Stereoscopic space map – semi-immersive configuration of 3Dstereoscopic tours in multi-display environments

Although large-scale stereoscopic 3D environments like CAVEs are a favorable location for group presentations, the perspective projection and stereoscopic optimization usually follows a navigator-centric approach. Therefore, these presentations are usually accompanied by strong side-effects, such as motion sickness which is often caused by a disturbed stereoscopic vision. The reason is that the stereoscopic visualization is usually optimized for the only head-tracked person in the CAVE – the navigator – ignoring the needs of the real target group – the audience. To overcome this misconception, this work proposes an alternative to the head tracking-based stereoscopic effect optimization. By using an interactive virtual overview map in 3D, the pre-tour and on-tour configuration of the stereoscopic effect is provided, partly utilizing our previously published interactive projection plane approach. This Stereoscopic Space Map is visualized by the zSpace 200®, whereas the virtual world is shown on a panoramic 330° CAVE2TM. A pilot expert study with eight participants was conducted using pre-configured tours through 3D models. The comparison of the manual and automatic stereoscopic adjustment showed that the proposed approach is an appropriate alternative to the nowadays commonly used head tracking-based stereoscopic adjustment

Omeprazole Inhibits Proliferation and Modulates Autophagy in Pancreatic Cancer Cells

BACKGROUND: Omeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Dose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results. CONCLUSIONS: We hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications

Electron microscopy of the ASPC-1 and MiaPaCa-2 cell lines treated or untreated with OMP.

<p>(A) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present. (E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).</p

Western Blot analysis with LC3B-antibody in ASPC-1 and MiaPaCa-2 cells.

<p>The experiment was repeated three times with nearly identical results. The LC3-I fractions can be clearly distinguished above the LC3-II signals. Bafilomycin A1 elevated slightly the LC3-II signal intensity compared to control in both cell lines. There was a marked and dose-dependent increase of the signal strength of both fractions when OMP was used. 5-FU alone did not relevantly influence the LC3-level. The corresponding β-Actin level is shown below the LC3-WB to confirm the correctness of this semiquantitative evaluation.</p

Mean inhibitory concentrations (IC₅₀) of OMP and 5-FU in various human pancreatic cancer cell lines.

<p>The IC₅₀ were determined by ATP-bioluminescence. All values are listed in µg/ml.</p><p>Abbreviations: conf. int. - 90% confidence intervals.</p

Gene expression analysis of membranal transport and apoptosis relevant genes in MiaPaCa-2 cells.

<p>mRNA quantification in MiaPaCa-2 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. While the mdr-1 mRNA is not signicantly changed, the vATPase mRNA is upregulated in the 5-FU+OMP group after 24 hours compared to control.</p

Identification of substances from a proton NMR spectrum of viable untreated MiaPaCa-2 cells.

<p>The cells were harvested from monolayer culture, kept and measured at 20°C. Measurement were performed by a 600 MHz Bruker spectrometer. For better visibility the part of the spectrum showing the protons of aliphatic groups is splitted into 2 parts - A and B. (A). aliphatic part I. The methyl and β- and γ- methylene groups of various fatty acids and amino acids are visible. In addition, isopropanol and tetrachlorethan (the external concentration standard) occured as pollutions. (B) Aliphatic part II. Phospholipid metabolites and the α-methylene groups of amino acids and lactate are visible. (C) Formula of OMP with numbering of the respective protons. The methyl groups (1–3, 9) and the methyl group (4) are covered by other metabolites in the aliphatic parts of the spectrum. In contrast, the aromatic protons are visible (H5, H8, H10). (D) Overlay of the aromatic parts of different spectra for intracellular identification of OMP. The singulet of the H5 proton and the dublets of the H8 and H10 protons can be identified when the medium and the cell spectra are compared to those without OMP treatment. Abbreviations: His - histidine, Tyr - tyrosine, Phe – phenylalanine, Leu, Ile, Val - Leucine, Isoleucine, Valine. Ala - Alanine. Glu - Glutamate, Gln – Glutamine, PC - phosphatidylcholine, Cho - Choline, GPC – glycerophosphocholine, Tau – taurine, Scyllo - scylloinositole.</p

Box-Whisker plots of lysosomal acidity quantified by AO fluorescence microscopy and image analysis.

<p>Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU, OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours.</p

Simplified metabolic network of the MiaPaCa-2 cell line as determined by proton NMR spectroscopy.

<p>The cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. Upon OMP, the PC/Cho ratios are significantly lower compared to control. Furthermore the acetate/FACH2 ratio is significantly decreased in the OMP group. The latter also showed a higher CH = CH level, the ratio to FACH2 is, however, decreased. Upon 5-FU and 5-FU+OMP, similar changes could be observed. Furthermore, in contrast to ASPC-1 cells, the Ala/Gln/AMP pathway is also involved. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline. Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.</p

Detection of growth-stimulatory effects of 5-FU and GEM in lower doses.

<p>Significant elevations of cell counts compared to the control groups could be observed upon 5-FU in ASPC-1 (at 0.5 µg/ml 5-FU), Panc-1 (at 0.5 and 1 µg/ml 5-FU) and PancTu-1 (at 0.125 and 0.25 µg/ml) cells. In contrast, no hormesis could be observed upon GEM.</p><p>Abbreviations: c-concentration, fu-unaffected fraction (cell count related to control).</p