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Pervasive salinity in soil and water is affecting agricultural yield and the health of millions of delta dwellers in Asia. This is also being exacerbated by climate change through increases in sea level and tropical storm surges. One consequence of this has been a widespread introduction of salt water shrimp farming. Here, we show, using field data and modeling, how changes in climate and land use are likely to result in increased salinization of shallow groundwater in SE Asian mega-deltas. We also explore possible adaptation options. We find that possible future increase of episodic inundation events, combined with salt water shrimp farming, will cause rapid salinization of groundwater in the region making it less suitable for drinking water and irrigation. However, modified land use and water management practices can mitigate the impacts on groundwater, as well as the overlying soil, from future salinization. The study therefore provides guidance for adaptation planning to reduce future salinization in Asian deltas.

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Drinking water in much of Asia, particularly in coastal and rural settings, is provided by a variety of sources, which are widely distributed and frequently managed at an individual or local community level. Coastal and near-inland drinking water sources in South and South East (SSE) Asia are vulnerable to contamination by seawater, most dramatically from tropical cyclone induced storm surges. This paper assesses spatial vulnerabilities to salinisation of drinking water sources due to meteorological variability and climate change along the (ca. 6000 km) coastline of SSE Asia. The risks of increasing climatic stresses are first considered, and then maps of relative vulnerability along the entire coastline are developed, using data from global scale land surface models, along with an overall vulnerability index. The results show that surface and near-surface drinking water in the coastal areas of the mega-deltas in Vietnam and Bangladesh-India are most vulnerable, putting more than 25 million people at risk of drinking 'saline' water. Climate change is likely to exacerbate this problem, with adverse consequences for health, such as prevalence of hypertension and cardiovascular diseases. There is a need for identifying locations that are most at risk of salinisation in order for policy makers and local officials to implement strategies for reducing these health impacts. To counter the risks associated with these vulnerabilities, possible adaptation measures are also outlined. We conclude that detailed and fine scale vulnerability assessments may become crucial for planning targeted adaptation programmes along these coasts.

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BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

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OBJECTIVE: To distinguish components of vulnerable atherosclerotic plaque by imaging their energy response using spectral CT and comparing images with histology. METHODS: After spectroscopic calibration using phantoms of plaque surrogates, excised human carotid atherosclerotic plaques were imaged using MARS CT using a photon-processing detector with a silicon sensor layer and microfocus X-ray tube (50 kVp, 0.5 mA) at 38-µm voxel size. The plaques were imaged, sectioned and re-imaged using four threshold energies: 10, 16, 22 and 28 keV; then sequentially stained with modified Von Kossa, Perl's Prussian blue and Oil-Red O, and photographed. Relative Hounsfield units across the energies were entered into a linear algebraic material decomposition model to identify the unknown plaque components. RESULTS: Lipid, calcium, iron and water-like components of plaque have distinguishable energy responses to X-ray, visible on spectral CT images. CT images of the plaque surface correlated very well with histological photographs. Calcium deposits (>1,000 µm) in plaque are larger than iron deposits (<100 µm), but could not be distinguished from each other within the same voxel using the energy range available. CONCLUSIONS: Spectral CT displays energy information in image form at high spatial resolution, enhancing the intrinsic contrast of lipid, calcium and iron within atheroma. KEY POINTS: Spectral computed tomography offers new insights into tissue characterisation. Components of vulnerable atherosclerotic plaque are spectrally distinct with intrinsic contrast. Spectral CT of excised atherosclerotic plaques can display iron, calcium and lipid. Calcium deposits are larger than iron deposits in atheroma. Spectral CT may help in the non-invasive detection of vulnerable plaques.

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BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

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OBJECTIVE: Spectral CT differs from dual-energy CT by using a conventional X-ray tube and a photon-counting detector. We wished to produce 3D spectroscopic images of mice that distinguished calcium, iodine and barium. METHODS: We developed a desktop spectral CT, dubbed MARS, based around the Medipix2 photon-counting energy-discriminating detector. The single conventional X-ray tube operated at constant voltage (75 kVp) and constant current (150 microA). We anaesthetised with ketamine six black mice (C57BL/6). We introduced iodinated contrast material and barium sulphate into the vascular system, alimentary tract and respiratory tract as we euthanised them. The mice were preserved in resin and imaged at four detector energy levels from 12 keV to 42 keV to include the K-edges of iodine (33.0 keV) and barium (37.4 keV). Principal component analysis was applied to reconstructed images to identify components with independent energy response, then displayed in 2D and 3D. RESULTS: Iodinated and barium contrast material was spectrally distinct from soft tissue and bone in all six mice. Calcium, iodine and barium were displayed as separate channels on 3D colour images at <55 microm isotropic voxels. CONCLUSION: Spectral CT distinguishes contrast agents with K-edges only 4 keV apart. Multi-contrast imaging and molecular CT are potential future applications.

