A 39 kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome

Transfected human apoptosis signal-regulating kinase 1 (ASK1) produces a 150 kDa protein. However, we have detected endogenous ASK1 predominantly as 39 and 50 kDa C-terminal and 75 and 110 kDa N-terminal fragments in a panel of nontransfected cancer cell lines and HUVEC endothelial cells. This suggests that in nonapoptotic cells, endogenous ASK1 protein is normally cleaved at a number of specific sites, some of which are in the kinase domain. Transfected ASK1 protein is known to be degraded by the proteasome. In contrast, the cleavage of endogenous ASK1 is independent of the proteasome as treatment with the proteasome inhibitor, lactacystin did not inhibit cleavage. Cisplatin treatment decreased the amount of 39 kDa C-terminal ASK1 fragments in mutant p53 cell lines suggesting a decrease in cleavage associated with apoptosis. Transfected ASK1 may, therefore, not accurately reflect the role of endogenous ASK1

Ozone depletion and chlorine loading potentials

The recognition of the roles of chlorine and bromine compounds in ozone depletion has led to the regulation or their source gases. Some source gases are expected to be more damaging to the ozone layer than others, so that scientific guidance regarding their relative impacts is needed for regulatory purposes. Parameters used for this purpose include the steady-state and time-dependent chlorine loading potential (CLP) and the ozone depletion potential (ODP). Chlorine loading potentials depend upon the estimated value and accuracy of atmospheric lifetimes and are subject to significant (approximately 20-50 percent) uncertainties for many gases. Ozone depletion potentials depend on the same factors, as well as the evaluation of the release of reactive chlorine and bromine from each source gas and corresponding ozone destruction within the stratosphere

Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines

Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. •Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner. •The technique is quick, affordable and eliminates sample manipulation. •The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines

Relative effects on stratospheric ozone of halogenated methanes and ethanes of social and industrial interest

Four atmospheric modeling groups have calculated relative effects of several halocarbons (chlorofluorocarbons (CFC's)-11, 12, 113, 114, and 115; hydrochlorofluorocarbons (HCFC's) 22, 123, 124, 141b, and 142b; hydrofluorocarbons (HFC's) 125, 134a, 143a, and 152a, carbon tetrachloride; and methyl chloroform) on stratospheric ozone. Effects on stratospheric ozone were calculated for each compound and normalized relative to the effect of CFC-11. These models include the representations for homogeneous physical and chemical processes in the middle atmosphere but do no account for either heterogeneous chemistry or polar dynamics which are important in the spring time loss of ozone over Antarctica. Relative calculated effects using a range of models compare reasonably well. Within the limits of the uncertainties of these model results, compounds now under consideration as functional replacements for fully halogenated compounds have modeled stratospheric ozone reductions of 10 percent or less of that of CFC-11. Sensitivity analyses examined the sensitivity of relative calculated effects to levels of other trace gases, assumed transport in the models, and latitudinal and seasonal local dependencies. Relative effects on polar ozone are discussed in the context of evolving information on the special processes affecting ozone, especially during polar winter-springtime. Lastly, the time dependency of relative effects were calculated

In vitro development of chemotherapy and targeted therapy drug-resistant cancer cell lines: a practical guide with case studies

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance

GP-SUM. Gaussian Processes Filtering of non-Gaussian Beliefs

This work studies the problem of stochastic dynamic filtering and state propagation with complex beliefs. The main contribution is GP-SUM, a filtering algorithm tailored to dynamic systems and observation models expressed as Gaussian Processes (GP), and to states represented as a weighted sum of Gaussians. The key attribute of GP-SUM is that it does not rely on linearizations of the dynamic or observation models, or on unimodal Gaussian approximations of the belief, hence enables tracking complex state distributions. The algorithm can be seen as a combination of a sampling-based filter with a probabilistic Bayes filter. On the one hand, GP-SUM operates by sampling the state distribution and propagating each sample through the dynamic system and observation models. On the other hand, it achieves effective sampling and accurate probabilistic propagation by relying on the GP form of the system, and the sum-of-Gaussian form of the belief. We show that GP-SUM outperforms several GP-Bayes and Particle Filters on a standard benchmark. We also demonstrate its use in a pushing task, predicting with experimental accuracy the naturally occurring non-Gaussian distributions.Comment: WAFR 2018, 16 pages, 7 figure

Theory and observations: Model simulations of the period 1955-1985

The main objective of the theoretical studies presented here is to apply models of stratospheric chemistry and transport in order to understand the processes that control stratospheric ozone and that are responsible for the observed variations. The model calculations are intended to simulate the observed behavior of atmospheric ozone over the past three decades (1955-1985), for which there exists a substantial record of both ground-based and, more recently, satellite measurements. Ozone concentrations in the atmosphere vary on different time scales and for several different causes. The models described here were designed to simulate the effect on ozone of changes in the concentration of such trace gases as CFC, CH4, N2O, and CO2. Changes from year to year in ultraviolet radiation associated with the solar cycle are also included in the models. A third source of variability explicitly considered is the sporadic introduction of large amounts of NO sub x into the stratosphere during atmospheric nuclear tests

ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair

Background: The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results: A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion: Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1