2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins

Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.

MR Angiography at 3 Tesla to Assess Proximal Internal Carotid Artery Stenoses: Contrast-Enhanced or 3D Time-of-Flight MR Angiography?

PURPOSE The aim of this study was to compare the diagnostic accuracy of 3D time-of-flight (TOF-MRA) and contrast-enhanced (CE-MRA) magnetic resonance angiography at 3 T for detection and quantification of proximal high-grade stenosis using multidetector computed tomography angiography (MDCTA) as reference standard. METHODS The institutional ethics committee approved this prospective study. A total of 41 patients suspected of having internal carotid artery (ICA) stenosis underwent both MDCTA and MRA. CE-MRA and TOF-MRA were performed using a 3.0-T imager with a dedicated eight-element cervical coil. ICA stenoses were measured according to the North American Symptomatic Carotid Endarterectomy Trial criteria and categorized as 0-25 % (minimal), 25-50 % (mild), 50-69 % (moderate), 70-99 % (high grade), and 100 % (occlusion). Sensitivity and specificity for the detection of high-grade ICA stenoses (70-99 %) and ICA occlusions were determined. In addition, intermodality agreement was assessed with κ-statistics for detection of high-grade ICA stenoses (70-99 %) and ICA occlusions. RESULTS A total of 80 carotid arteries of 41 patients were reviewed. Two previously stented ICAs were excluded from analysis. On MDCTA, 7 ICAs were occluded, 12 ICAs presented with and 63 without a high-grade ICA stenosis (70-99 %). For detecting 70-99 % stenosis, both 3D TOF-MRA and CE-MRA were 91.7 % sensitive and 98.5 % specific, respectively. Both MRA techniques were highly sensitive (100 %), and specific (CE-MRA, 100 %; TOF-MRA, 98.7 %) for the detection of ICA occlusion. However, TOF-MRA misclassified one high-grade stenosis as occlusion. Intermodality agreement for detection of 70-99 % ICA stenoses was excellent between TOF-MRA and CE-MRA [κ = 0.902, 95 % confidence interval (CI) = 0.769-1.000], TOF-MRA and MDCTA (κ = 0.902, 95 % CI = 0.769-1.000), and CE-MRA and MDCTA (κ = 0.902, 95 % CI = 0.769-1.000). CONCLUSION Both 3D TOF-MRA and CE-MRA at 3 T are reliable tools for detecting high-grade proximal ICA stenoses (70-99 %). 3D TOF-MRA might misclassify pseudo-occlusions as complete occlusions. If there are no contraindications for CE-MRA, CE-MRA is recommended as primary MR imaging modality.

Articulated Statistical Shape Model-Based 2D-3D Reconstruction of a Hip Joint

In this paper, reconstruction of three-dimensional (3D) patient-specific models of a hip joint from two-dimensional (2D) calibrated X-ray images is addressed. Existing 2D-3D reconstruction techniques usually reconstruct a patient-specific model of a single anatomical structure without considering the relationship to its neighboring structures. Thus, when those techniques would be applied to reconstruction of patient-specific models of a hip joint, the reconstructed models may penetrate each other due to narrowness of the hip joint space and hence do not represent a true hip joint of the patient. To address this problem we propose a novel 2D-3D reconstruction framework using an articulated statistical shape model (aSSM). Different from previous work on constructing an aSSM, where the joint posture is modeled as articulation in a training set via statistical analysis, here it is modeled as a parametrized rotation of the femur around the joint center. The exact rotation of the hip joint as well as the patient-specific models of the joint structures, i.e., the proximal femur and the pelvis, are then estimated by optimally fitting the aSSM to a limited number of calibrated X-ray images. Taking models segmented from CT data as the ground truth, we conducted validation experiments on both plastic and cadaveric bones. Qualitatively, the experimental results demonstrated that the proposed 2D-3D reconstruction framework preserved the hip joint structure and no model penetration was found. Quantitatively, average reconstruction errors of 1.9 mm and 1.1 mm were found for the pelvis and the proximal femur, respectively.

Mineralogical and isotopic record of diagenesis from the Opalinus Clay formation at Benken, Switzerland: Implications for the modeling of pore-water chemistry in a clay formation

