Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry

Snow crab (Chionoecetes opilio) proteins have been recognized as an important source of both food and occupational allergens. While snow crab causes a significant occupational allergy, only one novel allergen has recently been fully characterized. The muscle proteins from snow crab legs were profiled by SDS-PAGE. Several of these proteins were characterized using tandem mass spectrometry. Five proteins were identified; sarcoplasmic Ca-binding (20kDa), arginine kinase (40), troponin (23kDa) and α-actine (42kDa) and smooth endoplasmic reticulum Ca(2+)ATPase (113kDa). Immunoblotting using serum of sixteen allergic patients resulted in strong reactivity with the 40-kDa protein in seven patients (43%). This protein was purified by chromatography and subsequently de novo sequenced using matrix assisted laser desorption ionization and electrospray tandem mass spectrometry. We identified a second important allergen, arginine kinase, in snow crab, designated Chi o 3. Based on identity and homology analysis, using bioinformatics tools, a signature peptide was identified as a chemical surrogate for arginine kinase. The suitability of this signature peptide was tested for analytically representing the arginine kinase, by performing a multi-reaction monitoring tandem mass spectrometry approach on actual air filter samples collected from a simulated crab processing plant.     

N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiation

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.     

Effects of the Distribution of a Toxic Microcystis Bloom on the Small Scale Patchiness of Zooplankton

Toxic cyanobacterial blooms can strongly affect freshwater food web structures. However, little is known about how the patchy occurrence of blooms within systems affects the spatial distribution of zooplankton communities. We studied this by analysing zooplankton community structures in comparison with the spatially distinct distribution of a toxic Microcystis bloom in a small, shallow, eutrophic lake. While toxic Microcystis was present at all sites, there were large spatial differences in the level of cyanobacterial biomass and in the zooplankton communities; sites with persistently low cyanobacterial biomass displayed a higher biomass of adult Daphnia and higher zooplankton diversity than sites with persistently high cyanobacterial biomass. While wind was the most likely reason for the spatially distinct occurrence of the bloom, our data indicate that it was the differences in cyanobacterial biomass that caused spatial differences in the zooplankton community structures. Overall, our study suggests that even in small systems with extensive blooms ‘refuge sites’ exist that allow large grazers to persist, which can be an important mechanism for a successful re-establishment of the biodiversity in an ecosystem after periods of cyanobacterial blooms.     

Pharmacological Identification of a Guanidine-Containing β-Alanine Analogue with Low Micromolar Potency and Selectivity for the Betaine/GABA Transporter 1 (BGT1)

The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [³H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC₅₀ value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.     

Comparison of the Accuracy of Voxel Based Registration and Surface Based Registration for 3D Assessment of Surgical Change following Orthognathic Surgery

Purpose

Superimposition of two dimensional preoperative and postoperative facial images, including radiographs and photographs, are used to evaluate the surgical changes after orthognathic surgery. Recently, three dimensional (3D) imaging has been introduced allowing more accurate analysis of surgical changes. Surface based registration and voxel based registration are commonly used methods for 3D superimposition. The aim of this study was to evaluate and compare the accuracy of the two methods.

Materials and methods

Pre-operative and 6 months post-operative cone beam CT scan (CBCT) images of 31 patients were randomly selected from the orthognathic patient database at the Dental Hospital and School, University of Glasgow, UK. Voxel based registration was performed on the DICOM images (Digital Imaging Communication in Medicine) using Maxilim software (Medicim-Medical Image Computing, Belgium). Surface based registration was performed on the soft and hard tissue 3D models using VRMesh (VirtualGrid, Bellevue City, WA). The accuracy of the superimposition was evaluated by measuring the mean value of the absolute distance between the two 3D image surfaces. The results were statistically analysed using a paired Student t-test, ANOVA with post-hoc Duncan test, a one sample t-test and Pearson correlation coefficient test.

Results

The results showed no significant statistical difference between the two superimposition methods (p<0.05). However surface based registration showed a high variability in the mean distances between the corresponding surfaces compared to voxel based registration, especially for soft tissue. Within each method there was a significant difference between superimposition of the soft and hard tissue models.

Conclusions

There were no significant statistical differences between the two registration methods and it was unlikely to have any clinical significance. Voxel based registration was associated with less variability. Registering on the soft tissue in isolation from the hard tissue may not be a true reflection of the surgical change.     

Corticosteroids and Pediatric Septic Shock Outcomes: A Risk Stratified Analysis

Background

The potential benefits of corticosteroids for septic shock may depend on initial mortality risk.

Objective

We determined associations between corticosteroids and outcomes in children with septic shock who were stratified by initial mortality risk.

Methods

We conducted a retrospective analysis of an ongoing, multi-center pediatric septic shock clinical and biological database. Using a validated biomarker-based stratification tool (PERSEVERE), 496 subjects were stratified into three initial mortality risk strata (low, intermediate, and high). Subjects receiving corticosteroids during the initial 7 days of admission (n = 252) were compared to subjects who did not receive corticosteroids (n = 244). Logistic regression was used to model the effects of corticosteroids on 28-day mortality and complicated course, defined as death within 28 days or persistence of two or more organ failures at 7 days.

Results

Subjects who received corticosteroids had greater organ failure burden, higher illness severity, higher mortality, and a greater requirement for vasoactive medications, compared to subjects who did not receive corticosteroids. PERSEVERE-based mortality risk did not differ between the two groups. For the entire cohort, corticosteroids were associated with increased risk of mortality (OR 2.3, 95% CI 1.3–4.0, p = 0.004) and a complicated course (OR 1.7, 95% CI 1.1–2.5, p = 0.012). Within each PERSEVERE-based stratum, corticosteroid administration was not associated with improved outcomes. Similarly, corticosteroid administration was not associated with improved outcomes among patients with no comorbidities, nor in groups of patients stratified by PRISM.

Conclusions

Risk stratified analysis failed to demonstrate any benefit from corticosteroids in this pediatric septic shock cohort.