Rijkswaterstaat:Guardian of the Dutch Delta

Founded in 1798, Rijkswaterstaat, the Dutch government’s agency for infrastructural works, brought flood security, navigable waterways and highways to the Netherlands. It is an iconic institution within Dutch society, best known for its ‘battle against the water’. The Zuiderzee Works (1920–1968) and the Delta Works (1954–1997) brought worldwide acclaim. This chapter tells the story of a humble semi-military organization that developed into a formidable institution of civil engineers with a strong technocratic mission mystique. It also recounts the institutional crisis the agency experienced in the 1970s–1990s when it was too slow to adapt to major sociocultural and political changes. To ride the waves of change, it eventually developed several proactive adaptation strategies and reinvented its mission mystique in managerial terms. Adaptation to climate change now presents another key challenge, for which Rijkswaterstaat will have to develop a new ‘social license to operate’

Weight management interventions in adults with intellectual disabilities and obesity: a systematic review of the evidence

o evaluate the clinical effectiveness of weight management interventions in adults with intellectual disabilities (ID) and obesity using recommendations from current clinical guidelines for the first line management of obesity in adults. Full papers on lifestyle modification interventions published between 1982 to 2011 were sought by searching the Medline, Embase, PsycINFO and CINAHL databases. Studies were evaluated based on 1) intervention components, 2) methodology, 3) attrition rate 4) reported weight loss and 5) duration of follow up. Twenty two studies met the inclusion criteria. The interventions were classified according to inclusion of the following components: behaviour change alone, behaviour change plus physical activity, dietary advice or physical activity alone, dietary plus physical activity advice and multi-component (all three components). The majority of the studies had the same methodological limitations: no sample size justification, small heterogeneous samples, no information on randomisation methodologies. Eight studies were classified as multi-component interventions, of which one study used a 600 kilocalorie (2510 kilojoule) daily energy deficit diet. Study durations were mostly below the duration recommended in clinical guidelines and varied widely. No study included an exercise program promoting 225–300 minutes or more of moderate intensity physical activity per week but the majority of the studies used the same behaviour change techniques. Three studies reported clinically significant weight loss (≥ 5%) at six months post intervention. Current data indicate weight management interventions in those with ID differ from recommended practice and further studies to examine the effectiveness of multi-component weight management interventions for adults with ID and obesity are justified

Immunohistochemical, morphological and ultrastructural resemblance between dendritic cells and folliculo-stellate cells in normal human and rat anterior pituitaries

Immunolabeling of cryo-sections of human anterior pituitaries obtained at autopsy, and of cryo-sections of freshly prepared rat anterior pituitaries, with a panel of monoclonal antibodies against markers of the monocyte/dendritic cell/macrophage lineage, reveals in both species a characteristic pattern of immunopositive cells, among which many cells with dendritic phenotype are found. Cells characterized by marker expression of MHC-class II determinants and a dendritic morphology are present in both human and rat anterior pituitary. Markers characteristic of dendritic cells such as the L25 antigen and the OX62 antigen were present in anterior pituitaries from human and rat respectively. The population of MHC-class II expressing dendritic cells of the rat anterior pituitary is compared at the ultrastructural level with the folliculo-stellate cell population, which cell type has been previously characterized by its distinctive ultrastructure and immunopositivity for the S100 protein. Using immune-electron microscopy of rat anterior pituitaries fixed with periodate-lysine-paraformaldehyde, we were able to distinguish non-granulated cells expressing MHC-class II determinants, whereas no MHC-class II expression was found in the granulated endocrine cells. Using double immunolabeling of cryo-sections of these rat AP with 25 nm and 15 nm gold labels, we demonstrated an overlap between the populations of MHC-class II-expressing and S100 protein-expressing cells. Furthermore, MHC-class II-expressing and S100-positive cells showed ultrastructural characteristics that have been previously ascribed to folliculo-stellate cells. At the light microscopical level in the rat AP, a proportion of 10 to 20% of the S100-positive cells was found immunopositive for the MHC-class II marker OX6. In the hu

Identification of a possible role of thymine DNA glycosylase (TDG) in epigenome maintenance

