Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF

High-throughput methods for oligosaccharide analysis are required when searching for glycan-based biomarkers. Next to mass spectrometry-based methods, which allow fast and reproducible analysis of such compounds, further separation-based techniques are needed, which allow for quantitative analysis. Here, an optimized sample preparation method for N-glycan-profiling by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) was developed, enabling high-throughput glycosylation analysis. First, glycans are released enzymatically from denatured plasma glycoproteins. Second, glycans are labeled with APTS using 2-picoline borane as a nontoxic and efficient reducing agent. Reaction conditions are optimized for a high labeling efficiency, short handling times, and only limited loss of sialic acids. Third, samples are subjected to hydrophilic interaction chromatography (HILIC) purification at the 96-well plate format. Subsequently, purified APTS-labeled N-glycans are analyzed by CGE-LIF using a 48-capillary DNA sequencer. The method was found to be robust and suitable for high-throughput glycan analysis. Even though the method comprises two overnight incubations, 96 samples can be analyzed with an overall labor allocation time of 2.5 h. The method was applied to serum samples from a pregnant woman, which were sampled during first, second, and third trimesters of pregnancy, as well as 6 weeks, 3 months, and 6 months postpartum. Alterations in the glycosylation patterns were observed with gestation and time after delivery.     

Unique N-region determines low basal activity and limited inducibility of A-RAF kinase: the role of N-region in the evolutionary divergence of RAF kinase function in vertebrates

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. A prominent feature of RAF isoforms regards differences in basal and inducible kinase activities. To elucidate the nature of these differences, we studied the role of the nonconserved residues within the N-region (Negative-charge regulatory region). The nonconserved amino acids in positions -3 and +1 relative to the highly conserved serine 299 in A-RAF and serine 338 in C-RAF have so far not been considered as regulatory residues. Here we demonstrate the essential role of these residues in the RAF activation process. Substitution of tyrosine 296 in A-RAF to arginine led to a constitutively active kinase. In contrast, substitution of glycine 300 by serine (mimicking B- and C-RAF) acts in an inhibitory manner. Consistent with these data, the introduction of glycine in the analogous position of C-RAF (S339G mutant) led to a constitutively active C-RAF kinase. Based on the three-dimensional structure of the catalytic domain of B-RAF and using the sequences of the N-regions of A- and C-RAF, we searched by molecular modeling for the putative contact points between these two moieties. A tight interaction between the N-region residue serine 339 of C-RAF and arginine 398 of the catalytic domain was identified and proposed to inhibit the kinase activity of RAF proteins, because abrogation of this interaction contributes to RAF activation. Furthermore, tyrosine 296 in A-RAF favors a spatial orientation of the N-region segment, which enables a tighter contact to the catalytic domain, whereas a glutamine residue at this position in C-RAF abrogates this interaction. Considering this observation, we suggest that tyrosine 296, which is unique for A-RAF, is a major determinant of the low activating potency of this RAF isoform.     

B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding

Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.     

Thrombin causes the Enrichment of Rat Brain Primary Cultures with Ependymal Cells Via Protease-Activated Receptor 1

Ependymal cell culture models from rat have been developed over the last 20 years to facilitate biochemical studies on this least-studied glial cell type. The cell culture protocol calls for the presence of thrombin, which is essential for obtaining a high proportion of multiciliated ependymal cells. The serine protease appears to act via protease-activated receptor 1 to prevent the apoptosis of ependymal precursors and enhance their proliferation without affecting contaminating cells. Unciliated precursors differentiate into polyciliated ependymocytes by passing through a stage of monociliation. The message for protease-activated receptor (PAR) 1 is initially abundant in the cultures, but its level declines as the cells differentiate. Besides PAR 1, signalling through PAR 2 also promotes ciliation in rat brain primary cultures, albeit to a lesser degree than the thrombin receptor. Thrombin and other proteases may be involved in the regulation of ventricular wall development. This action would be mediated mainly by PAR1.     

