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Background: Optimal expression and proper function of key mitotic proteins facilitate control and repair processes that aim to prevent loss or gain of chromosomes, a hallmark of cancer. Altered expression of small regulatory microRNAs is associated with tumourigenesis and metastasis but the impact on mitotic signalling has remained unclear.Methods: Cell-based high-throughput screen identified miR-378a-5p as a mitosis perturbing microRNA. Transient transfections, immunofluorescence, western blotting, time-lapse microscopy, FISH and reporter assays were used to characterise the mitotic anomalies by excess miR-378a-5p. Analysis of microRNA profiles in breast tumours was performed.Results: Overexpression of miR-378a-5p induced numerical chromosome changes in cells and abrogated taxol-induced mitotic block via premature inactivation of the spindle assembly checkpoint. Moreover, excess miR-378a-5p triggered receptor tyrosine kinase-MAP kinase pathway signalling, and was associated with suppression of Aurora B kinase. In breast cancer in vivo, we found that high miR-378a-5p levels correlate with the most aggressive, poorly differentiated forms of cancer.Interpretation: Downregulation of Aurora B by excess miR-378a-5p can explain the observed microtubule drug resistance and increased chromosomal imbalance in the microRNA-overexpressing cells. The results suggest that breast tumours may deploy high miR-378a-5p levels to gain growth advantage and antagonise taxane therapy. [ABSTRACT FROM AUTHOR]

Background:Optimal expression and proper function of key mitotic proteins facilitate control and repair processes that aim to prevent loss or gain of chromosomes, a hallmark of cancer. Altered expression of small regulatory microRNAs is associated with tumourigenesis and metastasis but the impact on mitotic signalling has remained unclear.Methods:Cell-based high-throughput screen identified miR-378a-5p as a mitosis perturbing microRNA. Transient transfections, immunofluorescence, western blotting, time-lapse microscopy, FISH and reporter assays were used to characterise the mitotic anomalies by excess miR-378a-5p. Analysis of microRNA profiles in breast tumours was performed.Results:Overexpression of miR-378a-5p induced numerical chromosome changes in cells and abrogated taxol-induced mitotic block via premature inactivation of the spindle assembly checkpoint. Moreover, excess miR-378a-5p triggered receptor tyrosine kinase-MAP kinase pathway signalling, and was associated with suppression of Aurora B kinase. In breast cancer in vivo, we found that high miR-378a-5p levels correlate with the most aggressive, poorly differentiated forms of cancer.Interpretation:Downregulation of Aurora B by excess miR-378a-5p can explain the observed microtubule drug resistance and increased chromosomal imbalance in the microRNA-overexpressing cells. The results suggest that breast tumours may deploy high miR-378a-5p levels to gain growth advantage and antagonise taxane therapy. [ABSTRACT FROM AUTHOR]

Sagopilone, an optimized fully synthetic epothilone, is a microtubule-stabilizing compound that has shown high in vitro and in vivo activity against a broad range of human tumor models. We analyzed the differential mechanism of action of sagopilone in non-small cell lung cancer cell lines in vitro. Sagopilone inhibited proliferation of non-small cell lung cancer cell lines at lower nanomolar concentration. The treatment with sagopilone caused strong disturbances of cellular cytoskeletal organization. Two concentration-dependent phenotypes were observed. At 2.5 nM sagopilone or 4 nM paclitaxel an aneuploid phenotype occur whereas a mitotic arrest phenotype was induced by 40 nM sagopilone or paclitaxel. Interestingly, treatment with 2.5 nM of sagopilone effectively inhibited cell proliferation, but - compared to high concentrations (40 nM) - only marginally induced apoptosis. Treatment with a high versus a low concentration of sagopilone or paclitaxel regulates a non-overlapping set of genes, indicating that both phenotypes substantially differ from each other. Genes involved in G2/M phase transition and the spindle assembly checkpoint, like Cyclin B1 and BUBR1 were upregulated by treatment with 40 nM sagopilone. Unexpectedly, also genes involved in DNA damage response were upregulated under that treatment. In contrast, treatment of A549 cells with a low concentration of sagopilone revealed an upregulation of direct transcriptional target genes of TP53, like CDKN1A, MDM2, GADD45A, FAS. Knockdown of TP53, which inhibited the transcriptional induction of TP53 target genes, led to a significant increase in apoptosis induction in A549 cells when treated with a low concentration of sagopilone. The results indicate that activation of TP53 and its downstream effectors like CDKN1A by low concentrations of sagopilone is responsible for the relative apoptosis resistance of A549 cells and might represent a mechanism of resistance to sagopilone. [ABSTRACT FROM AUTHOR]