Thymine DNA glycosylase (TDG) was discovered as an enzyme capable of removing uracil (U) and thymine (T) from G/U and G/T mispairs, respectively. Owing to this ability, TDG was proposed to initiate restoration of C/G pairs at sites of cytosine or 5-methycytosine (5-meC) deamination. In addition to products of base deamination, the substrate spectrum of TDG covers a wide range of DNA base damages resulting from base oxidation and alkylation. TDG was also found to engage in physical and functional interactions with transcription factors, and more recent evidence supports additional interactions with the de novo DNA methyltransferases Dnmt3a and 3b in the context of gene transcription. Together with its biochemical properties, these observations suggest that TDG might be targeted to gene regulatory sequences as part of a macromolecular assembly to control their functional integrity. TDG may counteract the mutagenic effects of C and 5-meC deamination in CG-rich regions and/or be involved in the maintenance of CpG promoter methylation patterns. A tight regulation of CpG methylation at gene regulatory regions is critical for accurate gene expression, proper cellular differentiation and embryonic development. A somewhat surprising but in this context consistent finding was that, in contrast to other DNA glycosylases, TDG is essential for proper fetal development since a targeted knockout of the gene leads to embryonic lethality. To gain insights into the biological functions of TDG, we aimed to establish and apply biochemical fractionation procedures for high affinity purification and structural and functional characterization of TDG containing proteins complexes. The first part of the thesis was concerned with biochemical characterization of the protein interaction network of TDG in living mammalian cells. To this end, I applied different approaches allowing high affinity isolation of protein complexes from mammalian cells, such as the tandem affinity purification (TAP) method as well as immunoprecipitation of endogenous protein and of the TDGa isoform from TdgA overexpressing embryonic stem (ES) cells. These efforts, however, did not reveal any TDG interacting partners in subsequent mass spectrometry (MS) analyses. These results were surprising, as TDG was previously reported to interact with transcription factors and DNA methyltransferases. Remarkably, however, all previously identified protein interactors of TDG were discovered in screen with the respective partner proteins, and under conditions of simultaneous overexpression of both interacting proteins. The only proteins ever identified in screen with TDG were Sumo1 and Sumo3, which turned out to covalently modify the glycosylase. For this reason, we decided to pursue our search with classical cell fractionation experiments. We first did gel filtration experiments from total cell lysates and showed that TDG is indeed able to form distinct multiprotein complexes in undifferentiated mouse embryonic stem cells that may also contain the RNA helicase p68. Further subcellular fractionation experiments then revealed that TDG is present in all cell compartments, with a significant fraction of nuclear TDG being associated with chromatin, together with p68 and de novo DNA methyltransferases. Together with published findings, these results suggested that protein complexes containing TDG might act in a chromatin-associated context, at gene regulatory regions. The developmental phenotype of Tdg-/- knockout mice and the interactions of TDG with factors involved in developmental gene regulation (e.g. retinoic acid receptors RAR/RXR) implicate a function of TDG during early development and cell differentiation, at times governed by dynamic changes in gene expression, DNA methylation and histone modifications. Such changes have been studied using a well-established during in vitro differentiation of ES cells to lineage committed neuronal progenitors (NPs). We thus aimed to address the function of TDG as part of chromatin associated protein complexes during the process of retinoic acid induced differentiation of ES cells to NPs. In the second part of the thesis we made use of a this well-established in vitro differentiation system to examine the genome-wide localization of TDG to chromatin by TDG chromatin immunoprecipitation (ChIP) and to correlate TDG association to chromatin with gene expression and DNA methylation changes linked to cellular differentiation. TDG ChIP combined with high throughput sequencing showed that TDG associates with high preference to CpG islands in promoters of actively transcribed genes or genes poised for transcriptional activation. Such CpG rich sequences are normally unmethylated in mammalian genomes. Interestingly, we found TDG to localize to promoters of many genes controlling pluripotency (e.g. Oct4, Nanog) and developmental processes (e.g. Sfrp2, Tgfb2, Gata6), thus, supporting a function of TDG in cell differentiation and/or embryonic development. As different lines of circumstantial evidence have associated TDG with changes in CpG methylation following activation of hormone responsive gene promoters, we went on to further test genome-wide promoter methylation in Tdg+/- and Tdg-/- NPs making use of a combination of methylated DNA immunoprecipitation (MeDIP) and microarray technology. This showed that the loss of TDG does not affect global promoter DNA methylation. Nevertheless, there were a number of significant differences, suggesting that TDG might affect the CpG methylation pattern at some promoters. Also, owing to the limited resolution of the MeDIP method, however, we could not exclude an involvement of TDG in the control of DNA methylation of specific promoter CpGs. Additional bisulfite sequencing of promoters of TDG bound developmental genes (e.g. Sfrp2, Tgfb2) in NPs and differentiated mouse embryonic fibroblasts (MEFs) have indeed proved that loss of TDG affects local changes in DNA methylation at particular CpGs. Subsequent analysis of genome-wide gene expression profiles of ES cells and differentiated Tdg+/- and Tdg-/- NPs revealed that a limited number of genes (229) are differentially regulated in ES, whereas substantial differences in gene expression in were observed in NPs (1022 genes). This implicated a specific function of TDG in the regulation of cell differentiation triggered gene expression changes. Detailed analysis of the expression of the Pax6 gene, accurate regulation of which is essential for proper neuron development, showed that its promoter is bound by TDG and that its transcription is inappropriately regulated upon further differentiation of Tdg-/- NPs into the neuronal lineage. Whereas Tdg+/- NPs efficiently downregulated Pax6 (50x) and further differentiated into neuron-like cells, Tdg-/- NPs only partially downregulated Pax6 gene expression (6x) and underwent apoptosis at day 2 after plating in neuronal medium. This phenotype was complemented by expression of TDGa, clearly implicating TDG in the regulation of Pax6 expression during differentiation of ES cells to terminal neurons. We further observed misregulation of pluripotency genes (e.g. Oct4) regulated by TDG bound promoters during early differentiation of ES cells. In the absence of TDG, ES cells showed the tendency to enter spontaneous and/or RA induced differentiation, suggesting a role for TDG in the regulation of pluripotency. During RA induced differentiation we further observed the activation of the neuron specific gene Lrrtm2 exclusively in TDG proficient cells. In addition, ChIP experiments showed that transcription factors involved in the activation of the Lrrtm2 gene (e.g. COUP-TFI, RAR) are not recruited to the respective promoter in Tdg-/- cells, suggesting that TDG might act passively as a scaffold factor important for the recruitment of transcription factors to promoter regions. I set out to clarify the biological function of TDG by investigating its molecular interactions in mammalian cells. I found that TDG, as a DNA repair enzyme, associates tightly with chromatin, where it localizes with high preference to CpG island promoters of active genes and genes poised to be expressed. I also found that the loss of TDG causes misregulation of genes during cell differentiation and that this appears to be related to a function of TDG in establishing and/or maintaining CpG methylation pattern in gene regulatory sequences. These discoveries implicate a novel function of DNA repair, in the maintenance not only of the genome, but also the epigenome

