ABCC6 plays a significant role in the transport of nilotinib and dasatinib, and contributes to TKI resistance in vitro, in both cell lines and primary patient mononuclear cells

ATP Binding Cassette family efflux proteins ABCB1 and ABCG2 have previously been demonstrated to interact with Tyrosine Kinase Inhibitors (TKIs); however, evidence for the interaction of other potentially relevant drug transporters with TKIs is lacking. Through Taqman transporter array technology we assessed the impact of nilotinib on mRNA expression of ABC transporters, with ABCC6 identified as a transporter of interest. Additionally, increased expression of ABCC6 mRNA was observed during in vitro development of nilotinib resistance in BCR-ABL1-expressing cell lines. K562 cells exposed to gradually increasing concentrations of nilotinib (to 2 μM) expressed up to 57-fold higher levels of ABCC6 mRNA when compared with control cells (p = 0.002). Analogous results were observed in nilotinib resistant K562-Dox cells (up to 33-fold higher levels of ABCC6, p = 0.002). IC50 experiments were conducted on patient mononuclear cells in the absence and presence of three ABCC6 inhibitors: indomethacin, probenecid and pantoprazole. Results demonstrated that all three inhibitors significantly reduced nilotinib IC50 (p<0.001) indicating ABCC6 is likely involved in nilotinib transport. Cell line data confirmed these findings. Similar results were obtained for dasatinib, but not imatinib. Combined, these studies suggest that nilotinib and dasatinib are likely substrates of ABCC6 and to our knowledge, this is the first report of ABCC6 involvement in TKI transport. In addition, ABCC6 overexpression may also contribute to nilotinib and dasatinib resistance in vitro. With nilotinib and dasatinib now front line therapy options in the treatment of CML, concomitant administration of ABCC6 inhibitors may present an attractive option to enhance TKI efficacy.Laura N. Eadie, Phuong Dang, Jarrad M. Goyne, Timothy P. Hughes, Deborah L. Whit

Status of Turbulence Modeling for Hypersonic Propulsion Flowpaths

This report provides an assessment of current turbulent flow calculation methods for hypersonic propulsion flowpaths, particularly the scramjet engine. Emphasis is placed on Reynolds-averaged Navier-Stokes (RANS) methods, but some discussion of newer meth- ods such as Large Eddy Simulation (LES) is also provided. The report is organized by considering technical issues throughout the scramjet-powered vehicle flowpath including laminar-to-turbulent boundary layer transition, shock wave / turbulent boundary layer interactions, scalar transport modeling (specifically the significance of turbulent Prandtl and Schmidt numbers) and compressible mixing. Unit problems are primarily used to conduct the assessment. In the combustor, results from calculations of a direct connect supersonic combustion experiment are also used to address the effects of turbulence model selection and in particular settings for the turbulent Prandtl and Schmidt numbers. It is concluded that RANS turbulence modeling shortfalls are still a major limitation to the accuracy of hypersonic propulsion simulations, whether considering individual components or an overall system. Newer methods such as LES-based techniques may be promising, but are not yet at a maturity to be used routinely by the hypersonic propulsion community. The need for fundamental experiments to provide data for turbulence model development and validation is discussed

BCR-ABL1 genomic DNA PCR response kinetics during first-line imatinib treatment of chronic myeloid leukemia

Accurate quantification of minimal residual disease during treatment of chronic myeloid leukaemia guides clinical decisions. The conventional minimal residual disease method, RQ-PCR for BCR-ABL1 mRNA, reflects a composite of the number of circulating leukemic cells and the BCR-ABL1 transcripts per cell. BCR-ABL1 genomic DNA only reflects leukemic cell number. We used both methods in parallel to determine the relative contribution of the leukemic cell number to molecular response. BCR-ABL1 DNA PCR and RQ-PCR were monitored up to 24 months in 516 paired samples from 59 newly-diagnosed patients treated with first-line imatinib in the TIDEL-II study. In the first 3 months of treatment BCR-ABL1 mRNA values declined more rapidly than DNA. By 6 months the two measures aligned closely. The expression of BCR-ABL1 mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2 BCR-ABL1 was lower than that of e14a2 transcripts at multiple time points during treatment. BCR-ABL1 DNA was quantifiable in 48% of samples with undetectable BCR-ABL1 mRNA, resulting in minimal residual disease being quantifiable for an additional 5-18 months (median 12 months). These parallel studies show for the first time that the rapid decline in BCR-ABL1 mRNA over the first 3 months of treatment is due to a reduction in both cell number and transcript level per cell, whereas beyond 3 months falling levels of BCR-ABL1 mRNA are predominantly due to depletion of leukaemic cells.Ilaria S. Pagani, Phuong Dang, Ivar O. Kommers, Jarrad M. Goyne, Mario Nicola, Verity A. Saunders, Jodi Braley, Deborah L. White, David T. Yeung, Susan Branford, Timothy P. Hughes, and David M. Ros

