Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Ni²⁺-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni²⁺-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni²⁺-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.     

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms

Background and Aims

Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns.

Methods

Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden.

Key Results

The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden.

Conclusions

Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.Key words: Biogeography, Cypripedium calceolus, genome size, plastid microsatellites, population genetics, seed dispersal     

The monophyletic origin of a remarkable sexual system in akentrogonid rhizocephalan parasites: A molecular and larval structural study

We use sequences from the nuclear ribosomal genes, 18S and 28S to analyze the phylogeny of the Rhizocephala Akentrogonida including two species, Clistosaccus paguri and Chthamalophilus delagei, that are critical for understanding rhizocephalan evolution but have not previously been part of a molecularly based study. In addition we use light and scanning electron microscopy to compare the cypris larvae of C. paguri, Sylon hippolytes and two species of the family Thompsoniidae, since this larval stage offers a suite of characters for analyzing the evolution of these otherwise highly reduced parasites. The Rhizocephala Akentrogonida form a monophyletic group nested within a paraphyletic “Kentrogonida”. C. paguri and S. hippolytes are sistergroups confirming the monophyly of the Clistosaccidae that was originally based on similarities in the cypris larvae. We find numerous LM and SEM level similarities between the two species, many of which appear to be correlated with their specialized sexual system, where male cyprids use an antennule to implant cells into the virgin female parasite. Some of these traits are also found in cyprids of the thompsoniid species. We conclude that the special cypris morphology and the implantation of males by antennular penetration was present in the stem species to the Thompsoniidae and the Clistosaccidae and emphasize the power of larval characters in rhizocephalan systematics. C. delagei is a sister group to Boschmaella balani and the two are nested deep within the Akentrogonida. This confirms the monophyly of the Chthamalophilidae and falsifies the theory that C. delagei should represent the most primitive extant rhizocephalan. Instead, chthamalophilid rhizocephalans represent some of the most highly advanced members of the parasitic barnacles.     

Ocular Proteomics with Emphasis on Two-Dimensional Gel Electrophoresis and Mass Spectrometry

The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.     

Pollinator population size and pollination ecosystem service responses to enhancing floral and nesting resources

Modeling pollination ecosystem services requires a spatially explicit, process‐based approach because they depend on both the behavioral responses of pollinators to the amount and spatial arrangement of habitat and on the within‐ and between‐season dynamics of pollinator populations in response to land use. We describe a novel pollinator model predicting flower visitation rates by wild central‐place foragers (e.g., nesting bees) in spatially explicit landscapes. The model goes beyond existing approaches by: (1) integrating preferential use of more rewarding floral and nesting resources; (2) considering population growth over time; (3) allowing different dispersal distances for workers and reproductives; (4) providing visitation rates for use in crop pollination models. We use the model to estimate the effect of establishing grassy field margins offering nesting resources and a low quantity of flower resources, and/or late‐flowering flower strips offering no nesting resources but abundant flowers, on bumble bee populations and visitation rates to flowers in landscapes that differ in amounts of linear seminatural habitats and early mass‐flowering crops. Flower strips were three times more effective in increasing pollinator populations and visitation rates than field margins, and this effect increased over time. Late‐blooming flower strips increased early‐season visitation rates, but decreased visitation rates in other late‐season flowers. Increases in population size over time in response to flower strips and amounts of linear seminatural habitats reduced this apparent competition for pollinators. Our spatially explicit, process‐based model generates emergent patterns reflecting empirical observations, such that adding flower resources may have contrasting short‐ and long‐term effects due to apparent competition for pollinators and pollinator population size increase. It allows exploring these effects and comparing effect sizes in ways not possible with other existing models. Future applications include species comparisons, analysis of the sensitivity of predictions to life‐history traits, as well as large‐scale management intervention and policy assessment.     

Interleukin 21 therapy increases the density of tumor infiltrating CD8<Superscript>+ </Superscript>T cells and inhibits the growth of syngeneic tumors

Interleukin (IL)-21 is a recently discovered cytokine in early clinical development, which has shown anti-tumor activity in various animal models. In the present study, we examine the anti-tumor activity of IL-21 protein therapy in two syngeneic tumor models and its effect on the density of tumor infiltrating T cells. We treated mice bearing established subcutaneous B16 melanomas or RenCa renal cell carcinomas with intraperitoneal (i.p.) or subcutaneous (s.c.) IL-21 protein therapy and subsequently scored the densities of tumor infiltrating CD4⁺ and CD8⁺ T cells by immunohistochemistry. Whereas both routes of IL-21 administration significantly inhibited growth of small, established RenCa and B16 tumors, only s.c. therapy significantly inhibited the growth of large, established tumors. We found a greater bioavailability and significant drainage of IL-21 to regional lymph nodes following s.c. administration, which could account for the apparent increase in anti-tumor activity. Specific depletion of CD8⁺ T cells with monoclonal antibodies completely abrogated the anti-tumor activity, whereas NK1.1⁺ cell depletion did not affect tumor growth. In accordance, both routes of IL-21 administration significantly increased the density of tumor infiltrating CD8⁺ T cells in both B16 and RenCa tumors; and in the RenCa model s.c. administration of IL-21 led to a significantly higher density of tumor infiltrating CD8⁺ T cells compared to i.p. administration. The densities of CD4⁺ T cells were unchanged following IL-21 treatments. Taken together, these data demonstrate that IL-21 protein has anti-tumor activity in established syngeneic tumors, and we show that IL-21 therapy markedly increases the density of tumor infiltrating CD8⁺ T cells.     

Genetic structure of brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> L.) from the Hardangervidda mountain plateau (Norway) analyzed by microsatellite DNA: a basis for conservation guidelines

The Hardangervidda in southern Norway, the largest mountain plateau in Europe, has thousands of lakes and streams, mainly between 1000 and 1300 m above sea level, where brown trout is the only fish species. To describe the current genetic diversity of brown trout in this area, a total of 863 fish from 20 lakes were genotyped with eleven microsatellites. Most diversity is within lake populations, but diversity among geographical groups and populations within groups was significant, too. Neighbor-joining, principle coordinate analysis and Bayesian clustering show three major geographic groups in accordance with the river systems. Bias was caused by recent stocking in two lakes. Low/no genetic differentiation among some populations indicates that intermixing is common when lakes are well-connected, as was also shown by assignment test. We recommend preserving the genetic diversity of brown trout in this unique area by managing stocking in lake systems according to genetic structure.     

Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.