Ensemble Methods for APS In-Flight Particle Temperature and Velocity Prediction Considering Torch Electrodes Ageing.

The nonlinear relationship between the input process parameters and in-flight particle characteristics of the atmospheric plasma spray (APS) is of paramount importance for coating properties design and quality. It is also known that the ageing of torch electrodes affects this relationship. In recent years, machine learning algorithms have proven to be able to take into account such complex nonlinear interactions. This work illustrates the application of ensemble methods to predict the in-flight particle temperature and velocity during an APS process considering torch electrodes ageing. Experiments were performed to record simultaneously the input process parameters, the in-flight powder particle characteristics and the electrodes usage time. Random Forest (RF) and Gradient Boosting (GB) were used to rank and select the features for the APS process data recorded as the electrodes aged and the corresponding predictive models were compared. The time series aspect of the multivariate APS in-flight particle characteristics data is explored. Two strategies of time series embedding are considered. The first one simply embeds the attributes and the targets from the previous n time segments considered without any modification; whereas the second strategy first performs differencing to make the time series stationary before embedding. For the present application, RF is found to be more suitable than GB since RF can predict both the in-flight particle velocity and temperature simultaneously, properly considering the interactions between the two targets. On the other hand, GB can only predict these two targets one at a time. The superior performance of both embedded predictive models and the feature rankings of them suggest that it is better to consider the APS data as time series for the in-flight particle characteristic prediction. In particular, it is demonstrated that it is advantageous to first make the time series stationary using the traditional differencing technique, even when modeling using RF.

The Grapes checkpoint coordinates nuclear envelope breakdown and chromosome condensation.

Mutations in the embryonic Drosophila Grapes/Chk1 checkpoint result in an abbreviated interphase, chromosome condensation defects and metaphase delays. To clarify the relationship between these phenotypes, we simultaneously timed multiple nuclear and cytoplasmic events in mutant grp-derived embryos. These studies support a model in which grp disrupts an S-phase checkpoint, which results in progression into metaphase with incompletely replicated chromosomes. We also show that chromosome condensation is independent of the state of DNA replication in the early embryo. Therefore, grp condensation defects are not a direct consequence of entering metaphase with incompletely replicated chromosomes. Rather, initiation of chromosome condensation (ICC) occurs at the normal time in grp-derived embryos, but the shortened interval between ICC and metaphase does not provide sufficient time to complete condensation. Our results suggest that these condensation defects, rather than incomplete DNA replication, are responsible for the extensive metaphase delays observed in grp-derived embryos. This analysis provides an example of how the loss of a checkpoint can disrupt the timing of multiple events not directly monitored by that checkpoint. These results are likely to apply to vertebrate cells and suggest new strategies for destroying checkpoint-compromised cancer cells.

Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.

The positioning of centrosomes, or microtubule-organizing centres, within cells plays a critical part in animal development. Here we show that, in Drosophila embryos undergoing mitosis, the positioning of centrosomes within bipolar spindles and between daughter nuclei is determined by a balance of opposing forces generated by a bipolar kinesin motor, KLP61F, that is directed to microtubule plus ends, and a carboxy-terminal kinesin motor, Ncd, that is directed towards microtubule minus ends. This activity maintains the spacing between separated centrosomes during prometaphase and metaphase, and repositions centrosomes and daughter nuclei during late anaphase and telophase. Surprisingly, we do not observe a function for KLP61F in the initial separation of centrosomes during prophase. Our data indicate that KLP61F and Ncd may function by crosslinking and sliding antiparallel spindle microtubules in relation to one another, allowing KLP61F to push centrosomes apart and Ncd to pull them together.

The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity.

BACKGROUND: Cell cycle checkpoints maintain the fidelity of the somatic cell cycle by ensuring that one step in the cell cycle is not initiated until a previous step has been completed. The extent to which cell cycle checkpoints play a role in the initial rapid embryonic divisions of higher eukaryotes is unclear. The initial syncytial divisions of Drosophila embryogenesis provide an excellent opportunity to address this issue as they are amenable to both genetic and cellular analysis. In order to study the relevance of cell cycle checkpoints in early Drosophila embryogenesis, we have characterized the maternal-effect grapes (grp) mutation, which may affect feedback control during early syncytial divisions. RESULTS: The Drosophila grp gene encodes a predicted serine/threonine kinase and has significant homology to chk1/rad27, a gene required for a DNA damage checkpoint in Schizosaccharomyces pombe. Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage. During nuclear cycles 12 and 13, alignment of the chromosomes on the metaphase plate was disrupted in grp-derived embryos, and the embryos underwent a progression of cytological events that were indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation. The mutant embryos also failed to progress through a regulatory transition in Cdc2 activity that normally occurs during interphase of nuclear cycle 14. CONCLUSION: We propose that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions. This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. These results are discussed with respect to the proposed function of the chk1/rad27 gene.