Quantification of the 2-Deoxyribonolactone and Nucleoside 5 '-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of gamma-Radiation and Fe2+-EDTA

The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC−MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1′-oxidation and the nucleoside 5′-aldehyde of 5′-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC−MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5′-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin γ1I, was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5′-aldehyde per 106 nt per μM in accord with its established minor 1′- and major 5′-oxidation chemistry. Calicheamicin unexpectedly caused 1′-oxidation at a low level of 10 2-deoxyribonolactone per 106 nt per μM in addition to the expected predominance of 5′-oxidation at 560 nucleoside 5′-aldehyde per 106 nt per μM. The two hydroxyl radical-mediated DNA oxidants, γ-radiation and Fe2+−EDTA, produced nucleoside 5′-aldehyde at a frequency of 57 per 106 nt per Gy (G-value 74 nmol/J) and 3.5 per 106 nt per μM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, γ-radiation and Fe2+−EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 106 nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 106 nt per μM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5′-aldehyde of 9.7 and 73 lesions per 106 nt, respectively. γ-Irradiation of the cells caused increases of 0.045 and 0.22 lesions per 106 nt per Gy, respectively, which represents a 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by γ-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between γ-radiation and Fe2+−EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.National Institute of Environmental Health Sciences (ES002109)National Center for Research Resources (U.S.) (RR023783-01)National Center for Research Resources (U.S.) (RR017905-01)National Cancer Institute (U.S.) (CA103146

Skin-friction measurements in high-enthalpy hypersonic boundary layers

Skin-friction measurements are reported for high-enthalpy and high-Mach-number laminar, transitional and turbulent boundary layers. The measurements were performed in a free-piston shock tunnel with air-flow Mach number, stagnation enthalpy and Reynolds numbers in the ranges of 4.4-6.7, 3-13 MJ kg(-1) and 0.16 x 10(6)-21 x 10(6), respectively. Wall temperatures were near 300 K and this resulted in ratios of wall enthalpy to flow-stagnation enthalpy in the range of 0.1-0.02. The experiments were performed using rectangular ducts. The measurements were accomplished using a new skin-friction gauge that was developed for impulse facility testing. The gauge was an acceleration compensated piezoelectric transducer and had a lowest natural frequency near 40 kHz. Turbulent skin-friction levels were measured to within a typical uncertainty of +/-7%. The systematic uncertainty in measured skin-friction coefficient was high for the tested laminar conditions; however, to within experimental uncertainty, the skin-friction and heat-transfer measurements were in agreement with the laminar theory of van Driest (1952). For predicting turbulent skin-friction coefficient, it was established that, for the range of Mach numbers and Reynolds numbers of the experiments, with cold walls and boundary layers approaching the turbulent equilibrium state, the Spalding & Chi (1964) method was the most suitable of the theories tested. It was also established that if the heat transfer rate to the wall is to be predicted, then the Spalding & Chi (1964) method should be used in conjunction with a Reynolds analogy factor near unity. If more accurate results are required, then an experimentally observed relationship between the Reynolds analogy factor and the skin-friction coefficient may be applied

Shock tunnel skin friction measurement in a supersonic combustor

Shock tunnel measurements are reported of skin friction with supersonic hydrogen-air combustion in a constant area duct. A floating element skin friction gauge was used, in which the shear force was applied directly to a piezoceramic measuring element. The experiments were conducted at stagnation enthalpies of 5.7 and 6.8 MJ kg, a precombustion Mach number of approximately 4.5, and with a maximum duct Reynolds number of 1.3x10. The measurements showed that the skin friction coefficient was unaffected by supersonic combustion, and could be predicted by the correlation of Spalding and Chi. Measurements of heat transfer also established that Reynolds analogy could be used in both the fuel-off and fuel-on flows

Shock-tunnel skin-friction measurement in a supersonic combustor

Shock-tunnel measurements are reported of skin friction with supersonic hydrogen-air combustion in a constant area duct. A floating-element skin-friction gauge was used, in which the shear force was applied directly to a piezoceramic measuring element. The experiments were conducted at stagnation enthalpies of 5.7 and 6.8 MJ kg(-1), a precombustion Mach number of similar to 4.5, and with a maximum duct Reynolds number of 1.3 x 10(7). The measurements showed that, although supersonic combustion caused the skin friction to fluctuate with time, it did not affect the mean value of the skin friction coefficient, and this mean value could be predicted using existing turbulent: boundary-layer theory. Measurements of heat transfer also established that Reynolds analogy could be used in both the fuel-off and fuel-on flows