Ethylenedioxy-PIP2 Oxalate Reduces Ganglioside Storage in Juvenile Sandhoff Disease Mice

Sandhoff disease is an incurable neurodegenerative disorder caused by mutations in the lysosomal hydrolase β-hexosaminidase. Deficiency in this enzyme leads to excessive accumulation of ganglioside GM2 and its asialo derivative, GA2, in brain and visceral tissues. Small molecule inhibitors of ceramide-specific glucosyltransferase, the first committed step in ganglioside biosynthesis, reduce storage of GM2 and GA2. Limited brain access or adverse effects have hampered the therapeutic efficacy of the clinically approved substrate reduction molecules, eliglustat tartrate and the imino sugar NB-DNJ (Miglustat). The novel eliglustat tartrate analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1, 4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (EtDO-PIP2, CCG-203586 or “3h”), was recently reported to reduce glucosylceramide in murine brain. Here we assessed the therapeutic efficacy of 3h in juvenile Sandhoff (Hexb?/?) mice. Sandhoff mice received intraperitoneal injections of phosphate buffered saline (PBS) or 3h (60 mg/kg/day) from postnatal day 9 (p-9) to postnatal day 15 (p-15). Brain weight and brain water content was similar in 3h and PBS-treated mice. 3h significantly reduced total ganglioside sialic acid, GM2, and GA2 content in cerebrum, cerebellum and liver of Sandhoff mice. Data from the liver showed that 3h reduced the key upstream ganglioside precursor (glucosylceramide), providing evidence for an on target mechanism of action. No significant differences were seen in the distribution of cholesterol or of neutral and acidic phospholipids. These data suggest that 3h can be an effective alternative to existing substrate reduction molecules for ganglioside storage diseases.     

Microbial diversity and prevalence of foodborne pathogens in cheap and junk foods consumed by primary schoolchildren

Aerobic plate counts (APC), coliforms, Bacillus cereus, Escherichia coli and eight foodborne pathogens were tested in 1008 cheap and junk foods, including candies, dried cakes, chewing gum, chocolate, dried and seasoned seafood, ice cream, and sugary foods. APCs were positive for 342 samples (33·9%), and the majority of the counts were 2–3 log CFU g^?1 or ml^?1 (average: 1·10 log CFU g^?1 or ml^?1). Most samples (97·3%) contained no coliforms (average: 0·07 log CFU g^?1 or ml^?1). Bacillus cereus was detected in 68 samples (average: 0·14 log CFU g^?1 or ml^?1). Escherichia coli and Listeria monocytogenes were detected in 6 and 1 samples, respectively, whereas other foodborne pathogens were not isolated. The highest bacterial counts were associated with dried and seasoned seafood products and dried cakes, suggesting that appropriate regulations of these food types should be considered. Cheap and junk foods were produced mainly in developing countries, but there were no significant differences in the bacterial counts among different countries of origin. The presence of foodborne pathogens may pose a risk for children. These results suggest that there is cause for deeper concern about the safety of these foods and that effective countermeasures should be established to improve their microbiological safety.

Significance and Impact of the Study

Food safety is especially important for children, but only limited information is available about the microbiological quality of cheap and junk foods that are consumed frequently by primary schoolchildren (e.g. dried cakes, candies and chocolates). The present study investigated the microbial quality of cheap and junk foods, and our results indicate that these foods are a potential health risk for children, therefore, deeper concern about the safety of these foods and effective countermeasures should be established to improve their microbiological safety. The present study may contribute to the development of an appropriate child food safety management system.     

Crystal structure of human cytosolic aspartyl‐tRNA synthetase,a component of multi‐tRNA synthetase complex

Human cytosolic aspartyl‐tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi‐tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Ų which comprises 16.6% of the monomeric surface area. Our structure reveals the C‐terminal end of the N‐helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N‐helix for the transfer of tRNA^Asp to elongation factor 1α. From our analyses of the crystal structure and post‐translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.Proteins 2013; 81:1840–1846. © 2013 Wiley Periodicals, Inc.     

A faecal index of diet quality that predicts reproductive success in a marsupial folivore

Estimating the nutritional value of a herbivore’s diet is difficult because it requires knowing what the animal eats, the relative quality of each component and how these components interact in relation to animal physiology. Current methods are cumbersome and rely on many assumptions that are hard to evaluate. We describe a new method for estimating relative diet quality directly from faeces that avoids the problems inherent in other methods. We combine this method with near infrared reflectance spectroscopy (NIRS) to analyse many samples and thus provide a technique with immense value in ecological studies. The method stems from the correlation between the concentrations of dietary and faecal nitrogen in herbivores eating a tannin-free diet, but a weaker relationship in browsers that ingest substantial amounts of tannins, which form complexes with proteins. These complexes reduce the availability of nitrogen and may increase faecal nitrogen concentrations. Using the tannin-binding compound, polyethylene glycol, we showed that tannin-bound nitrogen is a significant and variable part of faecal nitrogen in wild common brushtail possums (Trichosurus vulpecula). We developed a technique to measure faecal available nitrogen and found that it predicted the reproductive success of female brushtail possums in northern Australia. Faecal available nitrogen combined with NIRS provides a powerful tool for estimating the relative nutritional value of the diets of browsing herbivores in many ecological systems. It is a better indicator of diet quality than other commonly used single-nutrient measures such as faecal nitrogen and foliage analysis paired with observed feeding behaviour.     

Motor Domain Phosphorylation Modulates Kinesin-1 Transport

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.     

Changes in nasal airflow and heat transfer correlate with symptom improvement after surgery for nasal obstruction

Surgeries to correct nasal airway obstruction (NAO) often have less than desirable outcomes, partly due to the absence of an objective tool to select the most appropriate surgical approach for each patient. Computational fluid dynamics (CFD) models can be used to investigate nasal airflow, but variables need to be identified that can detect surgical changes and correlate with patient symptoms. CFD models were constructed from pre- and post-surgery computed tomography scans for 10 NAO patients showing no evidence of nasal cycling. Steady-state inspiratory airflow, nasal resistance, wall shear stress, and heat flux were computed for the main nasal cavity from nostrils to posterior nasal septum both bilaterally and unilaterally. Paired t-tests indicated that all CFD variables were significantly changed by surgery when calculated on the most obstructed side, and that airflow, nasal resistance, and heat flux were significantly changed bilaterally as well. Moderate linear correlations with patient-reported symptoms were found for airflow, heat flux, unilateral allocation of airflow, and unilateral nasal resistance as a fraction of bilateral nasal resistance when calculated on the most obstructed nasal side, suggesting that these variables may be useful for evaluating the efficacy of nasal surgery objectively. Similarity in the strengths of these correlations suggests that patient-reported symptoms may represent a constellation of effects and that these variables should be tracked concurrently during future virtual surgery planning.     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.