Argillaceous rocks are considered to be a suitable geological barrier for the long-term containment of wastes. Their efficiency at retarding contaminant migration is assessed using reactive-transport experiments and modeling, the latter requiring a sound understanding of pore-water chemistry. The building of a pore-water model, which is mandatory for laboratory experiments mimicking in situ conditions, requires a detailed knowledge of the rock mineralogy and of minerals at equilibrium with present-day pore waters. Using a combination of petrological, mineralogical, and isotopic studies, the present study focused on the reduced Opalinus Clay formation (Fm) of the Benken borehole (30 km north of Zurich) which is intended for nuclear-waste disposal in Switzerland. A diagenetic sequence is proposed, which serves as a basis for determining the minerals stable in the formation and their textural relationships. Early cementation of dominant calcite, rare dolomite, and pyrite formed by bacterial sulfate reduction, was followed by formation of iron-rich calcite, ankerite, siderite, glauconite, (Ba, Sr) sulfates, and traces of sphalerite and galena. The distribution and abundance of siderite depends heavily on the depositional environment (and consequently on the water column). Benken sediment deposition during Aalenian times corresponds to an offshore environment with the early formation of siderite concretions at the water/sediment interface at the fluctuating boundary between the suboxic iron reduction and the sulfate reduction zones. Diagenetic minerals (carbonates except dolomite, sulfates, silicates) remained stable from their formation to the present. Based on these mineralogical and geochemical data, the mineral assemblage previously used for the geochemical model of the pore waters at Mont Terri may be applied to Benken without significant changes. These further investigations demonstrate the need for detailed mineralogical and geochemical study to refine the model of pore-water chemistry in a clay formation.

Benchmark problems for reactive transport modeling of the generation and attenuation of acid rock drainage

Acid rock drainage (ARD) is a problem of international relevance with substantial environmental and economic implications. Reactive transport modeling has proven a powerful tool for the process-based assessment of metal release and attenuation at ARD sites. Although a variety of models has been used to investigate ARD, a systematic model intercomparison has not been conducted to date. This contribution presents such a model intercomparison involving three synthetic benchmark problems designed to evaluate model results for the most relevant processes at ARD sites. The first benchmark (ARD-B1) focuses on the oxidation of sulfide minerals in an unsaturated tailing impoundment, affected by the ingress of atmospheric oxygen. ARD-B2 extends the first problem to include pH buffering by primary mineral dissolution and secondary mineral precipitation. The third problem (ARD-B3) in addition considers the kinetic and pH-dependent dissolution of silicate minerals under low pH conditions. The set of benchmarks was solved by four reactive transport codes, namely CrunchFlow, Flotran, HP1, and MIN3P. The results comparison focused on spatial profiles of dissolved concentrations, pH and pE, pore gas composition, and mineral assemblages. In addition, results of transient profiles for selected elements and cumulative mass loadings were considered in the intercomparison. Despite substantial differences in model formulations, very good agreement was obtained between the various codes. Residual deviations between the results are analyzed and discussed in terms of their implications for capturing system evolution and long-term mass loading predictions.

DAPK loss in colon cancer tumor buds: implications for migration capacity of disseminating tumor cells.

Defining new therapeutic strategies to overcome therapy resistance due to tumor heterogeneity in colon cancer is challenging. One option is to explore the molecular profile of aggressive disseminating tumor cells. The cytoskeleton-associated Death-associated protein kinase (DAPK) is involved in the cross talk between tumor and immune cells at the invasion front of colorectal cancer. Here dedifferentiated tumor cells histologically defined as tumor budding are associated with a high risk of metastasis and poor prognosis. Analyzing samples from 144 colorectal cancer patients we investigated immunhistochemical DAPK expression in different tumor regions such as center, invasion front, and buds. Functional consequences for tumor aggressiveness were studied in a panel of colon tumor cell lines using different migration, wound healing, and invasion assays. DAPK levels were experimentally modified by siRNA transfection and overexpression as well as inhibitor treatments. We found that DAPK expression was reduced towards the invasion front and was nearly absent in tumor buds. Applying the ECIS system with HCT116 and HCT116 stable lentiviral DAPK knock down cells (HCTshDAPK) we identified an important role for DAPK in decreasing the migratory capacity whereas proliferation was not affected. Furthermore, the migration pattern differed with HCTshDAPK cells showing a cluster-like migration of tumor cell groups. DAPK inhibitor treatment revealed that the migration rate was independent of DAPK's catalytic activity. Modulation of DAPK expression level in SW480 and DLD1 colorectal cancer cells significantly influenced wound closure rate. DAPK seems to be a major player that influences the migratory capability of disseminating tumor cells and possibly affects the dynamic interface between pro- and anti-survival factors at the invasion front of colorectal cancer. This interesting and new finding requires further evaluation.

Atomistic Modeling of Effect of Mg on Oxygen Vacancy Diffusion in α-Alumina

Oxygen diffusion plays an important role in grain growth and densification during the sintering of alumina ceramics and governs high-temperature processes such as creep. The atomistic mechanism for oxygen diffusion in alumina is, however, still debated; atomistic calculations not being able to match experimentally determined activation energies for oxygen vacancy diffusion. These calculations are, however, usually performed for perfectly pure crystals, whereas virtually every experimental alumina sample contains a significant fraction of impurity/dopants ions. In this study, we use atomistic defect cluster and nudged elastic band (NEB) calculations to model the effect of Mg impurities/dopants on defect binding energies and migration barriers. We find that oxygen vacancies can form energetically favorable clusters with Mg, which reduces the number of mobile species and leads to an additional 1.5 eV energy barrier for the detachment of a single vacancy from Mg. The migration barriers of diffusive jumps change such that an enhanced concentration of oxygen vacancies is expected around Mg ions. Mg impurities were also found to cause destabilization of certain vacancy configurations as well as enhanced vacancy–vacancy interaction.

