Search

While there has been recent success in the development of KRasG12C inhibitors, unmet needs for selective inhibitors of KRasG12D and the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by 19F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRasG12D. These differences in selectivity, combined with the high resolution of 19F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously.

Molecular switch proteins whose cycling between states is controlled by opposing regulators1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks3, the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN)4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches.

Aim To determine if a positive circumferential resection margin (CRM) can be predicted in patients with esophageal adenocarcinoma undergoing neo-adjuvant chemotherapy prior to surgery Methods 223 patients were included in this study. Multivariable analyses of clinico-pathological and computed tomography (CT) imaging predictors were performed to evaluate the likelihood of a positive CRM at initial staging and following neoadjuvant chemotherapy, with prediction models constructed. Area under curve (AUC) with 95% confidence interval (CI) from 1000 bootstrapping was assessed. Results Advanced clinical T-stage (T3-4) (odds ratio, OR 2.93 95%CI 1.03-9.48) and poor tumor differentiation (OR 2.84, 95%CI 1.39-6.01) were independently associated with an increased risk of a positive CRM. Non response to chemotherapy as estimated by CT independently corresponded with an increased risk of CRM positivity (OR 3.38 95% CI 1.43-8.50). Additional CT evidence of local invasion/pleural thickening and higher CT tumor volume (14cm3) improved the performance of a prediction model, including all above parameters; with AUC (c-index) of 0.76 (0.68-0.83). Conclusion Combining advanced clinical T-stage, poor tumor differentiation, CT non-response to chemotherapy, higher CT tumor volume and local invasion may be helpful in selecting patients for intensification of neo-adjuvant treatment prior to resection.

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized 13 C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real-time dynamic metabolic changes using a bioreactor system with live vorinostat-treated or control cells. We found a significant 40% decrease in the 13 C MRS-detectable ratio of hyperpolarized [1-13 C]lactate to hyperpolarized [1-13 C]pyruvate, [1-13 C]Lac/Pyr, and a 37% decrease in the pseudo-rate constant, kPL , for hyperpolarized [1-13 C]lactate production, in vorinostat-treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat-treated cells. In vivo 13 C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1-13 C]Lac/Pyr when comparing vorinostat-treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC-inhibited tumors. Thus, the 13 C MRSI-detectable decrease in hyperpolarized [1-13 C]lactate production could serve as a biomarker of response to HDAC inhibitors.

Large surface ships have traditionally used steel for their construction, which provides good blast resistance and predictable behaviour in use. This research project, however, considered the use of polymeric foam core sandwich panels for the construction of ship hulls, with the intention of reducing the radar signature of the vessel; increasing the maximum speed; reducing fuel consumption; and providing control over desired mechanical properties for specific applications. This project specifically considered the resistance of the sandwich composites to non-contact explosives, specifically sea mines and areal blast. This research project firstly presents air blast testing performed on polymeric foam core sandwich panels with glass fibre face-sheets. The foam polymer type, the effect of the face-sheet material and the effect of using a graded density foam core for blast wave attenuation were all investigated. When subjected to air blast loading it was found that by grading the core density a smoother back face-sheet displacement was witnessed. This was a significant discovery as protecting the back face-sheet is key to maintaining structural integrity of the ship hulls. In a comparison of foam polymers in the cores of the sandwich panel it was concluded that styrene acrylonitrile offers optimum fracture and adhesion properties. Furthermore, it was found that by interleaving high modulus polypropylene fibres between the glass fibre front face-sheets, front face-sheet cracking and delamination can be prevented, restricting water ingress into the sandwich panel if it were subjected to blast loading. Advances in using 3D high speed imaging for digital image correlation were also achieved, whereby the results were used to estimate core shear strain during deformation, and the kinetic energy and strain energy in the sandwich panel were estimated by comparison with blast wave pressure simulations. The residual flexural and edgewise compression properties of the air blasted sandwich panels were then determined to calculate the flexural stiffness and strength and edgewise stiffness and strength with varying damage. It was found that the construction of the sandwich panel properties were unaffected by the construction, and that debonding of the face-sheets and the core was the key performance reducing damage mechanism. Residual properties were determined for the sandwich materials, with the intention that these could be used in simulations to predict performance of the ship hulls after successfully withstanding a blast. In underwater blast scenarios it was discovered that using a graded density foam core reduced the central deflection of the sandwich panel due to the increase in energy absorption due to crushing of the stepwise increase in foam core density. This effect was greater in carbon fibre sandwich panels than in glass fibre panels, due to the increased stiffness of the face-sheets. The central deflection was calculated by averaging the strains measured on the two face-sheets with electronic strain gauges, which was a novel technique and took advantage of the symmetry present in the square sandwich panels. As the focus of this research project was on foam core properties, the polymeric foam materials were characterised in quasi-static and dynamic tension, and quasi-static and dynamic compression. Dynamic compression tests were performed using a split-Hopkinson pressure bar which was designed specifically for testing low density foam materials. Stress equilibrium was achieved in these tests using a textured polypropylene pulse shaper. Stress equilibrium was checked for by performing high speed digital image correlation during deformation, to ensure that core crushing did not begin at the incident bar. The final stage of the research project was to construct finite element simulations of the air blast tests, using the polymeric foam material properties measured in the quasi-static and dynamic tests. A large number of simulations were performed with varying charge sizes and stand-off distances, to determine a combination of blast peak pressure and impulse values at which the sandwich panels failed. Developments were achieved in the use of a brittle cracking material model and the effect of the boundary conditions on the simulations was also studied. This research project furthered the understanding of sandwich composite materials to air and underwater blast loading. Advancements were made into the use of stepwise graded density foam cores and in characterising foams at dynamic tensile and compressive rates. A major conclusion of the project is that the use of graded density cores can be utilised to prevent damage of the sandwich panel on the rear side and can be used to absorb blast energy due to core crushing. The results of the project aid in simulating the response of composite ship hulls to blast loading and better predict the usability of the ships after being subjected to a blast event. Open Access

