Search

Apoptosis resistance contributes to treatment failure in colorectal cancer (CRC). New treatments that reinstate apoptosis competency have potential to improve patient outcome but require predictive biomarkers to target them to responsive patient populations. Inhibitor of apoptosis proteins (IAPs) suppress apoptosis, contributing to drug resistance; IAP antagonists such as TL32711 have therefore been developed. We developed a systems biology approach for predicting response of CRC cells to chemotherapy and TL32711 combinations in vitro and in vivo. CRC cells responded poorly to TL32711 monotherapy in vitro; however, co-treatment with 5-Fluorouracil (5-FU) and oxaliplatin enhanced TL32711-induced apoptosis. Notably, cells from genetically identical populations responded highly heterogeneously, with caspases being activated both upstream and downstream of mitochondrial outer membrane permeabilisation (MOMP). These data, combined with quantities of key apoptosis regulators were sufficient to replicate in vitro cell death profiles by mathematical modelling. In vivo, apoptosis protein expression was significantly altered, and mathematical modelling for these conditions predicted higher apoptosis resistance that could nevertheless be overcome by combination of chemotherapy and TL32711. Subsequent experimental observations agreed with these predictions, and the observed effects on tumour growth inhibition correlated robustly with apoptosis competency. We therefore obtained insights into intracellular signal transduction kinetics and their population-based heterogeneities for chemotherapy/TL32711 combinations and provide proof-of-concept that mathematical modelling of apoptosis competency can simulate and predict responsiveness in vivo. Being able to predict response to IAP antagonist-based treatments on the background of cell-to-cell heterogeneities in the future might assist in improving treatment stratification approaches for these emerging apoptosis-targeting agents.

Genotoxic chemotherapy with temozolomide (TMZ) is a mainstay of treatment for glioblastoma (GBM); however, at best, TMZ provides only modest survival benefit to a subset of patients. Recent insight into the heterogeneous nature of GBM suggests a more personalized approach to treatment may be necessary to overcome cancer drug resistance and improve patient care. These include novel therapies that can be used both alone and with TMZ to selectively reactivate apoptosis within malignant cells. For this approach to work, reliable molecular signatures that can accurately predict treatment responsiveness need to be identified first. Here, we describe the first proof-of-principle study that merges quantitative protein-based analysis of apoptosis signaling networks with data- and knowledge-driven mathematical systems modeling to predict treatment responsiveness of GBM cell lines to various apoptosis-inducing stimuli. These include monotherapies with TMZ and TRAIL, which activate the intrinsic and extrinsic apoptosis pathways, respectively, as well as combination therapies of TMZ+TRAIL. We also successfully employed this approach to predict whether individual GBM cell lines could be sensitized to TMZ or TRAIL via the selective targeting of Bcl-2/Bcl-xL proteins with ABT-737. Our findings suggest that systems biology-based approaches could assist in personalizing treatment decisions in GBM to optimize cell death induction.

Due to the lack of effective treatments for glioblastoma (GBM), we here studied the responsiveness of GBM cell lines to the combination of death ligand, TRAIL and the IAP antagonist, TL32711 (Birinapant). Responses were highly heterogeneous, with synergistic apoptosis as well as treatment resistance observed. Caspase-8 and Bid, together with caspase-3, form a nonlinear signalling hub that efficiently induced apoptosis in responder cell lines. Cells resistant to TRAIL/TL32711 expressed low amounts of procaspase-8 and Bid and poorly activated caspase-3. We therefore hypothesised that improving caspase-8 activation or sensitising mitochondria to truncated Bid (tBid) could convert non-responder GBM cell lines to responders. Mathematical simulations of both strategies predicted mitochondrial sensitization to tBid would outperform enhancing caspase-8 activation. Indeed, antagonising Bcl-2 by ABT-199 allowed TRAIL/TL32711 response synergies to manifest in otherwise TRAIL resistant cell lines. These findings were further corroborated in experiments with a translationally relevant hexavalent TRAIL variant. Our study therefore demonstrates that a high caspase-8/Bid signature is associated with synergistic TRAIL/TL32711-induced apoptosis in GBM cells and outlines Bcl-2 antagonism as a highly potent intervention to sensitize highly TRAIL-resistant GBM cells to TRAIL/TL32711 combination treatment.

