Multivariate multiple test procedures based on nonparametric copula estimation

Multivariate multiple test procedures have received growing attention recently. This is due to the fact that data generated by modern applications typically are high‐dimensional, but possess pronounced dependencies due to the technical mechanisms involved in the experiments. Hence, it is possible and often necessary to exploit these dependencies in order to achieve reasonable power. In the present paper, we express dependency structures in the most general manner, namely, by means of copula functions. One class of nonparametric copula estimators is constituted by Bernstein copulae. We extend previous statistical results regarding bivariate Bernstein copulae to the multivariate case and study their impact on multiple tests. In particular, we utilize them to derive asymptotic confidence regions for the family‐wise error rate (FWER) of multiple test procedures that are empirically calibrated by making use of Bernstein copulae approximations of the dependency structure among the test statistics. This extends a similar approach by Stange et al. (2015) in the parametric case. A simulation study quantifies the gain in FWER level exhaustion and, consequently, power that can be achieved by exploiting the dependencies, in comparison with common threshold calibrations like the Bonferroni or ?idák corrections. Finally, we demonstrate an application of the proposed methodology to real‐life data from insurance.     

Metabolome analysis of biosynthetic mutants reveals a diversity of metabolic changes and allows identification of a large number of new compounds in Arabidopsis

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.     

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.     

A Holocene sequence of vegetation change at Lake Eteza, coastal KwaZulu-Natal, South Africa

Palynological and sedimentological data from a core extracted from Lake Eteza shed new light on the Holocene vegetation and climate history in KwaZulu-Natal and can be linked to regional and global climate change. A 2072 cm core with nineteen radiocarbon dates and chronological extrapolation to the bottom of the sequence suggests that sedimentation started ca. 10 200 cal yrs BP. Between ca. 10 200 and 6800 cal yrs BP pollen indicators point to a change from intermediately humid conditions to comparatively drier grassy environments. This is in good agreement with Sea Surface Temperature (SST) fluctuations from a core in the Mozambique Channel which influence precipitation in coastal KwaZulu-Natal, and the beginning of the Holocene Thermal Maximum ca. 10 500 cal yrs BP. The lower section of the core corresponds to gradually increasing Holocene sea levels along the coast and development of freshwater or estuarine conditions at Lake Eteza. The middle Holocene (ca. 6800-3600 cal yrs BP), when the sea level reached its highest stand and SST peak, indicate humid climatic conditions that favoured an increase of forest trees, e.g. Podocarpus, and undergrowth plants like Issoglossa. As a consequence of higher precipitation and increase of the water table, conditions were favourable for the spread of mangrove, swamp and possibly riverine forest. During the late Holocene after ca. 3600 cal yrs BP a decrease of Podocarpus and other trees as well as an increase of Chenopodiaceae/Amaranthaceae, grasses and Phoenix coincide with a return to lower sea levels and drier conditions. The decrease of all trees including Phoenix at ca. 700 cal yrs BP, accompanied by rapid sedimentation rates, possibly reflect forest clearing and upland erosion induced by activities of Iron Age settlers. A dry period at the globally recognized onset of the Little Ice Age might have contributed to these changes. Late Iron Age settlers have probably already introduced Zea mays, which was detected in the profile since ca. 210 BP. The appearance of neophytes like Pinus, Casuarina and pollen of Ambrosia-type in the youngest sediments indicates increased disturbance of European settlements and land use since ca. 100 cal yrs BP.     

Examination of Apoptosis Signaling in Pancreatic Cancer by Computational Signal Transduction Analysis

Background

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of cancer death. Changes in apoptosis signaling in pancreatic cancer result in chemotherapy resistance and aggressive growth and metastasizing. The aim of this study was to characterize the apoptosis pathway in pancreatic cancer computationally by evaluation of experimental data from high-throughput technologies and public data bases. Therefore, gene expression analysis of microdissected pancreatic tumor tissue was implemented in a model of the apoptosis pathway obtained by computational protein interaction prediction.

Methodology/Principal Findings

Apoptosis pathway related genes were assembled from electronic databases. To assess expression of these genes we constructed a virtual subarray from a whole genome analysis from microdissected native tumor tissue. To obtain a model of the apoptosis pathway, interactions of members of the apoptosis pathway were analysed using public databases and computational prediction of protein interactions. Gene expression data were implemented in the apoptosis pathway model. 19 genes were found differentially expressed and 12 genes had an already known pathophysiological role in PDAC, such as Survivin/BIRC5, BNIP3 and TNF-R1. Furthermore we validated differential expression of IL1R2 and Livin/BIRC7 by RT-PCR and immunohistochemistry. Implementation of the gene expression data in the apoptosis pathway map suggested two higher level defects of the pathway at the level of cell death receptors and within the intrinsic signaling cascade consistent with references on apoptosis in PDAC. Protein interaction prediction further showed possible new interactions between the single pathway members, which demonstrate the complexity of the apoptosis pathway.

Conclusions/Significance

Our data shows that by computational evaluation of public accessible data an acceptable virtual image of the apoptosis pathway might be given. By this approach we could identify two higher level defects of the apoptosis pathway in PDAC. We could further for the first time identify IL1R2 as possible candidate gene in PDAC.     

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.     

Inactivation of template-directed misfolding of infectious prion protein by ozone

Misfolded prions (PrP(Sc)) are well known for their resistance to conventional decontamination processes. The potential risk of contamination of the water environment, as a result of disposal of specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the water industry for inactivation of microbial contaminants and was tested in this study for its ability to inactivate prions (263K hamster scrapie = PrP(Sc)). Treatment variables included initial ozone dose (7.6 to 25.7 mg/liter), contact time (5 s and 5 min), temperature (4°C and 20°C), and pH (pH 4.4, 6.0, and 8.0). Exposure of dilute suspensions of the infected 263K hamster brain homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating properties of PrP(Sc), as measured by the protein misfolding cyclic amplification (PMCA) assay. The highest levels of prion inactivation (≥4 log(10)) were observed with ozone doses of 13.0 mg/liter, at pH 4.4 and 20°C, resulting in a CT (the product of residual ozone concentration and contact time) value as low as 0.59 mg · liter(-1) min. A comparison of ozone CT requirements among various pathogens suggests that prions are more susceptible to ozone degradation than some model bacteria and protozoa and that ozone treatment may be an effective solution for inactivating prions in water and wastewater.