Human clinical trials using 72 hours of mild hypothermia (32°C-34°C) after neonatal asphyxia have found substantially improved neurologic outcomes. As temperature changes differently modulate numerous metabolite fluxes and concentrations, we hypothesized that (1)H/(31)P nuclear magnetic resonance (NMR) spectroscopy of intracellular metabolites can distinguish different insults, treatments, and recovery stages. Three groups of superfused neonatal rat brain slices underwent 45 minutes oxygen-glucose deprivation (OGD) and then were: treated for 3 hours with mild hypothermia (32°C) that began with OGD, or similarly treated with hypothermia after a 15-minute delay, or not treated (normothermic control group, 37°C). Hypothermia was followed by 3 hours of normothermic recovery. Slices collected at different predetermined times were processed, respectively, for 14.1 Tesla NMR analysis, enzyme-linked immunosorbent assay (ELISA) cell-death quantification, and superoxide production. Forty-nine NMR-observable metabolites underwent a multivariate analysis. Separated clustering in scores plots was found for treatment and outcome groups. Final ATP (adenosine triphosphate) levels, severely decreased at normothermia, were restored equally by immediate and delayed hypothermia. Cell death was decreased by immediate hypothermia, but was equally substantially greater with normothermia and delayed hypothermia. Potentially important biomarkers in the (1)H spectra included PCr-(1)H (phosphocreatine in the (1)H spectrum), ATP-(1)H (adenosine triphosphate in the (1)H spectrum), and ADP-(1)H (adenosine diphosphate in the (1)H spectrum). The findings suggest a potential role for metabolomic monitoring during therapeutic hypothermia.

Biophysics is the study of the complex physical processes occurring in biological systems that are responsible for life. This dissertation addresses three important topics in biophysics: ionic transport, biomineralization, and force spectroscopy. Ionic transport involves the passage of ions through a special class of hollow, transmembrane proteins called ion channels which regulate the movement of charged species across nearly all biological membranes with varying degrees of specificity. Despite the fundamental importance of these channels to many physiological processes little is known about how channel structure and composition couple to determine its function. Deriving inspiration from these systems, a simple computational platform is developed to study the salient features of these channels in order to better understand the fundamental physics of these systems. The results of this work indicate that a converging-diverging region formed within the pore to create a single constriction is the most effective method to regulate the passage of ions through the pore. By controlling the geometry of the constriction the local potential and chemical gradients can be manipulated to tailor the channel for specific applications. The process of selective extraction and incorporation of local elements from the surrounding environment into functional structures under strict biological control is known as biomineralization. As an initial step to gain a more fundamental understanding of directed crystallization of zinc oxide molecular dynamics simulations were performed to study the conformational behavior of two experimentally derived biomimetic peptides in a precursor solution. Substantial differences in the conformational properties and affinity for zinc and hydroxide ions in solution were observed. These findings are in qualitative agreement with experimental observations. The mechanical response of biopolymers such as RNA and DNA to externally applied forces is a topic that has received wide interest both experimentally and theoretically. In the first of two separate force spectroscopy studies, the mechanical response of linear uncharged polymer chains of variable molecular weight subjected to repeated pulling-retraction cycles in poor solvent was investigated. It was found that the observed hysteresis in this system is highly dependent on the speed at which the chain is perturbed. In the second study, the force-induced globule-coil transition of a linear polyelectrolyte chain in poor solvent was examined. It was observed that the magnitude of the change in the degree of ionization of the chain at the transition is a strong function of counterion size and Coulombic strength.

