Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage

Background  

Since bacteria embedded in biofilms are far more difficult to eradicate than planktonic infections, it would be useful to know whether certain Staphylococcus aureus lineages are especially involved in strong biofilm formation. For this reason, in vitro biofilm formation of 228 clinical S. aureus isolates of distinct clonal lineages was investigated.     

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

Inferring meaningful pathways in weighted metabolic networks

An approach is presented for computing meaningful pathways in the network of small molecule metabolism comprising the chemical reactions characterized in all organisms. The metabolic network is described as a weighted graph in which all the compounds are included, but each compound is assigned a weight equal to the number of reactions in which it participates. Path finding is performed in this graph by searching for one or more paths with lowest weight. Performance is evaluated systematically by computing paths between the first and last reactions in annotated metabolic pathways, and comparing the intermediate reactions in the computed pathways to those in the annotated ones. For the sake of comparison, paths are computed also in the un-weighted raw (all compounds and reactions) and filtered (highly connected pool metabolites removed) metabolic graphs, respectively. The correspondence between the computed and annotated pathways is very poor (<30%) in the raw graph; increasing to approximately 65% in the filtered graph; reaching approximately 85% in the weighted graph. Considering the best-matching path among the five lightest paths increases the correspondence to 92%, on average. We then show that the average distance between pairs of metabolites is significantly larger in the weighted graph than in the raw unfiltered graph, suggesting that the small-world properties previously reported for metabolic networks probably result from irrelevant shortcuts through pool metabolites. In addition, we provide evidence that the length of the shortest path in the weighted graph represents a valid measure of the "metabolic distance" between enzymes. We suggest that the success of our simplistic approach is rooted in the high degree of specificity of the reactions in metabolic pathways, presumably reflecting thermodynamic constraints operating in these pathways. We expect our approach to find useful applications in inferring metabolic pathways in newly sequenced genomes.     

Biomechanical spinal growth modulation and progressive adolescent scoliosis – a test of the 'vicious cycle' pathogenetic hypothesis: Summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The text for this debate was written by Dr Ian A Stokes. It evaluates the hypothesis that in progressive scoliosis vertebral body wedging during adolescent growth results from asymmetric muscular loading in a "vicious cycle" (vicious cycle hypothesis of pathogenesis) by affecting vertebral body growth plates (endplate physes). A frontal plane mathematical simulation tested whether the calculated loading asymmetry created by muscles in a scoliotic spine could explain the observed rate of scoliosis increase by measuring the vertebral growth modulation by altered compression. The model deals only with vertebral (not disc) wedging. It assumes that a pre-existing scoliosis curve initiates the mechanically-modulated alteration of vertebral body growth that in turn causes worsening of the scoliosis, while everything else is anatomically and physiologically 'normal' The results provide quantitative data consistent with the vicious cycle hypothesis. Dr Stokes' biomechanical research engenders controversy. A new speculative concept is proposed of vertebral symphyseal dysplasia with implications for Dr Stokes' research and the etiology of AIS. What is not controversial is the need to test this hypothesis using additional factors in his current model and in three-dimensional quantitative models that incorporate intervertebral discs and simulate thoracic as well as lumbar scoliosis. The growth modulation process in the vertebral body can be viewed as one type of the biologic phenomenon of mechanotransduction. In certain connective tissues this involves the effects of mechanical strain on chondrocytic metabolism a possible target for novel therapeutic intervention.     

Metabolic Pathfinding Using RPAIR Annotation

Metabolic databases contain information about thousands of small molecules and reactions, which can be represented as networks. In the context of metabolic reconstruction, pathways can be inferred by searching optimal paths in such networks. A recurrent problem is the presence of pool metabolites (e.g., water, energy carriers, and cofactors), which are connected to hundreds of reactions, thus establishing irrelevant shortcuts between nodes of the network. One solution to this problem relies on weighted networks to penalize highly connected compounds. A more refined solution takes the chemical structure of reactants into account in order to differentiate between side and main compounds of a reaction. Thanks to an intensive annotation effort at KEGG, decompositions of reactions into reactant pairs (RPAIR) categorized by their role (main, trans, cofac, ligase, and leave) are now available.The goal of this article is to evaluate the impact of RPAIR data on pathfinding in metabolic networks. To this end, we measure the impact of different parameters concerning the construction of the metabolic network: mapping of reactions and reactant pairs onto a graph, use of selected categories of reactant pairs, weighting schemes for compounds and reactions, removal of highly connected metabolites, and reaction directionality. In total, we tested 104 combinations of parameters and identified their optimal values for pathfinding on the basis of 55 reference pathways from three organisms.The best-performing metabolic network combines the biochemical knowledge encoded by KEGG RPAIR with a weighting scheme penalizing highly connected compounds. With this network, we could recover reference pathways from Escherichia coli with an average accuracy of 93% (32 pathways), from Saccharomyces cerevisiae with an average accuracy of 66% (11 pathways), and from humans with an average accuracy of 70% (12 pathways). Our pathfinding approach is available as part of the Network Analysis Tools.     

Threat of rapid extermination of the lion (Panthera leo leo) in Waza National Park,Northern Cameroon

Lion populations in West and Central Africa are small and fragmented. In areas where park management is weak, threats will likely facilitate the extinction of the lion. Wildlife management requires knowledge of the population estimate. The population of lions in Waza National Park (Waza NP) was assessed by individual identification of members in the population. The population was assessed to comprise of 14–21 adult individual lions. The age structure was skewed towards adults; cubs comprised 22% of all lions identified while the sex ratio was 1 : 3. Two out of four collared lions were lost to illegal, retaliatory killings within 1 year; and probably two more males and one more female were also killed during this period. The lion population appears to have declined during the last 5 years with six lions dying per year, which is at a much higher rate than observed in the previous decades. Human‐livestock pressure has increased tremendously in this period, resulting to frequent human‐lion conflicts. To ensure the survival of the lion in Waza NP and in the entire region, management needs to intensify efforts to mitigate the pressure from humans and their livestock.     

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.     

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.