Finished Genome of the Fungal Wheat Pathogen Mycosphaerella graminicola Reveals Dispensome Structure, Chromosome Plasticity, and Stealth Pathogenesis.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicolawas sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed “mesosynteny” is very different from synteny seen between other organisms. A surprising feature of the M. graminicolagenome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors

Immunodetection of nmt55/p54(nrb) isoforms in human breast cancer

BACKGROUND: We previously identified and characterized a novel 55 kDa nuclear protein, termed nmt55/p54(nrb), whose expression was decreased in a subset of human breast tumors. The objective of this study was to determine if this reduced expression in human breast tumors was attributed to the regulation of mRNA transcription or the presence of altered forms of this protein. RESULTS: Northern blot analysis and ribonuclease protection assay indicated that nmt55/p54(nrb) mRNA is expressed at varying levels in estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast tumors suggesting that reduced expression of nmt55/p54(nrb) protein in ER- tumors was not due to transcriptional regulation. To determine if multiple protein isoforms are expressed in breast cancer, we utilized Western blot and immunohistochemical analyses, which revealed the expression of an nmt55/p54(nrb) protein isoform in a subset of ER+ tumors. This subset of ER+ human breast tumors expressed an altered form of nmt55/p54(nrb) that was undetectable with an amino-terminal specific antibody suggesting that this isoform contains alterations or modifications within the amino terminal domain. CONCLUSIONS: Our study indicates that nmt55/p54(nrb) protein is post-transcriptionally regulated in human breast tumors leading to reduced expression in ER- tumors and the expression of an amino terminal altered isoform in a subset of ER+ tumors. The potential involvement of nmt55/p54(nrb) in RNA binding and pre-mRNA splicing may be important for normal cell growth and function; thus, loss or alteration of protein structure may contribute to tumor growth and progression

