Imputation of single-nucleotide polymorphisms in inbred mice using local phylogeny

We present full-genome genotype imputations for 100 classical laboratory mouse strains, using a novel method. Using genotypes at 549,683 SNP loci obtained with the Mouse Diversity Array, we partitioned the genome of 100 mouse strains into 40,647 intervals that exhibit no evidence of historical recombination. For each of these intervals we inferred a local phylogenetic tree. We combined these data with 12 million loci with sequence variations recently discovered by whole-genome sequencing in a common subset of 12 classical laboratory strains. For each phylogenetic tree we identified strains sharing a leaf node with one or more of the sequenced strains. We then imputed high- and medium-confidence genotypes for each of 88 nonsequenced genomes. Among inbred strains, we imputed 92% of SNPs genome-wide, with 71% in high-confidence regions. Our method produced 977 million new genotypes with an estimated per-SNP error rate of 0.083% in high-confidence regions and 0.37% genome-wide. Our analysis identified which of the 88 nonsequenced strains would be the most informative for improving full-genome imputation, as well as which additional strain sequences will reveal more new genetic variants. Imputed sequences and quality scores can be downloaded and visualized online.     

Tissue-specific effects of allergic rhinitis in mouse nasal epithelia

Allergic rhinitis (AR) can cause significant olfactory loss, but few studies have specifically investigated AR effects on olfactory and nasal respiratory tissues per se. To address this, we used a murine AR protocol employing nasal allergen infusion for both sensitization and challenges. Seven- to 11-week BALB/c mice were bilaterally infused with 1% ovalbumin (OVA) in phosphate-buffered saline (PBS) or PBS alone for 6 or 11 weeks, given single bilateral PBS or OVA infusions 24 h before sacrifice, or left untreated. High OVA-specific IgE serum levels and eosinophil infiltration confirmed AR induction. Olfactory (OE) and respiratory (RE) epithelia showed distinctly different responses, most conspicuously, massive eosinophil infiltration of immediately RE-subjacent lamina propria. In OE, such infiltration was minimal. Significant RE hypertrophy and hyperplasia also occurred, although OE organization was generally maintained and extensive disruption localized despite a 20% reduction in sensory neurons and globose basal cells after 11 weeks OVA. Pronounced Bowman's gland hypertrophy crowded both OE and olfactory nerve bundles. Cellular proliferation was widely distributed in RE but in OE was localized to normally thinner OE and RE-proximal OE, suggesting possible indirect RE influences. Terminal deoxynucleotide transferase (TdT) nick end labeling was greater in OE than RE and, in contrast to other effects, occurred with acute infusions and chronic PBS alone, often unilaterally. Following chronic OVA, AR-related bilateral increases appeared superimposed on those. These findings indicate AR effects on olfactory function may be complex, reflecting various levels of RE/OE responses and interactions.     

Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.     

Complement Inhibitors from Scabies Mites Promote Streptococcal Growth – A Novel Mechanism in Infected Epidermis?

Background

Scabies is highly prevalent in socially disadvantaged communities such as indigenous populations and in developing countries. Generalized itching causes discomfort to the patient; however, serious complications can occur as a result of secondary bacterial pyoderma, commonly caused by Streptococcus pyogenes (GAS) or Staphylococcus aureus. In the tropics, skin damage due to scabies mite infestations has been postulated to be an important link in the pathogenesis of disease associated with acute rheumatic fever and heart disease, poststreptococcal glomerulonephritis and systemic sepsis. Treatment of scabies decreases the prevalence of infections by bacteria. This study aims to identify the molecular mechanisms underlying the link between scabies and GAS infections.

Methodology/Principal Findings

GAS bacteria were pre-incubated with blood containing active complement, phagocytes and antibodies against the bacteria, and subsequently tested for viability by plate counts. Initial experiments were done with serum from an individual previously exposed to GAS with naturally acquired anti-GAS antibodies. The protocol was optimized for large-scale testing of low-opsonic whole blood from non-exposed human donors by supplementing with a standard dose of heat inactivated human sera previously exposed to GAS. This allowed an extension of the dataset to two additional donors and four proteins tested at a range of concentrations. Shown first is the effect of scabies mite complement inhibitors on human complement using ELISA-based complement activation assays. Six purified recombinant mite proteins tested at a concentration of 50 µg/ml blocked all three complement activation pathways. Further we demonstrate in human whole blood assays that each of four scabies mite complement inhibitors tested increased GAS survival rates by 2–15 fold.

Conclusions/Significance

We propose that local complement inhibition plays an important role in the development of pyoderma in scabies infested skin. This molecular link between scabies and bacterial infections may provide new avenues to develop alternative treatment options against this neglected disease.     

Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract.

Duchenne muscular dystrophy is characterized by the absence of dystrophin from muscle cells. Dystrophic muscle cells are susceptible to oxidative stress. We tested the hypothesis that 3 wk of endurance exercise starting at age 21 days in young male mdx mice would blunt oxidative stress and improve dystrophic skeletal muscle function, and these effects would be enhanced by the antioxidant green tea extract (GTE). In mice fed normal diet, average daily running distance increased 300% from week 1 to week 3, and total distance over 3 wk was improved by 128% in mice fed GTE. Running, independent of diet, increased serum antioxidant capacity, extensor digitorum longus tetanic stress, and total contractile protein content, heart citrate synthase, and heart and quadriceps beta-hydroxyacyl-CoA dehydrogenase activities. GTE, independent of running, decreased serum creatine kinase and heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate synthase activity. These data suggest that both endurance exercise and GTE may be beneficial as therapeutic strategies to improve muscle function in mdx mice.     

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K_i = 1.2 nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.     

Intraspecific variation in movement patterns: modeling individual behaviour in a large marine predator

In large marine predators, foraging entails movement. Quantitative models reveal how behaviours can mediate individual movement, such that deviations from a random pattern may reveal specific search tactics or behaviour. Using locations for 52 grey seals fitted with satellite-linked recorders on Sable Island; we modeled movement as a correlated random walk (CRW) for individual animals, at two temporal scales. Mean move length, turning angle, and net squared displacement (R²_n: the rate of change in area over time) at successive moves over 3 to 10 months were calculated. The distribution of move lengths of individual animals was compared to a Lévy distribution to determine if grey seals use a Lévy flight search tactic. Grey seals exhibited three types of movement as determined by CRW model fit: directed movers – animals displaying directed long distance travel that were significantly underpredicted by the CRW (23% of animals); residents – animals remaining in the area surrounding Sable Island that were overpredicted by the model (29% of animals); and correlated random walkers – those (48% of animals) in which movement was predicted by the CRW model. Kernel home range size differed significantly among all three movement types, as did travel speed, mean move length, mean R²_n and total distance traveled. Sex and season of deployment were significant predictors of movement type, with directed movers more likely to be male and residents more likely to be female. Only 30% of grey seals fit a Lévy distribution, which suggests that food patches used by the majority of seals are not randomly distributed. Intraspecific variation in movement behaviour is an important characteristic in grey seal foraging ecology, underscoring the need to account for such variability in developing models of habitat use and predation.