Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Membrane localization diversity of TPK channels and their physiological role

Potassium (K) is one of the major nutrients that is essential for plant growth and development. The majority of cellular K⁺ resides in the vacuole and tonoplast K⁺ channels of the TPK (Two Pore K) family are main players in cellular K⁺ homeostasis. All TPK channels were previously reported to be expressed in the tonoplast of the large central lytic vacuole (LV) except for one isoform in Arabidopsis that resides in the plasma membrane. However, plant cells often contain more than one type of vacuole that coexist in the same cell. We recently showed that two TPK isoforms (OsTPKa and OsTPKb) from Oryza sativa localize to different vacuoles with OsTPKa predominantly found in the LV tonoplast and OsTPKb primarily in smaller compartments that resemble small vacuoles (SVs). Our study further revealed that it is the C-terminal domain that determines differential targeting of OsTPKa and OsTPKb. Three C-terminal amino acids were particularly relevant for targeting TPKs to their respective endomembranes. In this addendum we further evaluate how the different localization of TPKa and TPKb impact on their physiological role and how TPKs provide a potential tool to study the physiology of different types of vacuole.Key words: TPK channels, small vacuoles, vacuolar targeting, potassiumThe roles of plant vacuolar K⁺ channels are diverse and include potassium homeostasis, turgor regulation and responses to abiotic stress. Vacuolar K⁺-selective channels belong to two-pore K⁺ (TPK) channel families which have been found in genomes of many plant species such as Arabidopsis, poplar, Physcomitrella, Eucalyptus, barley, potato, rice and tobacco (Fig. 1). TPKs have structural similarity to mammalian “tandem P domain” channels with a secondary structure that contains four transmembrane domains and two pore regions (Fig. 2).^1–5 TPK channels have pore regions with a GYGD signature that endows K⁺ selectivity and a variable number of Ca²⁺ binding EF domains in the C terminus.^3–8 One of the best characterized members of the TPK family is AtTPK1 from Arabidopsis thaliana. AtTPK1 activity is voltage independent but sensitive to cytosolic Ca²⁺, cytosolic pH and N-terminal phosphorylation by 14-3-3 proteins.^5,6,8,9 In Arabidopsis, AtTPK1 expresses in the large lytic vacuole (LV) and plays roles in cellular K⁺ homeostasis, K⁺-release during stomatal closure and seed germination.^4,5 Other members of the Arabidopsis TPK family (AtTPK2, AtTPK3, AtTPK5) have been shown to localize to the LV but also showed some expression in smaller, vesicle-like, compartments.⁴ However, none of these isoforms appears to form functional channels in planta although our experiments with heterologous expression of AtTPK3 and AtTPK5 in the K⁺ uptake deficient E. coli LB2003 demonstrates complementation of bacterial growth phenotype (Isayenkov S, et al. unpublished results). Equally intriguing, is the plasma membrane localization of the Arabidopsis TPK4 isoform, in spite of its sequence being very similar to that of other TPKs.¹⁰Open in a separate windowFigure 1Phylogenetic tree of plant TPKs. The three main clusters of TPKs comprise: Cluster 1 with AtTPK1-like channels; Cluster 2 with AtTPK3/TPK5-like channels; Cluster 3 with barley HvTPKb. Bootstrap analysis was performed using ‘Molecular Evolutionary Genetics Analysis, MEGA4’ software available at www.megasoftware.net/mega4/megaOpen in a separate windowFigure 2Two-pore potassium channel secondary structure. TPK channels comprise four transmembrane domains (1–4) and two pore regions (P) per subunit. Functional channels are formed from two subunits. In most TPKs, both P regions contain a K⁺ selectivity signature, GYGD. However, the tobacco NtTPKa isoform has different motifs in the second P domain. In the N terminal region, TPKs have a 14-3-3 binding domain that impact on channel activity, with the binding of 14-3-3 protein leading to channel activation. C-termini of TPKs show a varying number of putative Ca²⁺ binding “EF hands” which may vary from zero to two.     

High host‐plant nitrogen content: a prerequisite for the evolution of ant–caterpillar mutualism?

The amount of nitrogen required to complete an insect's life cycle may vary greatly among species that have evolved distinct life history traits. Myrmecophilous caterpillars in the Lycaenidae family produce nitrogen-rich exudates from their dorsal glands to attract ants for protection, and this phenomenon has been postulated to shape the caterpillar's host-plant choice. Accordingly, it was postulated that evolution towards myrmecophily in Lycaenidae is correlated with the utilization of nitrogen-rich host plants. Although our results were consistent with the evolutionary shifts towards high-nutrient host plants serving as exaptation for the evolution of myrmecophily in lycaenids, the selection of nitrogen-rich host plants was not confined to lycaenids. Butterfly species in the nonmyrmecophilous family Pieridae also preferred nitrogen-rich host plants. Thus, we conclude that nitrogen is an overall important component in the caterpillar diet, independent of the level of myrmecophily, as nitrogen can enhance the overall insect fitness and survival. However, when nitrogen can be obtained through alternative means, as in socially parasitic lycaenid species feeding on ant brood, the selective pressure for maintaining the use of nutrient-rich host plants is relaxed, enabling the colonization of nitrogen-poor host plants.     

Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis

Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3⁺ T cells, CD38⁺ plasma cells and CD68⁺ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland–Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials.     

Climate‐based empirical models show biased predictions of butterfly communities along environmental gradients

A better understanding of the factors that mould ecological community structure is required to accurately predict community composition and to anticipate threats to ecosystems due to global changes. We tested how well stacked climate‐based species distribution models (S‐SDMs) could predict butterfly communities in a mountain region. It has been suggested that climate is the main force driving butterfly distribution and community structure in mountain environments, and that, as a consequence, climate‐based S‐SDMs should yield unbiased predictions. In contrast to this expectation, at lower altitudes, climate‐based S‐SDMs overpredicted butterfly species richness at sites with low plant species richness and underpredicted species richness at sites with high plant species richness. According to two indices of composition accuracy, the Sorensen index and a matching coefficient considering both absences and presences, S‐SDMs were more accurate in plant‐rich grasslands. Butterflies display strong and often specialised trophic interactions with plants. At lower altitudes, where land use is more intense, considering climate alone without accounting for land use influences on grassland plant richness leads to erroneous predictions of butterfly presences and absences. In contrast, at higher altitudes, where climate is the main force filtering communities, there were fewer differences between observed and predicted butterfly richness. At high altitudes, even if stochastic processes decrease the accuracy of predictions of presence, climate‐based S‐SDMs are able to better filter out butterfly species that are unable to cope with severe climatic conditions, providing more accurate predictions of absences. Our results suggest that predictions should account for plants in disturbed habitats at lower altitudes but that stochastic processes and heterogeneity at high altitudes may limit prediction success of climate‐based S‐SDMs.