Functional expression of gap junction gene Cx43 and the myogenic differentiation of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescence, slot-blot and dye-transfer methods. These cells grow rapidly and show aberrant and incomplete myogenic differentiation. To investigate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD single-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slowly and displays better myogenic differentiation. The expression of Cx43, its mRNA and the GJIC in RDL3 is comparable to that in normal myoblasts. Another subclone RDL6 which grows rapidly, but is poorly differentiated, expresses very low levels of Cx43 and its mRNA, and very weak GJIC. By using the calcium phosphate precipitate transfection technique, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduced into the RDL6 cells. Several stably transfected clones have been obtained. A stable Cx43-transfectant clone RDL6/C-4 expresses high level of Cx43 and its mRNA, and results in dramatic increase of GJIC. These cells grow slowly but display the enhanced myogenic differentiation. A correlation between the down-regulation of Cx43 gene expression and a reduced expression of myogenic differentiation in RD cells is is demonstrated. Forced expression of Cx43 not only inhibits cell growth but also correlates with the improved myogenic differentiation of RD cells.

SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC.

SOX2 is a well-known core transcription factor in embryonic stem cells (ESCs) and has an important role in the maintenance of pluripotency. Recently, SOX2 expression has also been reported in adult stem cells (ASCs), but the role of SOX2 in ASCs remains unknown. In this study, we examined the molecular mechanisms of SOX2 in human mesenchymal stem cells (hMSCs), a type of ASCs, by performing inhibition studies. SOX2 inhibition resulted in altered cell growth and differentiation capabilities. These changes coincided with a decrease in Dickkopf-1 (DKK1), a soluble inhibitor of WNT signaling. Chromatin immunoprecipitation and luciferase assays showed that SOX2 binds to DKK1 and has a positive regulatory role in transcription. The enforced expression of DKK1 in SOX2-inhibited hMSCs reversed the differentiation deformities, but could not abrogate the cell proliferation defect. Proliferation was regulated by c-MYC, whose expression can also be controlled by SOX2. Our study shows that SOX2 directly regulates DKK1 expression and, as a consequence, determines the differentiation lineage of hMSCs. Moreover, SOX2 also regulates proliferation by affecting c-MYC. Therefore, these results suggest that SOX2 might have a specific function by regulating DKK1 and c-MYC in the differentiation and growth of ASCs, which is separate from its roles in ESCs.

The correlation of blood pressure, age and endothelin-1 binding sites in aortic smooth muscle cells of rats.

Spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD) rats at the ages of 4, 8, 12, 16 and 24 weeks were used to examine the effects of age on the density of endothelin-1 (ET-1) binding sites in aortic smooth muscle cells and systolic blood pressure (SBP). The SBP of the 3 different rat strains was measured, and the maximum binding value (Bmax) and dissociation constant (Kd) of ET-1 binding sites in smooth muscle cells of the thoracic aorta were determined. The results showed that the SBP and Bmax values of SHR, WKY and SD rats increased with age; the SBP and Bmax value at each corresponding age were significantly higher in SHR than in WKY and SD rats, however, there was no significant difference between WKY and SD rats. The relationship of age vs SBP, age vs Bmax, and Bmax vs SBP showed significantly positive correlation in all 3 rat strains. The regression line in the Bmax of endothelin binding sites against SBP in the 3 different rat strains presented a similar slope. These results indicate that SBP, which increased with age, could be related to an increased density of ET-1 binding sites on vascular smooth muscle cells in these 3 different rat strains.

Truncated desmin in PtK2 cells induces desmin-vimentin-cytokeratin coprecipitation, involution of intermediate filament networks, and nuclear fragmentation: a model for many degenerative diseases.

The earliest expression of truncated desmin in transfected PtK2 cells results in the formation of dispersed microprecipitates containing not only the truncated desmin, but also endogenous vimentin and cytokeratin proteins. Desmin microprecipitates without vimentin or vimentin microprecipitates without desmin are not observed. The microprecipitates involving cytokeratin invariably are also positive for desmin and vimentin. Over time, the precipitates enlarge into 1- to 2-microns spheroids and then fuse into amorphous chimeric juxtanuclear masses that can occupy > 30% of the cell volume. Concurrently, first the vimentin and then the cytokeratin networks are resorbed. The chimeric precipitates are not recognized or marked for degradation by the lysosomal system. Ultimately the cell nucleus fragments and the cell dies. Similar protein complexes appear in many human and animal pathologies, suggesting that a similar protein-precipitation sequence initiated by the introduction of a mutationally or environmentally altered protein molecule is at work.

Increasing intracellular concentrations of thymosin beta 4 in PtK2 cells: effects on stress fibers, cytokinesis, and cell spreading.

Thymosin beta 4 (T beta 4) binds to G-actin in vitro and inhibits actin polymerization. We studied the effects of increasing T beta 4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane-associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane-associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of T beta 4 at a concentration (approximately 28 microM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular T beta 4 concentrations were increased by three different methods: 1) microinjection of an RSV vector containing a cDNA for T beta 4; 2) transfection with the same vector; and 3) microinjection of purified T beta 4 protein. The plasmid coding for T beta 4 was microinjected into PtK2 cells together with fluorescently labeled alpha-actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha-actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2-3 days showed disassembly of stress fibers as detected by rhodamine-phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure T beta 4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, similar microinjection of pure thymosin beta 4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow regressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these T beta 4-injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process.