High Sensitive Detection of Carbohydrate Binding Proteins in an ELISA-Solid Phase Assay Based on Multivalent Glyconanoparticles

Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers.     

Safety,Immunogenicity and Duration of Immunity Elicited by an Inactivated Bovine Ephemeral Fever Vaccine

Bovine ephemeral fever (BEF) is an economically important viral vector-borne cattle disease. Several live-attenuated, inactivated and recombinant vaccines have been tested, demonstrating varying efficacy. However, to the best of our knowledge, duration of immunity conferred by an inactivated vaccine has never been reported. In the last decade, Israel has faced an increasing number of BEF outbreaks. The need for an effective vaccine compatible with strains circulating in the Middle East region led to the development of a MONTANIDE™ ISA 206 VG (water-in-oil-in-water), inactivated vaccine based on a local strain. We tested the safety, immunogenicity and duration of immunity conferred by this vaccine. The induced neutralizing antibody (NA) response was followed for 493 days in 40 cows vaccinated by different protocols. The vaccine did not cause adverse reactions or a decrease in milk production. All cows [except 2 (6.7%) which did not respond to vaccination] showed a significant rise in NA titer of up to 1:256 following the second, third or fourth booster vaccination. Neutralizing antibody levels declined gradually to 1:16 up to 120 days post vaccination. This decline continued in cows vaccinated only twice, whereas cows vaccinated 3 or 4 times showed stable titers of approximately 1:16 for up to 267 days post vaccination. At least three vaccinations with the inactivated BEF vaccine were needed to confer long-lasting immunity. These results may have significant implications for the choice of vaccination protocol with inactivated BEF vaccines. Complementary challenge data should however be added to the above results in order to determine what is the minimal NA response conferring protection from clinical disease.     

Nucleus-independent chemical shift criterion for aromaticity in π-extended tetraoxa[8]circulenes

Recently synthesized π-extended symmetrical tetraoxa[8]circulenes that exhibit electroluminescent properties were calculated at the density functional theory (DFT) level using the quantum theory of atoms in molecules (QTAIM) approach to electron density distribution analysis. Nucleus-independent chemical shift (NICS) indices were used to characterize the aromaticity of the studied molecules. The tetraoxa[8]circulene molecules were found to consist of two antiaromatic perimeters (according to the Hückel “4n” antiaromaticity rule) that include 8 and 24 π-electrons. Conversely, NICS calculations demonstrated the existence of a common π-extended system (distributed like a flat ribbon) in the studied tetraoxa[8]circulene molecules. Thus, these symmetrical tetraoxa[8]circulene molecules provide examples of diatropic systems characterized by the presence of induced diatropic ring currents.

Multi-objective optimal control of dynamic bioprocesses using ACADO Toolkit

The optimal design and operation of dynamic bioprocesses gives in practice often rise to optimisation problems with multiple and conflicting objectives. As a result typically not a single optimal solution but a set of Pareto optimal solutions exist. From this set of Pareto optimal solutions, one has to be chosen by the decision maker. Hence, efficient approaches are required for a fast and accurate generation of the Pareto set such that the decision maker can easily and systematically evaluate optimal alternatives. In the current paper the multi-objective optimisation of several dynamic bioprocess examples is performed using the freely available ACADO Multi-Objective Toolkit (http://www.acadotoolkit.org). This toolkit integrates efficient multiple objective scalarisation strategies (e.g., Normal Boundary Intersection and (Enhanced) Normalised Normal Constraint) with fast deterministic approaches for dynamic optimisation (e.g., single and multiple shooting). It has been found that the toolkit is able to efficiently and accurately produce the Pareto sets for all bioprocess examples. The resulting Pareto sets are added as supplementary material to this paper.     

Population genetics of chamois in the contact zone between the Alps and the Dinaric Mountains: uncovering the role of habitat fragmentation and past management

The chamois is a habitat specialist ungulate occupying “continental archipelagos” of fragmented rocky habitats which are frequently restricted to high altitudes. It is not clear whether forest habitats separating such population fragments act as barriers to gene flow. We studied the genetic makeup of the chamois in a topographically diverse landscape at the contact zone of two mountain ranges in Slovenia. Based on sequences of mitochondrial DNA, all Slovenian populations belong to a Northern chamois (Rupicapra r. rupicapra) subspecies. The range of chamois in Slovenia encompasses three different regions, each with unique topography, habitat connectivity and abundance of chamois: the Alps, the Dinaric Mts., and the Pohorje Mts. The habitat of the chamois is extensive and more or less continuous in the Alps, but suboptimal and fragmented in the remaining regions. In agreement with neutral genetic theory, large Northern chamois populations tended to have higher allelic richness and observed heterozygosity. Spatial clustering bears the differentiation into four geographically associated clusters within Slovenia and also revealed a strong substructure within all mountain ranges with suboptimal chamois habitat. Surprisingly, some small Dinaric populations have stayed genetically isolated in restricted habitat patches, even if they are geographically very close to each other. The four clusters, each having a unique demographic history, should be regarded as independent units for management purposes.     