The morphology, thickness and surface pressure of the surfactant film of broncho-alveoalar lavage (BAL) fluid from patients with sarcoidosis were investigated during spontaneous adsorption of the BAL's surface active material at the air/aqueous buffer interface at 37 °C. The biochemical parameters of the BAL fluid determined were protein (Lowry), total phospholipids (from phosphate after ashing) and the individual phospholipids (HPLC). During the spontaneous adsorption of the pulmonary surfactant the surface pressure increased from initially 26 mN/m to 44 mN/m in the equilibrium state. Simultaneously to the increase of the surface pressure, a continuous increase of the reflectivity signal was observed by quantitative Brewster angle microscopy (BAM). The film thickness is calculated from the reflectivity values using an optical model. The effect of the uncertainty of the refractive index, which has to be estimated, is discussed. The BAM images show the inhomogeneous nature of the surfactant film with three distinct phases of different reflectivity, even at relatively low surface pressures. For the brightest phase, the thickness amounts to approximately 12 nm in the equilibrium state of adsorption. This suggests a multilamellar structure. Additionally, we found visual evidence for an adsorption mechanism involving the spreading of vesicles at the interface, in agreement with published results. Differences in the morphology and thickness of the pulmonary surfactant film reported in the literature are obviously due to the varying experimental conditions and materials. We think that the experimental conditions chosen in our study provide a more realistic view of the structure in the lungs in vivo. [ABSTRACT FROM AUTHOR]

Background:Epothilones are a novel group of microtubule (mt) targeting cancer drugs that bind to the β-subunit of the αβ-tubulin dimer. Epothilones inhibit cell proliferation and induce cell death by interfering with the normal mt function. In this study, we examined the consequences of altered expression of human β-tubulin isotypes in terms of the epothilone drug response in human lung and breast cancer cell lines.Methods:The β-tubulin isotypes TUBB2A-C, TUBB3 and TUBB were silenced or overexpressed in A549, A549EpoB40 and MCF7 cell lines in the presence or absence of epothilones. The drug effects on cell proliferation, mitosis and mt dynamics were determined using live cell microscopy and immunofluorescence assays.Results:Loss of TUBB3 enhanced the action of epothilones. TUBB3 knockdown increased the severity of drug-induced mitotic defects and resulted in stabilisation of the mt dynamics in cells. Moreover, exogenous expression of TUBB3 in the epothilone resistant cell line conferred the response to drug treatments. In contrast, reduced levels of TUBB2A-C or TUBB had not apparent effect on the cells' response to epothilones.Conclusion:Our results show that the expression of TUBB3 contributes to the cellular response to epothilones, putatively by having an impact on the mt dynamics. [ABSTRACT FROM AUTHOR]

Abstract: This paper addresses the topic of model based design of experiments for the identification of nonlinear dynamic systems. Data driven modeling decisively depends on informative input and output data obtained from experiments. Design of experiments is targeted to generate informative data and to reduce the experimentation effort as much as possible. Furthermore, design of experiments has to comply with constraints on the system inputs and the system output, in order to prevent damage to the real system and to provide stable operational conditions during the experiment. For that purpose a model based approach is chosen for the optimization of excitation signals in this paper. Two different modeling architectures, namely multilayer perceptron networks and local model networks are chosen and the experiment design is based on the optimization of the Fisher information matrix of the associated model architecture. The paper presents and discusses feasible problem formulations and solution approaches for the constrained dynamic design of experiments. In this context the effects of the Fisher information matrix in the static and the dynamic configurations are discussed. The effectiveness of the proposed method is demonstrated on a complex nonlinear dynamic engine simulation model and an analysis as well as a comparison of the presented model architectures for model based experiment design is given. [Copyright &y& Elsevier]