An online course in wind energy resource assessment has been developed by the Technical University of Denmark (DTU). The course builds upon a successful physical course, which the Department of Wind Energy at DTU has offered to the wind energy industry for more than 20 years. The course objectives are:1. To teach participants to use the Wind Atlas Analysis and Application Program (WAsP)2. To provide participants with enough theory about wind power meteorology to avoid the major pitfalls related to wind resource assessment.WAsP is the wind power industry-standard PC-software for wind resource assessment and siting of wind turbines and wind farms, with more than 4,000 licenses sold in more than 100 countries.

In this project, a more realistic representation of gusts, based on statistical analysis, will account for the variability observed in real-world gusts. The gustrepresentation will focus on temporal, spatial, and velocity scales that are relevant for modern wind turbines and which possibly affect the loads. Emphasis will be put on gust rise time and velocity jump, within the context of extreme as well as normal turbulence.

This report presents a test and validation of extreme wind calculation applying the Spectral Correction method as implemented in the WAsP Engineering 4 software package. The test and validation is based on four sites located in Denmark, one site located in the Netherlands and one site located in the USA. Calculations have been carried out using wind data from on-site meteorological masts as well as long-term reference wind data.

Parameterizing the effect of vertically-distributed vegetation through an effective roughness (z0,eff) – whereby momentum loss through a three-dimensional foliage volume is represented as momentum loss over an area at one vertical level – can facilitate the use of forest data in flow models, to any level of detail, and simultaneously reduce computational cost. Results of numerical experiments and comparison with observations show that a modelling approach based on z0,eff can estimate wind speed and turbulence levels over forested areas, at heights of interest for wind energy applications (∼ 60 m and higher), but only above flat terrain. Caution must be exercised in the application of such a model to zones of forest edges. Advanced flow models capable of incorporating local (distributed) drag forces are recommended for complex terrain covered by forest.

Using the Wind Atlas methodology to predict the average wind speed at one location from measured climatological wind frequency distributions at another nearby location we analyse the relative prediction errors using a linearized flow model (IBZ) and a more physically correct fully non-linear 3D flow model (CFD) for a number of sites in very complex terrain (large terrain slopes). We first briefly describe the Wind Atlas methodology as implemented in WAsP and the specifics of the “classical” model setup and the new setup allowing the use of the CFD computation engine. We discuss some known shortcomings of the linear orographic flow model (BZ) and possible modifications that could be considered, including the established RIX method.

We present a method for generalizing wind climatologies generated from mesoscale model output (e.g. the Weather, Research and Forecasting (WRF) model.) The generalization procedure is based on Wind Atlas framework of WAsP and KAMM/WAsP, and been extensively in wind resources assessment in DTU Wind Energy for many years (Mortensen et al 2005, Badger et al 2010). The method is verified by two distinct sets of WRF simulations: A single WRF wind atlas over Denmark, and a series of WRF simulations with the Vortex MAPS system with 29 sites in many wind climate regimes. Comparison is made between generalized wind climatologies estimated by the microscale model WAsP and the methodology presented here. For the Danish wind measurements the mean absolute error in the ‘raw’ wind speeds is 9.2%, while the mean absolute error in the generalized wind speeds is 4.1%. The generalization procedure has been installed and automated in the Vortex downscaling chain for the MAPS calculation, which are computed at 3 km horizontal resolution grid. For the MAPS-derived wind speeds the mean absolute error in the ‘raw’ wind speeds is 17.3%, while the mean absolute error in thegeneralized winds is 10.7%.

Background: The role of adjuvant therapy in patients with oesophagogastric adenocarcinoma treated by neoadjuvant chemotherapy (NAC) and surgery is contentious. In UK practice, surgical resection margin status is often used to classify patients into receiving adjuvant treatment. This study aimed to assess any survival benefit of adjuvant therapy in patients with clear resection margins. Methods: This was a retrospective collaborative cohort study combining two prospectively collected UK institutional databases of patients with oesophageal adenocarcinoma. Multivariable Cox regression and propensity matched analyses were used to compare overall and recurrence-free survival according to the adjuvant treatment. Results: Of 374 patients with clear resection margins, 221 patients (59%) had no adjuvant treatment, 137 patients (37%) had adjuvant chemotherapy and 16 patients (4%) had adjuvant chemoradiotherapy. For patients who had received NAC (290, 76%), when adjuvant chemotherapy was compared to no adjuvant treatment, hazard ratios (HRs) favoured adjuvant chemotherapy but did not reach independent significance (overall survival [OS] HR 0.65 95% confidence interval [CI] 0.40–1.06; p.0.087). Responders to NAC (Mandard 1–3) were seemingly more likely to demonstrate a survival benefit from adjuvant chemotherapy (HR 0.42 95% CI 0.15–1.11; p.1.081). Conclusions: Although no independent survival benefit was observed, the point estimates favoured adjuvant treatment, predominantly in patients with chemo-responsive tumours. Refereed/Peer-reviewed