Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer

Consistency Analysis of Redundant Probe Sets on Affymetrix Three-Prime Expression Arrays and Applications to Differential mRNA Processing

Affymetrix three-prime expression microarrays contain thousands of redundant probe sets that interrogate different regions of the same gene. Differential expression analysis methods rarely consider probe redundancy, which can lead to inaccurate inference about overall gene expression or cause investigators to overlook potentially valuable information about differential regulation of variant mRNA products. We investigated the behaviour and consistency of redundant probe sets in a publicly-available data set containing samples from mouse brain amygdala and hippocampus and asked how applying filtering methods to the data affected consistency of results obtained from redundant probe sets. A genome-based filter that screens and groups probe sets according to their overlapping genomic alignments significantly improved redundant probe set consistency. Screening based on qualitative Present-Absent calls from MAS5 also improved consistency. However, even after applying these filters, many redundant probe sets showed significant fold-change differences relative to each other, suggesting differential regulation of alternative transcript production. Visual inspection of these loci using an interactive genome visualization tool (igb.bioviz.org) exposed thirty putative examples of differential regulation of alternative splicing or polyadenylation across brain regions in mouse. This work demonstrates how P/A-call and genome-based filtering can improve consistency among redundant probe sets while at the same time exposing possible differential regulation of RNA processing pathways across sample types

On the Wegener granulomatosis associated region on chromosome 6p21.3

BACKGROUND: Wegener granulomatosis (WG) belongs to the heterogeneous group of systemic vasculitides. The multifactorial pathophysiology of WG is supposedly caused by yet unknown environmental influence(s) on the basis of genetic predisposition. The presence of anti-neutrophil cytoplasmic antibodies (ANCA) in the plasma of patients and genetic involvement of the human leukocyte antigen system reflect an autoimmune background of the disease. Strong associations were revealed with WG by markers located in the major histocompatibility complex class II (MHC II) region in the vicinity of human leukocyte antigen (HLA)-DPB1 and the retinoid X receptor B (RXRB) loci. In order to define the involvement of the 6p21.3 region in WG in more detail this previous population-based association study was expanded here to the respective 3.6 megabase encompassing this region on chromosome 6. The RXRB gene was analysed as well as a splice-site variation of the butyrophilin-like (BTNL2) gene which is also located within the respective region. The latter polymorphism has been evaluated here as it appears as a HLA independent susceptibility factor in another granulomatous disorder, sarcoidosis. METHODS: 150–180 German WG patients and a corresponding cohort of healthy controls (n = 100–261) were used in a two-step study. A panel of 94 microsatellites was designed for the initial step using a DNA pooling approach. Markers with significantly differing allele frequencies between patient and control pools were individually genotyped. The RXRB gene was analysed for single strand conformation polymorphisms (SSCP) and restriction fragment length polymorphisms (RFLP). The splice-site polymorphism in the BTNL2 gene was also investigated by RFLP analysis. RESULTS: A previously investigated microsatellite (#1.0.3.7, Santa Cruz genome browser (UCSC) May 2004 Freeze localisation: chr6:31257596-34999883), which was used as a positive control, remained associated throughout the whole two-step approach. Yet, no additional evidence for association of other microsatellite markers was found in the entire investigated region. Analysis of the RXRB gene located in the WG associated region revealed associations of two variations (rs10548957 p(allelic )= 0.02 and rs6531 p(allelic )= 5.20 × 10(-5), OR = 1.88). Several alleles of markers located between HLA-DPB1, SNP rs6531 and microsatellite 1.0.3.7 showed linkage disequilibrium with r(2 )values exceeding 0.10. Significant differences were not demonstrable for the sarcoidosis associated splice-site variation (rs2076530 p(allelic )= 0.80) in our WG cohort. CONCLUSION: Since a microsatellite flanking the RXRB gene and two intragenic polymorphisms are associated significantly with WG on chromosome 6p21.3, further investigations should be focussed on extensive fine-mapping in this region by densely mapping with additional markers such as SNPs. This strategy may reveal even deeper insights into the genetic contributions of the respective region for the pathogenesis of WG

Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Experiments in Globalisation, Food Security and Land Use Decision Making

The globalisation of trade affects land use, food production and environmentsaround the world. In principle, globalisation can maximise productivity andefficiency if competition prompts specialisation on the basis of productive capacity.In reality, however, such specialisation is often constrained by practical or politicalbarriers, including those intended to ensure national or regional food security.These are likely to produce globally sub-optimal distributions of land uses. Bothoutcomes are subject to the responses of individual land managers to economicand environmental stimuli, and these responses are known to be variable and often(economically) irrational. We investigate the consequences of stylised food securitypolicies and globalisation of agricultural markets on land use patterns under avariety of modelled forms of land manager behaviour, including variation inproduction levels, tenacity, land use intensity and multi-functionality. We find that asystem entirely dedicated to regional food security is inferior to an entirelyglobalised system in terms of overall production levels, but that several forms ofbehaviour limit the difference between the two, and that variations in land useintensity and functionality can substantially increase the provision of food and otherecosystem services in both cases. We also find emergent behaviour that results inthe abandonment of productive land, the slowing of rates of land use change andthe fragmentation or, conversely, concentration of land uses following changes indemand levels

European agricultural landscapes, common agricultural policy and ecosystem services: a review

Since the 1950s, intensification and scale enlargement of agriculture have changed agricultural landscapes across Europe. The intensification and scale enlargement of farming was initially driven by the large-scale application of synthetic fertilizers, mechanization and subsidies of the European Common Agricultural Policy (CAP). Then, after the 1990s, a further intensification and scale enlargement, and land abandonment in less favored areas was caused by globalization of commodity markets and CAP reforms. The landscape changes during the past six decades have changed the flows and values of ecosystem services. Here, we have reviewed the literature on agricultural policies and management, landscape structure and composition, and the contribution of ecosystem services to regional competitiveness. The objective was to define an analytical framework to determine and assess ecosystem services at the landscape scale. In contrast to natural ecosystems, ecosystem service flows and values in agricultural landscapes are often a result of interactions between agricultural management and ecological structures. We describe how land management by farmers and other land managers relates to landscape structure and composition. We also examine the influence of commodity markets and policies on the behavior of land managers. Additionally, we studied the influence of consumer demand on flows and values of the ecosystem services that originate from the agricultural landscape