes, whether dominant, recessive, or maternal, could play a major role in interspecific gene flow. Here we report on the gene flow between foxtail millet (Setaria italica), an autogamous crop, and its weedy relative, S. viridis, growing within or beside fields containing the three kinds of inherited herbicide resistance. Over the 6-year study, in the absence of herbicide selection, the maternal chloroplast-inherited resistance was observed at a 2 × 10⁻⁶ frequency in the weed populations. Resistant weed plants were observed 60 times as often, at 1.2 × 10⁻⁴ in the case of the nuclear recessive resistance, and 190 times as often, at 3.9 × 10⁻⁴ in the case of the dominant resistance. Because the recessive gene was not expressed in the first-generation hybrids, it should be more effective than dominant genes in reducing gene flow under normal agricultural conditions where herbicides are sprayed because interspecific hybrids cannot gain from beneficial genes.