Netherlands Twin Register: a focus on longitudinal research.

In 1986 we began The Netherlands Twin Register (NTR) by recruiting young twins and multiples a few weeks or months after birth. Currently we register around 50% of all newborn multiples in The Netherlands. Their parents receive a questionnaire at registration and afterwards when the children are 2, 3, 5, 7, 10 and 12 years of age. Teachers are asked to rate the behavior of the children at ages 7, 10 and 12 years. Adolescent and young-adult twins were recruited through City Councils in the early 1990s. These twins, their parents and siblings participate in longitudinal survey studies that include items about health, fertility, lifestyle, addiction, personality and psychopathology, religion, socioeconomic status, and educational attainment. The total number of twins and multiples registered with the NTR is currently over 60,000. Subgroups of twins and siblings take part in studies of cognitive development, brain function and neuropsychological indices of attention processes, and molecular genetic studies of classical and behavioral cardiovascular risk factors. DNA samples are currently collected in selected twin families for two large linkage studies, which aim to find QTLs for anxious depression and for nicotine addiction. Sisters who are mothers of DZ twins contribute DNA samples for a linkage study of DZ twinning. Large cohorts of phenotyped family members from the general population are very valuable for genetic epidemiological studies and permit selection of informative families for gene finding studies.     

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.     

Improving the safety of a conditional-live human immunodeficiency virus type 1 vaccine by controlling both gene expression and cell entry

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because faster-replicating pathogenic virus variants may evolve after vaccination. We previously presented a conditional-live HIV-1 variant of which replication can be switched off as an alternative vaccination strategy. To improve the safety of such a vaccine, we constructed a new HIV-1 variant that depends not only on doxycycline for gene expression but also on the T20 peptide for cell entry. Replication of this virus can be limited to the level required to induce the immune system by transient administration of doxycycline and T20. Subsequent withdrawal of these inducers efficiently blocks viral replication and evolution.     

Skeletal muscle stem cells

Satellite cells are myogenic stem cells responsible for the post-natal growth, repair and maintenance of skeletal muscle. This review focuses on the basic biology of the satellite cell with emphasis on its role in muscle repair and parallels between embryonic myogenesis and muscle regeneration. Recent advances have altered the long-standing view of the satellite cell as a committed myogenic stem cell derived directly from the fetal myoblast. The experimental basis for this evolving perspective will be highlighted as will the relationship between the satellite cell and other newly discovered muscle stem cell populations. Finally, advances and prospects for cell-based therapies for muscular dystrophies will be addressed.     

Microsatellites and 16S sequences corroborate phenotypic evidence of trans-Andean variation in the parasitoid Microctonus hyperodae (Hymenoptera: Braconidae)

Eight South American geographical populations of the parasitoid Microctonus hyperodae Loan were collected in South America (Argentina, Brazil, Chile and Uruguay) and released in New Zealand for biological control of the weevil Listronotus bonariensis (Kuschel), a pest of pasture grasses and cereals. DNA sequencing (16S, COI, 28S, ITS1, beta-tubulin), RAPD, AFLP, microsatellite, SSCP and RFLP analyses were used to seek markers for discriminating between the South American populations. All of the South American populations were more homogeneous than expected. However, variation in microsatellites and 16S gene sequences corroborated morphological, allozyme and other phenotypic evidence of trans-Andes variation between the populations. The Chilean populations were the most genetically variable, while the variation present on the eastern side of the Andes mountains was a subset of that observed in Chile.     

Dam inactivation in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>: prevalence among diverse hyperinvasive lineages

Background  

DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of Neisseria meningitidis, as a consequence of the insertion of a restriction endonuclease-encoding gene, the 'dam replacing gene' (drg) at the dam locus. Here, we report the results of a survey to assess the prevalence of drg in a globally representative panel of disease-associated meningococci.     

Moroccan specimens of Microctonus aethiopoides spice our understanding of genetic variation in this internationally important braconid parasitoid of adult weevils

Microctonus aethiopoides Loan (Hymenoptera: Braconidae) was introduced from Morocco to Australia and New Zealand for biological control of the lucerne pest, Sitona discoideus. Previous research has indicated that M. aethiopoides intraspecific genetic variation is more strongly associated with weevil host species than geographic origin. Cytochrome c oxidase subunit 1 (COI) sequences from parasitoids dissected from weevils collected during a survey of lucerne-growing areas in Morocco allowed us to further test this hypothesis. As found previously, there were two strong clades in M. aethiopoides with no geographical basis to this structure. Earlier research suggested that intraspecific variability within M. aethiopoides was related to weevil host genus (Sitona vs. Hypera), and the analysis confirmed that one of the clades corresponded strongly with the host Sitona discoideus. The other clade, however, previously characterised by parasitoids from Hypera postica also included parasitoids dissected from Charagmus spp., which is a sister genus to Sitona. It is suggested that food plant associations of the host weevils might have had an influence on the evolutionary history of the parasitoid.     

Spider: an R package for the analysis of species identity and evolution, with particular reference to DNA barcoding

Spider: SPecies IDentity and Evolution in R is a new R package implementing a number of useful analyses for DNA barcoding studies and associated research into species delimitation and speciation. Included are functions essential for generating important summary statistics from DNA barcode data, assessing specimen identification efficacy, and for testing and optimizing divergence threshold limits. In terms of investigating evolutionary and taxonomic questions, techniques for assessing diagnostic nucleotides and probability of reciprocal monophyly are also provided. Additionally, a sliding window function offers opportunities to analyse information across a gene, essential for marker design in degraded DNA studies. Spider capitalizes on R's extensible ethos and offers an integrated platform ideal for the analysis of both nucleotide and morphological data. The program can be obtained from the comprehensive R archive network (CRAN, http://cran.r-project.org) and from the R-Forge package development site (http://spider.r-forge.r-project.org/).     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Calcareous dinoflagellate cyst distributions in surface sediments from upwelling areas off NW Africa, and their relationships with environmental parameters of the upper water column

Only very few studies have focussed on the spatial distribution and ecology of calcareous dinoflagellate cysts (dinocysts) in upwelling areas. Here, distributions of individual calcareous dinocyst species in surface sediment samples from the coastal upwelling zone off NW Africa and their relationships with known environmental parameters of the (sub-)surface waters have been analysed in order to enhance our knowledge on their modern distribution patterns and to determine the ecological significance and palaeoenvironmental reconstruction potential of each calcareous dinocyst species within this exemplary high-productivity region. In addition to calculating relative cyst abundances, well-constrained sedimentation rates have allowed the calculation of dinocyst accumulation rates (cysts cm^− 2 ka^− 1) for most of the surface sample positions, thus providing a much more accurate estimation of actual calcareous dinocyst production in the upper water column than could be warranted in similar studies so far. Distinct differences in the geographic distribution patterns of nine species were observed. In general, high accumulation rates of calcareous dinocysts were found around and south of 29°N and offshore the westernmost Canary Island La Palma, with slightly lower accumulation rates in the upwelling-influenced areas off Cape Yubi and in the upwelling filament area off Cape Ghir. Multivariate ordination techniques were applied in order to compare the cyst accumulation rates of individual species with physical parameters (temperature, salinity, density, mixed layer depth) and the trophic state (nitrate, phosphate and chlorophyll concentrations) of the overlying (sub-)surface waters. All species were found to relate significantly to one or more of the environmental parameters, partly confirming previous results on cyst ecology but also providing new information which will be useful for future palaeoenvironmental reconstructions within upwelling areas.