N-glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues

In recent years, the glycoconjugates of many parasitic nematodes have attracted interest due to their immunogenic and immunomodulatory nature. Previous studies with the porcine roundworm parasite Ascaris suum have focused on its glycosphingolipids, which were found, in part, to be modified by phosphorylcholine. Using mass spectrometry and western blotting, we have now analyzed the peptide N-glycosidase A-released N-glycans of adults of this species. The presence of hybrid bi- and triantennary N-glycans, some modified by core alpha1,6-fucose and peripheral phosphorylcholine, was demonstrated by LC/electrospray ionization (ESI)-Q-TOF-MS/MS, as was the presence of paucimannosidic N-glycans, some of which carry core alpha1,3-fucose, and oligomannosidic oligosaccharides. Western blotting verified the presence of protein-bound phosphorylcholine and core alpha1,3-fucose, whereas glycosyltransferase assays showed the presence of core alpha1,6-fucosyltransferase and Lewis-type alpha1,3-fucosyltransferase activities. Although, the unusual tri- and tetrafucosylated glycans found in the model nematode Caenorhabditis elegans were not found, the vast majority of the N-glycans found in A. suum represent a subset of those found in C. elegans; thus, our data demonstrate that the latter is an interesting glycobiological model for parasitic nematodes.     

Evolution after gene duplication: models, mechanisms, sequences, systems, and organisms

Gene duplication is postulated to have played a major role in the evolution of biological novelty. Here, gene duplication is examined across levels of biological organization in an attempt to create a unified picture of the mechanistic process by which gene duplication can have played a role in generating biodiversity. Neofunctionalization and subfunctionalization have been proposed as important processes driving the retention of duplicate genes. These models have foundations in population genetic theory, which is now being refined by explicit consideration of the structural constraints placed upon genes encoding proteins through physical chemistry. Further, such models can be examined in the context of comparative genomics, where an integration of gene-level evolution and species-level evolution allows an assessment of the frequency of duplication and the fate of duplicate genes. This process, of course, is dependent upon the biochemical role that duplicated genes play in biological systems, which is in turn dependent upon the mechanism of duplication: whole genome duplication involving a co-duplication of interacting partners vs. single gene duplication. Lastly, the role that these processes may have played in driving speciation is examined.     

The db mutation improves memory in younger mice in a model of Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, while obesity is a major global public health problem associated with the metabolic disorder type 2 diabetes mellitus (T2DM). Chronic obesity and T2DM have been identified as invariant risk factors for dementia and late-onset AD, while their impacts on the occurrence and development of AD remain unclear. As shown in our previous study, the diabetic mutation (db, Lepr^db/db) induces mixed or vascular dementia in mature to middle-aged APP^ΔNL/ΔNL x PS1^P264L/P264L knock-in mice (db/AD). In the present study, the impacts of the db mutation on young AD mice at 10 weeks of age were evaluated. The db mutation not only conferred young AD mice with severe obesity, impaired glucose regulation and activated mammalian target of rapamycin (mTOR) signaling pathway in the mouse cortex, but lead to a surprising improvement in memory. At this young age, mice also had decreased cerebral Aβ content, which we have not observed at older ages. This was unlikely to be related to altered Aβ synthesis, as both β- and γ-secretase were unchanged. The db mutation also reduced the cortical IL-1β mRNA level and IBA1 protein level in young AD mice, with no significant effect on the activation of microglia and astrocytes. We conclude that the db mutation could transitorily improve the memory of young AD mice, a finding that may be partially explained by the relatively improved glucose homeostasis in the brains of db/AD mice compared to their counterpart AD mice, suggesting that glucose regulation could be a strategy for prevention and treatment of neurodegenerative diseases like AD.     

High-density tissue-like cultivation of JAR choriocarcinoma cells for the in vitro production of human xylosyltransferase

Human xylosyltransferase is the chain-initiating enzyme involved in the biosynthesis of glycosaminoglycans. Large amounts of xylosyltransferase are required to study the biochemical properties of the native enzyme. To achieve this goal a scale-up of animal cell culture systems was inevitable due to the small amounts of the enzyme present in tissues, e.g. only 0.5 microg XT can be obtained from a chick embryo. JAR choriocarcinoma cells cultured with 10% fetal calf serum were found to secrete xylosyltransferase with relatively high activities (1.10 mU l(-1)). To reduce contaminating proteins JAR cells were adapted to serum-free conditions. Xylosyltransferase activities up to 0.22 mU l(-1) were determined in the harvested cell culture supernatant. Scaling-up of JAR cell culture in the hybrid hollow fiber bioreactor Tecnomouse resulted in the production of 15.8 mU or 270 microg XT in 0.5 l of XT-enriched cell culture supernatant using 57 l of serum-free cell culture medium. The XT activity per ml harvest solution was 200-280-fold higher in this cell culture supernatant than in cell culture flasks. In addition, the specific XT activity of the bioreactor product was 6 microU mg(-1) of total protein, which is 2-fold higher than that obtained under static culture conditions. This study clearly demonstrates the successful high-density, tissue-like cultivation of JAR choriocarcinoma cells in a hollow fiber bioreactor resulting in an effective production of native human xylosyltransferase.     

Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10⁴ times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.     

How to Handle Speciose Clades? Mass Taxon-Sampling as a Strategy towards Illuminating the Natural History of Campanula (Campanuloideae)

Background

Speciose clades usually harbor species with a broad spectrum of adaptive strategies and complex distribution patterns, and thus constitute ideal systems to disentangle biotic and abiotic causes underlying species diversification. The delimitation of such study systems to test evolutionary hypotheses is difficult because they often rely on artificial genus concepts as starting points. One of the most prominent examples is the bellflower genus Campanula with some 420 species, but up to 600 species when including all lineages to which Campanula is paraphyletic. We generated a large alignment of petD group II intron sequences to include more than 70% of described species as a reference. By comparison with partial data sets we could then assess the impact of selective taxon sampling strategies on phylogenetic reconstruction and subsequent evolutionary conclusions.

Methodology/Principal Findings

Phylogenetic analyses based on maximum parsimony (PAUP, PRAP), Bayesian inference (MrBayes), and maximum likelihood (RAxML) were first carried out on the large reference data set (D680). Parameters including tree topology, branch support, and age estimates, were then compared to those obtained from smaller data sets resulting from “classification-guided” (D088) and “phylogeny-guided sampling” (D101). Analyses of D088 failed to fully recover the phylogenetic diversity in Campanula, whereas D101 inferred significantly different branch support and age estimates.

Conclusions/Significance

A short genomic region with high phylogenetic utility allowed us to easily generate a comprehensive phylogenetic framework for the speciose Campanula clade. Our approach recovered 17 well-supported and circumscribed sub-lineages. Knowing these will be instrumental for developing more specific evolutionary hypotheses and guide future research, we highlight the predictive value of a mass taxon-sampling strategy as a first essential step towards illuminating the detailed evolutionary history of diverse clades.     

The Density of Knobs on Plasmodium falciparum-Infected Erythrocytes Depends on Developmental Age and Varies among Isolates

Background

The virulence of Plasmodium falciparum malaria is related to the parasite’s ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density.

Methodology/Principal Findings

We used atomic force microscopy to characterize knobs on the surface of P. falciparum-infected erythrocytes. Fourteen ex vivo isolates from Ghanaian children with malaria and 10 P. falciparum isolates selected in vitro for expression of a particular PfEMP1 protein (VAR2CSA) were examined. Knob density increased from ∼20 h to ∼35 h post-invasion, with significant variation among isolates. The knob density ex vivo, which was about five-fold higher than following long-term in vitro culture, started to decline within a few months of culture. Although knob diameter and height varied among isolates, we did not observe significant time-dependent variation in these dimensions.

Conclusions/Significance

The density of knobs on the P. falciparum-IE surface depends on time since invasion, but is also determined by the infecting isolate in a time-independent manner. This is the first study to quantitatively evaluate knob densities and dimensions on different P. falciparum isolates, to examine ex vivo isolates from humans, and to compare ex vivo and long-term in vitro-cultured isolates. Our findings contribute to the understanding of the interaction between P. falciparum parasites and the infected host.     

Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import

Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.     

Fast corroding,thin magnesium coating displays antibacterial effects and low cytotoxicity

Bacterial colonisation and biofilm formation are characteristics of implant-associated infections. In search of candidates for improved prosthetic materials, fast corroding Mg-based coatings on titanium surfaces were examined for their cytotoxic and antimicrobial properties. Human osteoblasts and Staphylococcus epidermidis were each cultured on cylindrical Ti samples coated with a thin layer of Mg/Mg₄₅Zn₅Ca, applied via magnetron sputtering. Uncoated titanium samples served as controls. S. epidermidis was quantified by counting colony forming units. The biofilm-bound fraction was isolated via ultrasonic treatment, and the planktonic fraction via centrifugation. Biofilm-bound S. epidermidis was significantly decreased by approximately four to five orders of magnitude in both Mg- and Mg₄₅Zn₅Ca-coated samples after seven days compared to the control. The osteoblast viability was within the tolerance threshold of 70% stated in DIN EN ISO 10993-5:2009-10 for Mg (~80%) but not for Mg₄₅Zn₅Ca (~25%). Accordingly, Mg-coated titanium was identified as a promising candidate for an implant material with antibacterial properties and low cytotoxicity levels. The approach of exploiting fast corrosion contrasts with existing methods, which have generally focused on reducing corrosion.