New quenched-in face-centred cubic fluorite-type materials were synthesised in the Bi2O3–Yb2O3–PbO system. After annealing in air at 500 °C for up to 200 h, each material underwent a conductivity-lowering structural transformation, thus making them unsuitable for use as solid electrolytes in solid-oxide fuel cells. For example, (BiO1.5)0.80(YbO1.5)0.17(PbO)0.03 underwent a fluorite- to Bi17Yb7O36-type orthorhombic transformation, indicative of long-range cation ordering, and (BiO1.5)0.80(YbO1.5)0.11(PbO)0.09 underwent a fluorite- to β-Bi2O3-type tetragonal transformation, indicative of long-range 〈001〉 oxide-ion vacancy ordering.

Glioblastoma (GBM) is the most common primary brain tumor with no available cure. As previously described, seliciclib, a first-generation cyclin-dependent kinase (CDK) inhibitor, down-regulates the anti-apoptotic protein, Mcl-1, in GBM, thereby sensitizing GBM cells to the apoptosis-inducing effects of the death receptor ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we have assessed the efficacy of seliciclib when delivered in combination with the antibody against human death receptor 5, drozitumab, in clinically relevant patient-derived xenograft (PDX) models of GBM. A reduction in viability and significant levels of apoptosis were observed in vitro in human GBM neurospheres following treatment with seliciclib plus drozitumab. While the co-treatment strategy induced a similar effect in PDX models, the dosing regimen required to observe seliciclib-targeted responses in the brain, resulted in lethal toxicity in 45% of animals. Additional studies showed that the second-generation CDK inhibitor, CYC065, with improved potency in comparison to seliciclib, induced a significant decrease in the size of human GBM neurospheres in vitro and was well tolerated in vivo, upon administration at clinically relevant doses. This study highlights the continued need for robust pre-clinical assessment of promising treatment approaches using clinically relevant models.

Apoptosis signaling crucially depends on caspase activities. Caspase-2 shares features of both initiator and effector caspases. Opinions are divided on whether caspase-2 activity is established during apoptosis initiation or execution in response to DNA damage, death receptor stimulation, or heat shock. So far, approaches towards measuring caspase-2 activity were restricted to analyses in cell homogenates and extracts, yielded inconsistent results, and were often limited in sensitivity, thereby contributing to controversies surrounding the role of caspase-2 during apoptosis. Furthermore, caspases overlap in substrate specificities, and caspase-8 as well as effector caspases may cleave the optimal VDVAD recognition motif as well. We therefore generated a highly sensitive Förster resonance energy transfer (FRET) substrate to determine the relative contribution of these caspases to VDVADase activity non-invasively inside living cells. We observed limited proteolysis of the substrate during apoptosis initiation in response to death receptor stimulation by FasL, TNFα and TRAIL. However, this activity was attributable to caspase-8 rather than caspase-2. Likewise, no caspase-2-specific activity was detected during apoptosis initiation in response to genotoxic stress (cisplatin, 5-FU), microtubule destabilization (vincristine), or heat shock. The contribution of caspase-2 to proteolytic activities during apoptosis execution was insignificant. Since even residual, ectopically introduced caspase-2 activity could readily be detected inside living cells in our measurements, we conclude, in contrast to several previous studies, that caspase-2 activity does not contribute to apoptosis in the scenarios investigated, and that instead caspase-8 and effector caspases are the most significant VDVADases during canonical apoptosis signaling.

New quenched-in fluorite-type materials with composition (BiO 1.5)0.94-x(LaO1.5)0.06(PbO) x, x = 0.02, 0.03, 0.04 and 0.05, were synthesised by solid state reaction. The new materials undergo a number of phase transformations during heating between room temperature and 750 °C, as indicated by differential thermal analysis. Variable temperature X-ray diffraction performed on the material (BiO1.5)0.92(LaO1.5) 0.06(PbO)0.02 revealed that the quenched-in fcc fluorite-type material first undergoes a transformation to a β-Bi 2O3-type tetragonal phase around 400 °C. In the range 450-700 °C, α-Bi2O3-type monoclinic, Bi 12PbO19-type bcc and β1/β 2-type rhombohedral phases, and what appeared to be a ε-type monoclinic phase, were observed, before a single-phase fluorite-type material was regained at 750 °C. © 2010 Elsevier Ltd © 2011 Elsevier Ltd. All rights reserved.

Quenched-in fcc fluorite-type materials in the Bi 2 O 3 –Er 2 O 3 –PbO system were annealed in air at 500 and 600 °C for up to 2000 h. Each material experienced a conductivity-lowering structural transformation, thus making them unsuitable for use in SOFCs. For example, the materials (BiO 1.5 ) 0.80 (ErO 1.5 ) 0.20− x (PbO) x , x = 0.03, 0.06 and 0.09, underwent a fluorite-type to tetragonal transformation during annealing at 500 °C due to 〈100〉 oxide-ion vacancy ordering, and the rate of conductivity decay at 500 °C increased with increasing Pb 2+ /Er 3+ ratio.