Expansion of Microsatellites on Evolutionary Young Y Chromosome

Sex chromosomes are an ideal system to study processes connected with suppressed recombination. We found evidence of microsatellite expansion, on the relatively young Y chromosome of the dioecious plant sorrel (Rumex acetosa, XY1Y2 system), but no such expansion on the more ancient Y chromosomes of liverwort (Marchantia polymorpha) and human. The most expanding motifs were AC and AAC, which also showed periodicity of array length, indicating the importance of beginnings and ends of arrays. Our data indicate that abundance of microsatellites in genomes depends on the inherent expansion potential of specific motifs, which could be related to their stability and ability to adopt unusual DNA conformations. We also found that the abundance of microsatellites is higher in the neighborhood of transposable elements (TEs) suggesting that microsatellites are probably targets for TE insertions. This evidence suggests that microsatellite expansion is an early event shaping the Y chromosome where this process is not opposed by recombination, while accumulation of TEs and chromosome shrinkage predominate later.     

Low Genetic Diversity in Wide-Spread Eurasian Liver Fluke Opisthorchis felineus Suggests Special Demographic History of This Trematode Species

Opisthorchis felineus or Siberian liver fluke is a trematode parasite (Opisthorchiidae) that infects the hepato-biliary system of humans and other mammals. Despite its public health significance, this wide-spread Eurasian species is one of the most poorly studied human liver flukes and nothing is known about its population genetic structure and demographic history. In this paper, we attempt to fill this gap for the first time and to explore the genetic diversity in O. felineus populations from Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Analysis of marker DNA fragments from O. felineus mitochondrial cytochrome c oxidase subunit 1 and 3 (cox1, cox3) and nuclear rDNA internal transcribed spacer 1 (ITS1) sequences revealed that genetic diversity is very low across the large geographic range of this species. Microevolutionary processes in populations of trematodes may well be influenced by their peculiar biology. Nevertheless, we suggest that lack of population genetics structure observed in O. felineus can be primarily explained by the Pleistocene glacial events and subsequent sudden population growth from a very limited group of founders. Rapid range expansion of O. felineus through Asian and European territories after severe bottleneck points to a high dispersal potential of this trematode species.     

Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term “laccase-like multi-copper oxidase” (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.     

Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.     

Connections Between Benthic Populations and Local Strandings of the Southern Bull Kelp Durvillaea Antarctica Along the Continental Coast of Chile1

Floating seaweeds are important dispersal vectors in marine ecosystems. However, the relationship between benthic populations and stranded seaweeds has received little attention. After detachment, a fraction of floating specimens returns to the shore, resulting in strandings that fluctuate in space and time. It has been hypothesized that the availability of stranded seaweeds is related to their benthic abundance on adjacent coasts. Using the large fucoid Durvillaea antarctica, we tested whether stranded biomasses are higher at sites with dense adjacent benthic populations. Benthic abundance of D. antarctica along the continental coast of Chile was estimated using three approximations: (i) availability of potentially suitable habitat (PSH), (ii) categorical visual abundance estimates in the field, and (iii) abundance measurements in the intertidal zone. Higher PSH for D. antarctica was observed between 31° S–32° S and 40° S–42° S than between 33° S and 39° S. Lowest benthic biomasses were estimated for the northern latitudes (31° S–32° S). Regression models showed that the association between stranded biomass and PSH was highest when only the extent of rocky shore 10 km to the south of each beach was included, suggesting relatively short-distance dispersal and asymmetrical transport of floating kelps, which is further supported by low proportions of rafts with Lepas spp. (indicator of rafting). The results indicate that stranded biomasses are mostly subsidized by nearby benthic populations, which can partly explain the low genetic connectivity among populations in the study region. Future studies should also incorporate other local factors (e.g., winds, currents, wave-exposure) that influence stranding dynamics.