Mutation Bias Favors Protein Folding Stability in the Evolution of Small Populations

Mutation bias in prokaryotes varies from extreme adenine and thymine (AT) in obligatory endosymbiotic or parasitic bacteria to extreme guanine and cytosine (GC), for instance in actinobacteria. GC mutation bias deeply influences the folding stability of proteins, making proteins on the average less hydrophobic and therefore less stable with respect to unfolding but also less susceptible to misfolding and aggregation. We study a model where proteins evolve subject to selection for folding stability under given mutation bias, population size, and neutrality. We find a non-neutral regime where, for any given population size, there is an optimal mutation bias that maximizes fitness. Interestingly, this optimal GC usage is small for small populations, large for intermediate populations and around 50% for large populations. This result is robust with respect to the definition of the fitness function and to the protein structures studied. Our model suggests that small populations evolving with small GC usage eventually accumulate a significant selective advantage over populations evolving without this bias. This provides a possible explanation to the observation that most species adopting obligatory intracellular lifestyles with a consequent reduction of effective population size shifted their mutation spectrum towards AT. The model also predicts that large GC usage is optimal for intermediate population size. To test these predictions we estimated the effective population sizes of bacterial species using the optimal codon usage coefficients computed by dos Reis et al. and the synonymous to non-synonymous substitution ratio computed by Daubin and Moran. We found that the population sizes estimated in these ways are significantly smaller for species with small and large GC usage compared to species with no bias, which supports our prediction

Impairment of Rat Fetal Beta-Cell Development by Maternal Exposure to Dexamethasone during Different Time-Windows

Glucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during different time-windows of gestation for the development of the fetal endocrine pancreas

Aphids acquired symbiotic genes via lateral gene transfer

<p>Abstract</p> <p>Background</p> <p>Aphids possess bacteriocytes, which are cells specifically differentiated to harbour the obligate mutualist <it>Buchnera aphidicola </it>(γ-Proteobacteria). <it>Buchnera </it>has lost many of the genes that appear to be essential for bacterial life. From the bacteriocyte of the pea aphid <it>Acyrthosiphon pisum</it>, we previously identified two clusters of expressed sequence tags that display similarity only to bacterial genes. Southern blot analysis demonstrated that they are encoded in the aphid genome. In this study, in order to assess the possibility of lateral gene transfer, we determined the full-length sequences of these transcripts, and performed detailed structural and phylogenetic analyses. We further examined their expression levels in the bacteriocyte using real-time quantitative RT-PCR.</p> <p>Results</p> <p>Sequence similarity searches demonstrated that these fully sequenced transcripts are significantly similar to the bacterial genes <it>ldcA </it>(product, LD-carboxypeptidase) and <it>rlpA </it>(product, rare lipoprotein A), respectively. <it>Buchnera </it>lacks these genes, whereas many other bacteria, including <it>Escherichia coli</it>, a close relative of <it>Buchnera</it>, possess both <it>ldcA </it>and <it>rlpA</it>. Molecular phylogenetic analysis clearly demonstrated that the aphid <it>ldcA </it>was derived from a rickettsial bacterium closely related to the extant <it>Wolbachia </it>spp. (α-Proteobacteria, Rickettsiales), which are intracellular symbionts of various lineages of arthropods. The evolutionary origin of <it>rlpA </it>was not fully resolved, but it was clearly demonstrated that its double-ψ β-barrel domain is of bacterial origin. Real-time quantitative RT-PCR demonstrated that <it>ldcA </it>and <it>rlpA </it>are expressed 11.6 and 154-fold higher in the bacteriocyte than in the whole body, respectively. LdcA is an enzyme required for recycling murein (peptidoglycan), which is a component of the bacterial cell wall. As <it>Buchnera </it>possesses a cell wall composed of murein but lacks <it>ldcA</it>, a high level of expression of the aphid <it>ldcA </it>in the bacteriocyte may be essential to maintain <it>Buchnera</it>. Although the function of RlpA is not well known, conspicuous up-regulation of the aphid <it>rlpA </it>in the bacteriocyte implies that this gene is also essential for <it>Buchnera</it>.</p> <p>Conclusion</p> <p>In this study, we obtained several lines of evidence indicating that aphids acquired genes from bacteria via lateral gene transfer and that these genes are used to maintain the obligately mutualistic bacterium, <it>Buchnera</it>.</p