Modeling cancer driver events in vitro using barrier bypass-clonal expansion assays and massively parallel sequencing.

The information on candidate cancer driver alterations available from public databases is often descriptive and of limited mechanistic insight, which poses difficulties for reliable distinction between true driver and passenger events. To address this challenge, we performed in-depth analysis of whole-exome sequencing data from cell lines generated by a barrier bypass-clonal expansion (BBCE) protocol. The employed strategy is based on carcinogen-driven immortalization of primary mouse embryonic fibroblasts and recapitulates early steps of cell transformation. Among the mutated genes were almost 200 COSMIC Cancer Gene Census genes, many of which were recurrently affected in the set of 25 immortalized cell lines. The alterations affected pathways regulating DNA damage response and repair, transcription and chromatin structure, cell cycle and cell death, as well as developmental pathways. The functional impact of the mutations was strongly supported by the manifestation of several known cancer hotspot mutations among the identified alterations. We identified a new set of genes encoding subunits of the BAF chromatin remodeling complex that exhibited Ras-mediated dependence on PRC2 histone methyltransferase activity, a finding that is similar to what has been observed for other BAF subunits in cancer cells. Among the affected BAF complex subunits, we determined Smarcd2 and Smarcc1 as putative driver candidates not yet fully identified by large-scale cancer genome sequencing projects. In addition, Ep400 displayed characteristics of a driver gene in that it showed a mutually exclusive mutation pattern when compared with mutations in the Trrap subunit of the TIP60 complex, both in the cell line panel and in a human tumor data set. We propose that the information generated by deep sequencing of the BBCE cell lines coupled with phenotypic analysis of the mutant cells can yield mechanistic insights into driver events relevant to human cancer development.

Stabilisation effects of phosphane ligands in the homogeneous approach of sunlight induced hydrogen production.

Most of the systems for photochemical hydrogen production are not stable and suffer from decomposition. With bis(bidentate) tetraphosphane ligands the stability increases enormously, up to more than 1000 h. This stability was achieved with a system containing osmium(ii) as a light harvesting antenna and palladium(ii) as a water reduction catalyst connected with a bis(bidentate) phosphane ligand in one molecule with the chemical formula [Os(bpy)2(dppcb)Pd(dppm)](PF6)4. With the help of electrochemical measurements as well as photophysical data and its single crystal X-ray structure, the electron transfer between the two active metal centres (light harvesting antenna, water reduction catalyst) was analysed. The distance between the two active metal centres was determined to be 7.396(1) Å. In a noble metal free combination of a copper based photosensitiser and a cobalt diimine-dioxime complex as water reduction catalyst a further stabilisation effect by the phosphane ligands is observed. With the help of triethylamine as a sacrificial donor in the presence of different monophosphane ligands it was possible to produce hydrogen with a turnover number of 1176. This completely novel combination is also able to produce hydrogen in a wide pH-range from pH = 7.0 to 12.5 with the maximum production at pH = 11.0. The influence of monophosphane ligands with different Tolman cone angles was investigated. Monophosphane ligands with a large Tolman cone angle (>160°) could not stabilise the intermediate of the cobalt based water reduction catalyst and so the turnover number is lower than for systems with an addition of monophosphane ligands with a Tolman cone angle smaller than 160°. The role of the monophosphane ligand during sunlight-induced hydrogen production was analysed and these results were confirmed with DFT calculations. Furthermore the crystal structures of two important Co(i) intermediates, which are the catalytic active species during the catalytic pathway, were obtained. The exchange of PPh3 with other tertiary phosphane ligands can have a major impact on the activity, depending on the coordination properties. By an exchange of monophosphane ligands with functionalised phosphane ligands (hybrid ligands) the hydrogen production was raised 2.17 times.

Evaluation of selected 3D virtual screening tools for the prospective identification of peroxisome proliferator-activated receptor (PPAR) γ partial agonists.

The peroxisome proliferator-activated receptor (PPAR) γ regulates the expression of genes involved in adipogenesis, lipid homeostasis, and glucose metabolism, making it a valuable drug target. However, full activation of the nuclear receptor is associated with unwanted side effects. Research therefore focuses on the discovery of novel partial agonists, which show a distinct protein-ligand interaction pattern compared to full agonists. Within this study, we employed pharmacophore- and shape-based virtual screening and docking independently and in parallel for the identification of novel PPARγ ligands. The ten top-ranked hits retrieved with every method were further investigated with external in silico bioactivity profiling tools. Subsequent biological testing not only confirmed the binding of nine out of the 29 selected test compounds, but enabled the direct comparison of the method performances in a prospective manner. Although all three methods successfully identified novel ligands, they varied in the numbers of active compounds ranked among the top-ten in the virtual hit list. In addition, these compounds were in most cases exclusively predicted as active by the method which initially identified them. This suggests, that the applied programs and methods are highly complementary and cover a distinct chemical space of PPARγ ligands. Further analyses revealed that eight out of the nine active molecules represent novel chemical scaffolds for PPARγ, which can serve as promising starting points for further chemical optimization. In addition, two novel compounds, identified with docking, proved to be partial agonists in the experimental testing.

Validation of hyperbaric oxygen treatment software for use with monoplace chambers.