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This study confirms that the Medipix2 x-ray detector enables spectroscopic bio-medical plain radiography. We show that the detector has the potential to provide new, useful information beyond the limited spectroscopic information of modem dual-energy computed tomography (CT) scanners. Full spectroscopic 3D-imaging is likely to be the next major technological advance in computed tomography, moving the modality towards molecular imaging applications. This paper focuses on the enabling technology which allows spectroscopic data collection and why this information is useful. In this preliminary study we acquired the first spectroscopic images of human tissue and other biological samples obtained using the Medipix2 detector. The images presented here include the clear resolution of the 1.4mm long distal phalanx of a 20-week-old miscarried foetus, showing clear energy-dependent variations. The opportunities for further research using the forthcoming Medipix3 detector are discussed and a prototype spectroscopic CT scanner (MARS, Medipix All Resolution System) is briefly described.

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A mathematical model simulating the hydrological and biochemical processes occurring in landfilled waste is presented and demonstrated. The model combines biochemical and hydrological models into an integrated representation of the landfill environment. Waste decomposition is modelled using traditional biochemical waste decomposition pathways combined with a simplified methodology for representing the rate of decomposition. Water flow through the waste is represented using a statistical velocity model capable of representing the effects of waste heterogeneity on leachate flow through the waste. Given the limitations in data capture from landfill sites, significant emphasis is placed on improving parameter identification and reducing parameter requirements. A sensitivity analysis is performed, highlighting the model's response to changes in input variables. A model test run is also presented, demonstrating the model capabilities. A parameter perturbation model sensitivity analysis was also performed. This has been able to show that although the model is sensitive to certain key parameters, its overall intuitive response provides a good basis for making reasonable predictions of the future state of the landfill system. Finally, due to the high uncertainty associated with landfill data, a tool for handling input data uncertainty is incorporated in the model's structure. It is concluded that the model can be used as a reasonable tool for modelling landfill processes and that further work should be undertaken to assess the model's performance.

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A landfill is a very complex heterogeneous environment and as such it presents many modelling challenges. Attempts to develop models that reproduce these complexities generally involve the use of large numbers of spatially dependent parameters that cannot be properly characterised in the face of data uncertainty. An alternative method is presented, which couples a simplified microbial degradation model with a stochastic hydrological and contaminant transport model. This provides a framework for incorporating the complex effects of spatial heterogeneity within the landfill in a simplified manner, along with other key variables. A methodology for handling data uncertainty is also integrated into the model structure. Illustrative examples of the model's output are presented to demonstrate effects of data uncertainty on leachate composition and gas volume prediction.

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This paper describes a 12-month experiment designed to study the extent of upward migration of (125)I (as a surrogate for (129)I) from near-surface groundwater, through a 50-cm column of soil and into perennial ryegrass. The water table was established at a depth of 45 cm below the soil surface. By 3 months, (125)I had migrated about half way up the soil column. After this, it tended to accumulate just above this mid-point, with only very small amounts being transported to the upper 20 cm of soil. This behaviour seemed to be explained well by soil moisture and redox conditions. The experiment indicated that (125)I was mobile only within the saturated/low redox zone at the base of the soil column and accumulated in the zone of transition between anoxic and oxic soil conditions. Uptake of (125)I by the ryegrass was found to be low.

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Overexpression of ornithine decarboxylase (ODC) is an important oncogenic event in tumorigenesis. Although ODC was one of the first genes described whose product is inducible by 12-O-tetradecanoylphorbol-13-acetate (TPA), the mechanisms of ODC transcriptional regulation have remained elusive. In this study, we systematically analyzed the rat ODC core promoter region for novel TPA response elements. Analysis of linker scanning mutants of the ODC promoter from the TATA box to the transcription start site demonstrated that mutation of the TATA box reduced the TPA induction ratio by 40%, while the basal ODC promoter activity was not significantly changed. A novel region between nt - 20 to - 10 was shown to be critical for both basal promoter activity and induction by TPA. Random mutagenesis of this region showed that conversion of the GC-rich wild-type sequence into a T-rich sequence could either substantially increase the basal promoter activity and decrease the TPA induction ratio or dramatically reduce the basal promoter activity, depending on the T content. Mutant R5, containing an ATTT sequence at nt - 15 to - 12, caused a more than twofold increase of basal promoter activity and 80% reduction of TPA induction ratio. We suggest that this region interacts with components of the general transcription machinery and that the strength of this interaction is mediated by the T-content in this region.

