Alternatives to animal testing in the safety evaluation of products

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.     

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> adherence to human cells

Background  

Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.     

Primary antibody-forming cells and secondary B cells are generated from separate precursor cell subpopulations

Two precursor cell subpopulations have been isolated from the spleen cells of nonimmune mice. The major B cell subpopulation binds high levels of the J11D monoclonal antibody and, upon T cell-dependent antigenic stimulation, gives rise to primary antibody-forming cell clones but not secondary B cells. A minority of the 10%-14% of Ia+ precursors that bind low levels of J11D (J11Dlo) also generate antibody-forming cell clones after primary stimulation. However, over 70% of J11Dlo precursors yield no primary antibody-forming cell clones but instead give rise to secondarily responsive B cells. The existence of a distinct precursor cell subpopulation that is responsible for the generation of B cell memory is further evidenced by the distribution of variable region clonotypes among J11Dlo primary precursors, which resembles the clonotype patterns of secondary B cells, and by the accumulation of somatic mutations in their clonal progeny.     

Penalized Multimarker vs. Single-Marker Regression Methods for Genome-Wide Association Studies of Quantitative Traits

The data from genome-wide association studies (GWAS) in humans are still predominantly analyzed using single-marker association methods. As an alternative to single-marker analysis (SMA), all or subsets of markers can be tested simultaneously. This approach requires a form of penalized regression (PR) as the number of SNPs is much larger than the sample size. Here we review PR methods in the context of GWAS, extend them to perform penalty parameter and SNP selection by false discovery rate (FDR) control, and assess their performance in comparison with SMA. PR methods were compared with SMA, using realistically simulated GWAS data with a continuous phenotype and real data. Based on these comparisons our analytic FDR criterion may currently be the best approach to SNP selection using PR for GWAS. We found that PR with FDR control provides substantially more power than SMA with genome-wide type-I error control but somewhat less power than SMA with Benjamini–Hochberg FDR control (SMA-BH). PR with FDR-based penalty parameter selection controlled the FDR somewhat conservatively while SMA-BH may not achieve FDR control in all situations. Differences among PR methods seem quite small when the focus is on SNP selection with FDR control. Incorporating linkage disequilibrium into the penalization by adapting penalties developed for covariates measured on graphs can improve power but also generate more false positives or wider regions for follow-up. We recommend the elastic net with a mixing weight for the Lasso penalty near 0.5 as the best method.     

The complete mitochondrial DNA sequence of the shark Mustelus manazo: evaluating rooting contradictions to living bony vertebrates

A remarkable example of a misleading mitochondrial protein tree is presented, involving ray-finned fishes, coelacanths, lungfishes, and tetrapods, with sea lampreys as an outgroup. In previous molecular phylogenetic studies on the origin of tetrapods, ray-finned fishes have been assumed as an outgroup to the tetrapod/lungfish/coelacanth clade, an assumption supported by morphological evidence. Standard methods of molecular phylogenetics applied to the protein-encoding genes of mitochondria, however, give a bizarre tree in which lamprey groups with lungfish and, therefore, ray-finned fishes are not the outgroup to a tetrapod/lungfish/coelacanth clade. All of the dozens of published phylogenetic methods, including every possible modification to maximum likelihood known to us (such as inclusion of site heterogeneity and exclusion of potentially misleading hydrophobic amino acids), fail to place the ray-finned fishes in a biologically acceptable position. A likely cause of this failure may be the use of an inappropriate outgroup. Accordingly, we have determined the complete mitochondrial DNA sequence from the shark, Mustelus manazo, which we have used as an alternative and more proximal outgroup than the lamprey. Using sharks as the outgroup, lungfish appear to be the closest living relative of tetrapods, although the possibility of a lungfish/coelacanth clade being the sister group of tetrapods cannot be excluded.