Mutation discovery in the mouse using genetically guided array capture and resequencing

Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (~200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. M. D’Ascenzo and C. Meacham contributed equally to this work.     

School Variation in Asthma: Compositional or Contextual?

Background

Childhood asthma prevalence and morbidity have been shown to vary by neighborhood. Less is known about between-school variation in asthma prevalence and whether it exists beyond what one might expect due to students at higher risk of asthma clustering within different schools. Our objective was to determine whether between-school variation in asthma prevalence exists and if so, if it is related to the differential distribution of individual risk factors for and correlates of asthma or to contextual influences of schools.

Methods

Cross-sectional analysis of 16,640 teens in grades 7–12 in Wave 1 (data collected in 1994–5) of the National Longitudinal Study of Adolescent Health. Outcome was current diagnosis of asthma as reported by respondents'' parents. Two-level random effects models were used to assess the contribution of schools to the variance in asthma prevalence before and after controlling for individual attributes.

Results

The highest quartile schools had mean asthma prevalence of 21.9% compared to the lowest quartile schools with mean asthma prevalence of 7.1%. In our null model, the school contributed significantly to the variance in asthma ( = 0.27, CI: 0.20, 0.35). Controlling for individual, school and neighborhood attributes reduced the between-school variance modestly ( = 0.19 CI: 0.13–0.29).

Conclusion

Significant between-school variation in current asthma prevalence exists even after controlling for the individual, school and neighborhood factors. This provides evidence for school level contextual influences on asthma. Further research is needed to determine potential mechanisms through which schools may influence asthma outcomes.     

Maize Inbreds Exhibit High Levels of Copy Number Variation (CNV) and Presence/Absence Variation (PAV) in Genome Content

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.     

Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 1

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to target focal adhesion kinase (FAK). A number of these pyrrolopyrimides exhibited low micromolar inhibitory activities against focal adhesion kinase, and their preliminary SAR was established via systematic chemical modifications. The 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines represent a new class of kinase inhibitors.     

TLR2 Mediates Recognition of Live Staphylococcus epidermidis and Clearance of Bacteremia

Background

Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown.

Methodology/Principal Findings

We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24–48 h) were markedly impaired in TLR2-deficient mice.

Conclusions/Significance

TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.     

Neurexin in Embryonic Drosophila Neuromuscular Junctions

Background

Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described.

Methodology/Principal Findings

We use a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). Our results surprisingly suggest that neurexin in embryos is present both pre and postsynaptically. Presynaptic neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, we find that loss of neurexin only affects receptors containing the subunit GluRIIA.

Conclusions/Significance

Our study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and our results provide new insights into the role of neurexin in synapse development.     

A finite element analysis of masticatory stress hypotheses

Understanding how the skull transmits and dissipates forces during feeding provides insights into the selective pressures that may have driven the evolution of primate skull morphology. Traditionally, researchers have interpreted masticatory biomechanics in terms of simple global loading regimes applied to simple shapes (i.e., bending in sagittal and frontal planes, dorsoventral shear, and torsion of beams and cylinders). This study uses finite element analysis to examine the extent to which these geometric models provide accurate strain predictions in the face and evaluate whether simple global loading regimes predict strains that approximate the craniofacial deformation pattern observed during mastication. Loading regimes, including those simulating peak loads during molar chewing and those approximating the global loading regimes, were applied to a previously validated finite element model (FEM) of a macaque (Macaca fascicularis) skull, and the resulting strain patterns were compared. When simple global loading regimes are applied to the FEM, the resulting strains do not match those predicted by simple geometric models, suggesting that these models fail to generate accurate predictions of facial strain. Of the four loading regimes tested, bending in the frontal plane most closely approximates strain patterns in the circumorbital region and lateral face, apparently due to masseter muscle forces acting on the zygomatic arches. However, these results indicate that no single simple global loading regime satisfactorily accounts for the strain pattern found in the validated FEM. Instead, we propose that FE models replace simple cranial models when interpreting bone strain data and formulating hypotheses about craniofacial biomechanics.     

Making human nasal cilia beat in the cold: a real time assay for cell signalling

Human nasal epithelium must adapt to cold climates, and yet, in vitro, human ciliary beat frequency (CBF) is zero at 4 degrees C. Similarly, hibernating mammals do not die of pneumonia despite a core body temperature as low as 6 degrees C, implying that cilia continue to beat. Here, we show that protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) regulate the profile of human nasal CBF in response to rising temperature from 4 degrees C. Onset of ciliary beat was at 10 degrees C in Medium 199, 7 degrees C in the presence of the PKC activator phorbol 12-myristate 13-acetate (PMA), the calcium ionophore ionomycin, or the CAMKII blocker myristoylated autocamtide-2 related inhibitory peptide (MACI), and 6 degrees C for the myristoylated peptide PKC inhibitor EGF-R Fragment 651-658 (MyrPKCI). During cell warming to 32 degrees C, the thermal profile was sigmoid in all solutions except those containing MACI+PMA. Surprisingly, cilia continued to beat despite 4 degrees C and were significantly more responsive to rising temperature with either MACI+PMA, or MACI+MyrPKCI. Our data suggest that CaMKII and PKC regulate the thermal slope and profile of CBF in vitro, and that when these protein kinases are manipulated, cilia can continue to beat despite hypothermia. These findings may relate to adaptive responses to cold climates.     

The Association of Early Childhood Cognitive Development and Behavioural Difficulties with Pre-Adolescent Problematic Eating Attitudes

Objectives

Few studies have prospectively investigated associations of child cognitive ability and behavioural difficulties with later eating attitudes. We investigated associations of intelligence quotient (IQ), academic performance and behavioural difficulties at 6.5 years with eating attitudes five years later.

Methods

We conducted an observational cohort study nested within the Promotion of Breastfeeding Intervention Trial, Belarus. Of 17,046 infants enrolled at birth, 13,751 (80.7%) completed the Children''s Eating Attitude Test (ChEAT) at 11.5 years, most with information on IQ (n = 12,667), academic performance (n = 9,954) and behavioural difficulties (n = 11,098) at 6.5 years. The main outcome was a ChEAT score ≥85^th percentile, indicative of problematic eating attitudes.

Results

Boys with higher IQ at 6.5 years reported fewer problematic eating attitudes, as assessed by ChEAT scores ≥85^th percentile, at 11.5 years (OR per SD increase in full-scale IQ = 0.87; 0.79, 0.94). No such association was observed in girls (1.01; 0.93, 1.10) (p for sex-interaction = 0.016). In both boys and girls, teacher-assessed academic performance in non-verbal subjects was inversely associated with high ChEAT scores five years later (OR per unit increase in mathematics ability = 0.88; 0.82, 0.94; and OR per unit increase in ability for other non-verbal subjects = 0.86; 0.79, 0.94). Behavioural difficulties were positively associated with high ChEAT scores five years later (OR per SD increase in teacher-assessed rating = 1.13; 1.07, 1.19).

Conclusion

Lower IQ, worse non-verbal academic performance and behavioural problems at early school age are positively associated with risk of problematic eating attitudes in early adolescence.