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We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

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The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

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Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC(1)) hybrids produced from backcrossing F(1) hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC(1) hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC(1) hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC(1) hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianus CDKN2X alleles in BC(1) hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC(1) hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

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A putative Ets site with a core of GGAA located at nt -88 to -85 of the rat ornithine decarboxylase (ODC) gene was characterized by site-directed mutagenesis and transient expression assays. Mutation of this site, when in pODClux2m, which contains a cluster of four Sp1-binding sites, resulted in a 2.6-fold increase in basal promoter activity in untreated cells, whereas the ratio of activity in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells relative to the ratio in untreated cells (the induction ratio) remained largely unchanged. However, when the mutation was in pODClux168, which contains only a single Sp1-binding site (GC box V), it caused little alteration to either basal promoter activity or TPA induction ratio. A protein of 55-60 kDa was found specifically bound to this site, as shown by ultraviolet cross-linking assay. In competition assay and methylation interference assay, this protein was shown to occupy the GGAA core, although it showed no antigenic relation to c-Ets-1 in an supershift assay. We suggest that this protein binds specifically to the GGAA core and functions to inhibit activation of the ODC promoter by distal elements, including the upstream Sp1 sites.

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Altered regulation of ornithine decarboxylase (ODC) is frequently observed in epidermal tumors. We have shown that the transcription factor Sp1 is one of the regulators of ODC expression and that Sp3 antagonizes this Sp1-mediated activation of ODC expression. These results led us to examine the levels and binding activity of Sp1 and Sp3 in nuclear extracts prepared from cultured murine keratinocytes, transformed keratinocyte cell lines and epidermal tumors. Here we show that the Sp1 DNA-binding activity is higher in established keratinocyte cell line extracts than in primary keratinocyte extracts. Sp1 message levels and Sp1 DNA-binding activity was found to be low in 20-week papillomas and high in squamous cell carcinomas. These results suggest that increased levels of Sp1 and enhanced Sp1 DNA binding activity are correlated with epidermal tumor progression. Based on these results, we propose that increased Sp1 DNA binding may augment the proliferative capacity of tumor cells through overexpression of Sp1-responsive genes, possibly including ODC.

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We compared the Sp1 binding activity of Rat2 fibroblasts in nuclear extracts prepared from quiescent cells and cells stimulated with 20% serum. Increased DNA-binding activity was observed in extracts from serum-stimulated cells when an Sp1 oligonucleotide was used as radiolabeled probe in electrophoretic mobility shift assays. This increase in Sp1 DNA-binding activity is not due to changes in the amount of Sp1 in the nucleus as shown by immunoblot analysis. The transcriptional activity of a reporter construct containing six Sp1 sites upstream of a minimal adenovirus promoter or an Sp1-dependent promoter such as ornithine decarboxylase (ODC) containing Sp1 sites was enhanced following serum stimulation in transient transfection assays. Dephosphorylation of the nuclear extracts with potato acid phosphatase abolished the Sp1 DNA-binding activity, demonstrating a possible correlation between phosphorylation of Sp1 and DNA-binding activity. These results implicate a potential role for Sp1 in mediating signal transduction pathways in response to mitogenic signals.

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A series of in-air measurements showed that collimator scatter (Sc) did not change significantly for 6MV photons when the centre of the field was moved away from the central axis. This result enabled a model to be developed for the off-axis Effective Output Factor (EOF) which was then verified for 6MV and 18MV photons on a Varian 2100c accelerator and for 6MV photons on a Varian 600c accelerator. Thus off-axis output factors may be predicted, for a range of rectangular asymmetric fields, using only the Primary Off-Centre Ratio (POCR) in air and the on-axis output factor. Depth doses were also investigated off-axis and found to have no clinically significant differences compared with on-axis depth doses, for depths less than 7.5 cm for 6MV and 12.5 cm for 18MV photons. The model is simple to implement and avoids the need for a measurement for each patient, thus saving accelerator time.

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Ornithine decarboxylase (ODC) expression is important for proliferation and is elevated in many tumor cells. We previously showed that Sp1 is a major positive regulator of ODC transcription. In this paper we have investigated transcriptional regulation of rat ODC by the closely related factor Sp3. While over-expression of Sp1 caused a dramatic activation of the ODC promoter, over-expression of Sp3 caused little or no activation in either Drosophila SL2 cells (lacking endogenous Sp1 or Sp3) or in H35 rat hepatoma cells. Furthermore, co-transfection studies demonstrated that Sp3 abolished trans -activation of the ODC promoter by Sp1. DNase I footprint studies and electrophoretic mobility shift assays demonstrated that both recombinant Sp1 and Sp3 bind specifically to several sites within the ODC promoter also protected by nuclear extracts, including overlapping GC and CT motifs located between -116 and -104. This CT element is a site of negative ODC regulation. Mutation of either element reduced binding, but mutation of both sites was required to eliminate binding of either Sp1 or Sp3. These results demonstrate that ODC is positively regulated by Sp1 and negatively regulated by Sp3, suggesting that the ratio of these transcription factors may be an important determinant of ODC expression during development or transformation.

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Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression.

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