Selected anthropometric variables and aerobic fitness as predictors of cardiovascular disease risk in children

The aim of this study was to assess the suitability of body mass index, waist circumference, waist-to-height ratio and aerobic fitness as predictors of cardiovascular risk factor clustering in children. A cross-sectional study was conducted with 290 school boys and girls from 6 to 10 years old, randomly selected. Blood was collected after a 12-hour fasting period. Blood pressure, waist circumference (WC), height and weight were evaluated according to international standards. Aerobic fitness (AF) was assessed by the 20-metre shuttle-run test. Clustering was considered when three of these factors were present: high systolic or diastolic blood pressure, high low-density lipoprotein (LDL) cholesterol, high triglycerides, high plasma glucose, high insulin concentrations and low high-density lipoprotein (HDL) cholesterol. A ROC curve identified the cut-off points of body mass index (BMI), WC, waist-to-height ratio (WHtR) and AF as predictors of risk factor clustering. BMI, WC and WHR resulted in significant areas under the ROC curves, which was not observed for AF. The anthropometric variables were good predictors of cardiovascular risk factor clustering in both sexes, whereas aerobic fitness should not be used to identify cardiovascular risk factor clustering in these children.     

Polycomb group protein Bmi1 negatively regulates IL-10 expression in activated macrophages

Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.     

Metabolically engineered soybean seed with enhanced threonine levels: biochemical characterization and seed‐specific expression of lysine‐insensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii

Threonine (Thr) is one of a few limiting essential amino acids (EAAs) in the animal feed industry, and its level in feed rations can impact production of important meat sources, such as swine and poultry. Threonine as well as EAAs lysine (Lys) and methionine (Met) are all synthesized via the aspartate family pathway. Here, we report a successful strategy to produce high free threonine soybean seed via identification of a feedback‐resistant aspartate kinase (AK) enzyme that can be over‐expressed in developing soybean seed. Towards this goal, we have purified and biochemically characterized AK from the enteric bacterium Xenorhabdus bovienii (Xb). Site‐directed mutagenesis of XbAK identified two key regulatory residues Glu‐257 and Thr‐359 involved in lysine inhibition. Three feedback‐resistant alleles, XbAK_T359I, XbAK_E257K and XbAK_E257K/T359I, have been generated. This study is the first to kinetically characterize the XbAK enzyme and provide biochemical and transgenic evidence that Glu‐257 near the catalytic site is a critical residue for the allosteric regulation of AK. Furthermore, seed‐specific expression of the feedback‐resistant XbAK_T359I or XbAK_E257K allele results in increases of free Thr levels of up to 100‐fold in R₁ soybean seed when compared to wild‐type. Expression of feedback‐sensitive wild‐type AK did not substantially impact seed Thr content. In addition to high Thr, transgenic seed also showed substantial increases in other major free amino acid (FAA) levels, resulting in an up to 3.5‐fold increase in the total FAA content. The transgenic seed was normal in appearance and germinated well under greenhouse conditions.     

Molecular phylogeny of the Astrophorida (Porifera, Demospongiae(p)) reveals an unexpected high level of spicule homoplasy

Background

The Astrophorida (Porifera, Demospongiae ^p) is geographically and bathymetrically widely distributed. Systema Porifera currently includes five families in this order: Ancorinidae, Calthropellidae, Geodiidae, Pachastrellidae and Thrombidae. To date, molecular phylogenetic studies including Astrophorida species are scarce and offer limited sampling. Phylogenetic relationships within this order are therefore for the most part unknown and hypotheses based on morphology largely untested. Astrophorida taxa have very diverse spicule sets that make them a model of choice to investigate spicule evolution.

Methodology/Principal Findings

With a sampling of 153 specimens (9 families, 29 genera, 89 species) covering the deep- and shallow-waters worldwide, this work presents the first comprehensive molecular phylogeny of the Astrophorida, using a cytochrome c oxidase subunit I (COI) gene partial sequence and the 5′ end terminal part of the 28S rDNA gene (C1-D2 domains). The resulting tree suggested that i) the Astrophorida included some lithistid families and some Alectonidae species, ii) the sub-orders Euastrophorida and Streptosclerophorida were both polyphyletic, iii) the Geodiidae, the Ancorinidae and the Pachastrellidae were not monophyletic, iv) the Calthropellidae was part of the Geodiidae clade (Calthropella at least), and finally that v) many genera were polyphyletic (Ecionemia, Erylus, Poecillastra, Penares, Rhabdastrella, Stelletta and Vulcanella).

Conclusion

The Astrophorida is a larger order than previously considered, comprising ca. 820 species. Based on these results, we propose new classifications for the Astrophorida using both the classical rank-based nomenclature (i.e., Linnaean classification) and the phylogenetic nomenclature following the PhyloCode, independent of taxonomic rank. A key to the Astrophorida families, sub-families and genera incertae sedis is also included. Incongruences between our molecular tree and the current classification can be explained by the banality of convergent evolution and secondary loss in spicule evolution. These processes have taken place many times, in all the major clades, for megascleres and microscleres.     

High-density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.