4D/3D reduction of dualities: mirrors on the circle

We engineer a brane picture for the reduction of Seiberg dualities from 4D to 3D, valid also in the presence of orientifold planes. We obtain effective 3D dualities on the circle by T-duality, geometrizing the non-perturbative superpotential which is an affine Toda potential. When reducing to pure 3D, we define a double-scaling limit which creates a sector of interacting singlets, giving a unified mechanism for the brane reduction of dualities.

2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli.

The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IIC(glc)) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IIC(glc) was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IIC(glc) into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IIC(glc), which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IIC(glc) lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.

Comparison of 2.5D and 3D Quantification of Femoral Head Coverage in Normal Control Subjects and Patients with Hip Dysplasia.

Hip dysplasia is characterized by insufficient femoral head coverage (FHC). Quantification of FHC is of importance as the underlying goal of the surgery to treat hip dysplasia is to restore a normal acetabular morphology and thereby to improve FHC. Unlike a pure 2D X-ray radiograph-based measurement method or a pure 3D CT-based measurement method, previously we presented a 2.5D method to quantify FHC from a single anteriorposterior (AP) pelvic radiograph. In this study, we first quantified and compared 3D FHC between a normal control group and a patient group using a CT-based measurement method. Taking the CT-based 3D measurements of FHC as the gold standard, we further quantified the bias, precision and correlation between the 2.5D measurements and the 3D measurements on both the control group and the patient group. Based on digitally reconstructed radiographs (DRRs), we investigated the influence of the pelvic tilt on the 2.5D measurements of FHC. The intraclass correlation coefficients (ICCs) for absolute agreement was used to quantify interobserver reliability and intraobserver reproducibility of the 2.5D measurement technique. The Pearson correlation coefficient, r, was used to determine the strength of the linear association between the 2.5D and the 3D measurements. Student's t-test was used to determine whether the differences between different measurements were statistically significant. Our experimental results demonstrated that both the interobserver reliability and the intraobserver reproducibility of the 2.5D measurement technique were very good (ICCs > 0.8). Regression analysis indicated that the correlation was very strong between the 2.5D and the 3D measurements (r = 0.89, p < 0.001). Student's t-test showed that there were no statistically significant differences between the 2.5D and the 3D measurements of FHC on the patient group (p > 0.05). The results of this study provided convincing evidence demonstrating the validity of the 2.5D measurements of FHC from a single AP pelvic radiograph and proved that it could serve as a surrogate for 3D CT-based measurements. Thus it may be possible to use this method to avoid a CT scan for the purpose of estimating 3D FHC in diagnosis and post-operative treatment evaluation of patients with hip dysplasia.

Diabetes, use of antidiabetic drugs, and the risk of glioma

BACKGROUND Prior epidemiologic studies suggest inverse relations between diabetes and glioma risk, but the underlying mechanisms, including use of antidiabetic drugs, are unknown. METHODS We therefore performed a matched case-control analysis using the Clinical Practice Research Datalink (CPRD). We identified incident glioma cases diagnosed between 1995 and 2012 and matched each case with 10 controls on age, gender, calendar time, general practice, and years of active history in the CPRD. We performed conditional logistic regression to estimate odds ratios (ORs) with 95% CIs, adjusted for body mass index and smoking. RESULTS We identified 2005 cases and 20 050 controls. Diabetes was associated with decreased risk of glioma (OR = 0.74; 95% CI = 0.60-0.93), particularly glioblastoma (OR = 0.69; 95% CI = 0.51-0.94). Glioblastoma risk reduction was markedly pronounced among diabetic men (OR = 0.60; 95% CI = 0.40-0.90), most apparently for those with diabetes of long-term duration (OR for >5 vs 0 y = 0.46; 95% CI = 0.26-0.82) or poor glycemic control (OR for HbA1c ≥8 vs <6.5% = 0.20; 95% CI = 0.06-0.70). In contrast, the effect of diabetes on glioblastoma risk was absent among women (OR = 0.85; 95% CI = 0.53-1.36). No significant associations with glioma were found for use of metformin (OR for ≥30 vs 0 prescriptions = 0.72; 95% CI = 0.38-1.39), sulfonylureas (OR = 0.71; 95% CI = 0.39-1.30), or insulin (OR = 0.79; 95% CI = 0.37-1.69). CONCLUSIONS Antidiabetic treatment appears to be unrelated to glioma, but long-term diabetes duration and increased HbA1c both show decreased glioma risk. Stronger findings in men than women suggest low androgen levels concurrent with diabetes as a biologic mechanism.