The mechanochemical milling of arsenopyrite, pyrite and pyrrhotite in the presence of SO2 has demonstrated the potential for inducing gas/solid reactions at low temperature. Both reactants and milled products were characterised by powder X-ray diffractometry and by scanning electron microscopy of polished sections. The gas compositions were determined by mass spectrometry. Milling in an atmosphere of SO2 results in the conversion of arsenopyrite to pyrite via an intermediate pyrrhotite phase. Milling pyrite or pyrrhotite results in a continuous cyclic process where pyrite decomposes to pyrrhotite, which then undergoes resulfurisation back to pyrite.

Charge contrast images (CCI) of synthetic gibbsite obtained on an environmental scanning electron microscope gives information on the crystallization process. Furthermore, X-ray mapping of the same grains shows that impurities are localized during the initial stages of growth and that the resulting composition images have features similar to these observed in CCI. This suggests a possible correlation between impurity distributions and the emission detected during CCI. X-ray line profiles, simulating the spatial distribution of impurities derived from the Monte Carlo program CASINO, have been compared with experimental line profiles and give an estimate of the localization. The model suggests that a main impurity, Ca, is depleted from the solution within approximately 3–4 μm of growth.

Charge contrast images (CCI) of synthetic gibbsite obtained on an environmental scanning electron microscope gives information on the crystallization process. Furthermore, X-ray mapping of the same grains shows that impurities are localized during the initial stages of growth and that the resulting composition images have features similar to these observed in CCI. This suggests a possible correlation between impurity distributions and the emission detected during CCI. X-ray line profiles, simulating the spatial distribution of impurities derived from the Monte Carlo program CASINO, have been compared with experimental line profiles and give an estimate of the localization. The model suggests that a main impurity, Ca, is depleted from the solution within approximately 3–4 μm of growth.

The decomposition of monazite mineral with calcium oxide and calcium chloride during mechanical milling has been investigated. Various milling times, gaseous atmospheres and charge ratios were tested to optimize the reaction conditions. Powder X-ray diffractometry, scanning electron microscopy and energy dispersive spectrometry were used to characterize the decomposition products. It has been found that the monazite was effectively decomposed in 12 h, under an inert argon atmosphere with a 15:1 charge ratio, producing rare earth element oxychloride and oxide, calcium chlorophosphate (chlorapatite) and thorium dioxide. About 95% of the monazite milled under these conditions may be extracted into 6 M nitric acid solution. Lowering the charge ratio or changing the milling atmosphere to air slows down the reaction rate and reduces the dissolution fraction. When sufficient air (oxygen) was present in the vial during milling, a cerium and thorium dioxide solid solution was formed, which is detrimental to acid leach.

Fluorite-type fcc phases have been synthesised in the system Bi 2 O 3 –Er 2 O 3 –PbO by solid state reaction, and a partial air-quenchable domain of the fluorite-type phase has been established. Some of these materials display high oxide ion conductivities, notably (BiO 1.5 ) 0.80 (ErO 1.5 ) 0.11 (PbO) 0.09 and (BiO 1.5 ) 0.85 (ErO 1.5 ) 0.12 (PbO) 0.03 , which have conductivities of 0.49 and 0.72 S cm − 1 at 750 °C, respectively, placing them among the most conductive Bi 2 O 3 -based materials. Conductivity was found to increase with increasing Pb 2+ /Er 3+ ratio and decreasing (Er 3+ + Pb 2+ )/Bi 3+ ratio. Positional disorder in the oxide ion sublattice was characterised by neutron powder diffraction. At room temperature, the oxide ion sublattice appeared to be completely disordered, with oxide ions only in 32 f and 48 i sites, and changes in occupancy with increasing Pb 2+ /Er 3+ and (Er 3+ + Pb 2+ )/Bi 3+ ratios were not significant. At 700 °C, there appeared to be oxide ions in 8 c sites for the material (BiO 1.5 ) 0.80 (ErO 1.5 ) 0.11 (PbO) 0.09 , with a correspondingly smaller occupancy of the 32 f sites, whilst the occupancy of the 48 i sites had not changed significantly.

An iterative process of serial sectioning and charge contrast imaging has been applied to gibbsite particles embedded in epoxy resin. The features observed in the two-dimensional sections have been reconstructed into three-dimensional (3-D) objects, so that the volumetric relationships in the microstructure are visualised. The 3-D objects confirm that the image detail present in charge contrast images of gibbsite is related to the microstructure and the processes occurring during the crystallisation of gibbsite. Speculative explanations for what these microstructures may represent are presented.

