Potential novel candidate polymorphisms identified in genome-wide association study for breast cancer susceptibility

Previous genome-wide association studies (GWAS) have shown several risk alleles to be associated with breast cancer. However, the variants identified so far contribute to only a small proportion of disease risk. The objective of our GWAS was to identify additional novel breast cancer susceptibility variants and to replicate these findings in an independent cohort. We performed a two-stage association study in a cohort of 3,064 women from Alberta, Canada. In Stage I, we interrogated 906,600 single nucleotide polymorphisms (SNPs) on Affymetrix SNP 6.0 arrays using 348 breast cancer cases and 348 controls. We used single-locus association tests to determine statistical significance for the observed differences in allele frequencies between cases and controls. In Stage II, we attempted to replicate 35 significant markers identified in Stage I in an independent study of 1,153 cases and 1,215 controls. Genotyping of Stage II samples was done using Sequenom Mass-ARRAY iPlex platform. Six loci from four different gene regions (chromosomes 4, 5, 16 and 19) showed statistically significant differences between cases and controls in both Stage I and Stage II testing, and also in joint analysis. The identified variants were from EDNRA, ROPN1L, C16orf61 and ZNF577 gene regions. The presented joint analyses from the two-stage study design were not significant after genome-wide correction. The SNPs identified in this study may serve as potential candidate loci for breast cancer risk in a further replication study in Stage III from Alberta population or independent validation in Caucasian cohorts elsewhere.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Independent evolution of functional MHC class II DRB genes in New World bat species

Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressed MHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.     

Accurate,Fully-Automated NMR Spectral Profiling for Metabolomics

Many diseases cause significant changes to the concentrations of small molecules (a.k.a. metabolites) that appear in a person’s biofluids, which means such diseases can often be readily detected from a person’s “metabolic profile"—i.e., the list of concentrations of those metabolites. This information can be extracted from a biofluids Nuclear Magnetic Resonance (NMR) spectrum. However, due to its complexity, NMR spectral profiling has remained manual, resulting in slow, expensive and error-prone procedures that have hindered clinical and industrial adoption of metabolomics via NMR. This paper presents a system, BAYESIL, which can quickly, accurately, and autonomously produce a person’s metabolic profile. Given a 1D ¹H NMR spectrum of a complex biofluid (specifically serum or cerebrospinal fluid), BAYESIL can automatically determine the metabolic profile. This requires first performing several spectral processing steps, then matching the resulting spectrum against a reference compound library, which contains the “signatures” of each relevant metabolite. BAYESIL views spectral matching as an inference problem within a probabilistic graphical model that rapidly approximates the most probable metabolic profile. Our extensive studies on a diverse set of complex mixtures including real biological samples (serum and CSF), defined mixtures and realistic computer generated spectra; involving > 50 compounds, show that BAYESIL can autonomously find the concentration of NMR-detectable metabolites accurately (~ 90% correct identification and ~ 10% quantification error), in less than 5 minutes on a single CPU. These results demonstrate that BAYESIL is the first fully-automatic publicly-accessible system that provides quantitative NMR spectral profiling effectively—with an accuracy on these biofluids that meets or exceeds the performance of trained experts. We anticipate this tool will usher in high-throughput metabolomics and enable a wealth of new applications of NMR in clinical settings. BAYESIL is accessible at http://www.bayesil.ca.     

Translocations of Chromosome End-Segments and Facultative Heterochromatin Promote Meiotic Ring Formation in Evening Primroses

Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled end-segments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories.     

Structural and Biophysical Characterization of the Syk Activation Switch

Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.     

Caste-specific visual adaptations to distinct daily activity schedules in Australian Myrmecia ants

Animals are active at different times of the day and their activity schedules are shaped by competition, time-limited food resources and predators. Different temporal niches provide different light conditions, which affect the quality of visual information available to animals, in particular for navigation. We analysed caste-specific differences in compound eyes and ocelli in four congeneric sympatric species of Myrmecia ants, with emphasis on within-species adaptive flexibility and daily activity rhythms. Each caste has its own lifestyle: workers are exclusively pedestrian; alate females lead a brief life on the wing before becoming pedestrian; alate males lead a life exclusively on the wing. While workers of the four species range from diurnal, diurnal-crepuscular, crepuscular-nocturnal to nocturnal, the activity times of conspecific alates do not match in all cases. Even within a single species, we found eye area, facet numbers, facet sizes, rhabdom diameters and ocelli size to be tuned to the distinct temporal niche each caste occupies. We discuss these visual adaptations in relation to ambient light levels, visual tasks and mode of locomotion.