We briefly review the nature and provenance of extremely weathered ilmenite, then investigate its fate when it is 10 % of a blended feed in the reduction kilns of the Becher process. There it is bound, unintentionally and intermittently, in sinter, which is discarded. It takes with it discrete aluminosilicate grains as well as contaminants, adsorbed and occluded within the ilmenite. Two distinct sintering mechanisms are identified. A layered wall accretion forms purely by solid state reaction, and is thickest early in the kiln. It occasionally detaches under its own weight or is eroded, and is tolerated between scheduled shutdowns for maintenance. However, if the weathered ilmenite and accompanying silica escape earlier wall accretion, a transitory aluminosilicate melt may later promote sintering of this material within the bed, adhering catastrophically to constrictions and obstructions on the kiln wall. The mechanisms and controlling factors are discussed.

Charge Contrast Imaging (CCI), is a recently reported technique allowing the imaging of the internal microstructure of non and poorly conductive materials. In previous work, the relationship between controlled growth events in batch-precipitated gibbsite, Al(OH)3and the appearance of contrasting bands in CCIs has been empirically correlated. The data presented was spatial, based on the number and dimensions of growth rings, however no interpretation of the contrast variation was made. Suggestions that the contrast may be related to charge trapping, which in turn is related to conductivity pathways and impurity concentrations indicates that the technique may provide insight into crystallisation processes. The current work examines the spatial distribution of impurity elements and their effect on the contrast observed within growth layers in charge contrast images of gibbsite.Analyses for Ca, Fe and Si, the main impurities in the solutions before and after precipitation indicate a preferential partitioning of Ca and to a lesser extent Si and Fe into gibbsite.

Three-dimensional reconstructions are a useful visualisation aid for understanding the volumetric relationships between features in an object. Typically, three dimensional reconstructions have evolved with regards to biological applications, where the collection and manipulation of the data has matured to a point where volumetric rendering has become almost routine. Two common data collection procedures involve either virtual sectioning using CLSM, where layers within the object are imaged successively without mechanical sectioning, or alternatively the projection of the object is imaged, via TEM while being rotated through a range of angles. This paper describes a novel application of three dimensional imaging by collecting 2 dimensional serial section images using a recently described technique for imaging materials using a VPSEM, known as Charge Contrast Imaging.Figure 1 shows a typical image of a polished grain of gibbsite. Previous work has correlated the appearance of the concentric rings to laboratory controlled precipitation events, thus showing the technique provides information on the precipitation history

Lattice images of the niobium oxides, structures based on the linkage of octahedral groups in continuous networks, occasionally contain features recognizable as dislocations. Since lattice imaging enables the micro-structure to be resolved in great detail, at the level of local structural organization, it is possible to determine the configuration, and also to infer the chemical composition, of dislocated areas. By treating the niobium oxide ‘block’ structures as superstructures of the ReO3(DO9) type, the topology of dislocations can be expressed by relations between the insertion (or deletion) of one or more half-planes of cations, or of oxygen atoms only, changes in the number of crystallographic shear plane interfaces between blocks or columns, changes in (idealized) dimensions and any requisite distortion in the third dimension. Mapping the structure around a dislocation, from the lattice image, is directly equivalent to plotting the Burgers’ circuit. In this way, the precise nature of a dislocating perturbation and its implications for the local chemical composition of the crystal can be directly identified. The method is exemplified by analysis of dislocations and of related extended defects of several types, associated with twinning phenomena, semicoherent intergrowth between different ReO3-type superstructures and arrays building up a low angle boundary. The essential features of the analysis are not restricted to structures of the niobium oxide type, but can be extended to other types of polyhedron networks.

The formation of mixed phases in the system MgF2+ Nb2O5 has been examined, on the grounds that similarity in ionic radii between Mg and Ti, and between F and O, should lead to the analogues of the known titanium–niobium block structure oxides. Electron diffraction and direct electron microscope lattice imaging methods show that, in addition to the compound already described as MgNb14O35F2, with the (5 × 3)∞ structure, phases iso-structural with H-Nb2O5, N-Nb2O5, TiNb24O62, and Ti2Nb10O29 are formed. By compensatory substitution, each of these structures represents a solid solution series and mixtures with compositions on the MgF2–Nb2O5 tie line yield multiphase products. Regular intergrowth structures may be formed between the basic structural types.