Hyperbaric oxygen (HBO2) therapy is increasingly used in the treatment of a wide variety of medical conditions. However, for monoplace chambers, there is some uncertainty when sufficiently high oxygen concentrations are attained, because most chambers are not instrumented to measure oxygen. To remedy this, Microsoft Excel-based software, HBO O2 Smart Guide, was developed to simulate the atmosphere ofmonoplace chambers during treatment. Based upon chamber dimensions, patient weight, oxygen purge rates, desired pressurization, and HBO2 time, the program calculates oxygen concentration, consumption and exposure for each treatment. Software testing was conducted using four different chambers instrumented with an oxygen analyzer. Two purge rate profiles were used: constant, and biphasic (a high initial purge rate was changed to a lower plateau rate when pressurization was reached). Comparison of measured and calculated times to reach 95% oxygen concentration within the chambers demonstrated the software was accurate within 1%. The HBO O2 Smart Guide enables optimum purge profiles to be simulated with resultant potential improvements in HBO2 treatment efficacy, calculation of effective oxygen exposures (actual time during prescribed treatment during which patient breathes > or = 95% oxygen) to enable more accurate comparison of treatment profiles and outcomes, and cost savings in oxygen usage. This software will enable clinicians to provide more consistent HBO2 treatments.

Cigarette smoke induces direct DNA damage in the human B-lymphoid cell line Raji.

Human lymphoid cells (Raji) were exposed to water-soluble compounds from cigarette smoke (CS) generated in a smoking machine. DNA damage, as detected by alkaline single-cell microelectrophoresis (COMET assay), was induced in a time- and concentration-dependent manner in the cells. Most of the rapidly induced DNA damage was attributable to direct-acting compounds since cytochrome P450-related metabolic activities (ethoxy- and pentoxyresorufin-O-deethylases and coumarin-7-hydroxylase) were absent or very low. In addition, induction of DNA damage could be inhibited only slightly by beta-naphthoflavone and coumarin. Vitamin C enhanced DNA damage in Raji cells probably by redox cycling of catechol and hydroquinone present in CS implicating reactive oxygen intermediates as another source of DNA damage. N-acetylcysteine, a radical scavenger and glutathione precursor, reduced DNA damage in Raji cells when exposure to CS was followed by 2 h post-incubation in culture medium. Unrepaired DNA damage caused by CS persisted longer than gamma-irradiation-induced DNA damage. Among the CS constituents, acrolein, but not formaldehyde and acetaldehyde, induced DNA damage although less intensely than CS itself. At 50 and 100 microM concentrations, acrolein also inhibited repair of gamma- irradiation-induced DNA damage in the COMET assay. Inhibition of DNA synthesis by acrolein at 50 microM was demonstrated by an immunochemical assay for bromo-deoxyuridine incorporation; however, inhibition of a representative repair enzyme, 8-oxoguanosine hydrolase, by either CS or acrolein was not observed. The present results further confirm the presence of direct-acting genotoxic components and inhibitors of DNA repair in the gas phase of tobacco smoke, that may contribute to DNA damage and smoking-associated cancers of the upper aerodigestive tract.

An example of intron junctional sliding in the gene families encoding squalene monooxygenase homologues in Arabidopsis thaliana and Brassica napus.

Sequences of three Arabidopsis thaliana and two Brassica napus cDNAs encoding squalene monooxygenase homologues (Sqp1 and Sqp2) are reported. Southern analysis confirmed that these cDNAs are derived from small gene families in both species. Expression analysis indicates that Sqp1 genes in B. napus are strongly expressed in leaves but not roots or developing seeds. Comparison of cDNA and genomic sequences indicate that the 3' splice site of an intron in these genes has undergone junctional sliding. The evolutionary significance of this phenomenon is discussed.

Comparison of Dehydrin Gene Expression and Freezing Tolerance in Bromus inermis and Secale cereale Grown in Controlled Environments, Hydroponics, and the Field.

There have been very few reports on the expression of stress-responsive genes in field-grown material. A barley dehydrin cDNA was used to investigate the expression of dehydrin-like transcripts after low-temperature and abscisic acid-induced acclimation of bromegrass (Bromus inermis Leyss) suspension cells and of bromegrass and rye (Secale cereale) plants grown in the field and under controlled environmental conditions. Field-acclimated plants accumulated high levels of dehydrin transcripts and were very freezing tolerant. Plants grown in pots and hydroponics under controlled environments also accumulated dehydrin transcripts and showed increased freezing tolerance. Simulation of a combined drought and freezing stress in pots resulted in expression of dehydrin-like transcripts comparable to those observed in field-acclimated material.

Determination of oxolinic acid residues in salmon muscle tissue by liquid chromatography with fluorescence detection.

The present paper describes a method for determination of oxolinic acid in salmon muscle tissue. Tissue (0.5-2 g) mixed with 2 g anhydrous sodium sulfate is extracted twice with ethyl acetate, centrifuged, and the extract evaporated. The residue is partitioned in a mixture of hexane and 0.01M oxalic acid and the aqueous phase chromatographed using fluorescence detection at 327 nm excitation and 369 nm emission. Calibration and standard curves are linear from 10-200 ppb and 100-2000 ppb at different sensitivity settings. Recoveries ranged from 71-83% in spiked blanks, with a CV of 4-10.3% over a 2-week period. Preliminary results in treated salmon were variable, possibly because some fish refused to eat medicated feed.

[Effect of nitroimidazole therapy on the demonstration of gonococci]. / Einfluss der Nitroimidazol-Therapie auf den Gonokokken-Nachweis.

The minimal inhibitory concentration of metronidazole and tinidazole lies between 4000 micrograms/ml and 7.8 micrograms/ml. Drug levels are present in this range for 2--5 hours in genital discharge following single dose oral therapy with 2 g tinidazole or 1.5 g metronidazole. Therefore, 5-nitroimidazole derivatives exert a bacteriostatic effect in the genital region at least on the more sensitive strains. As shown in our experiments, otherwise reliable laboratory procedures for the demonstration of gonococci may yield false negative results, if specimens for culture are obtained at an inappropriate time.

[Experimental contribution to the theme of pharyngeal gonorrhea]. / Experimenteller Beitrag zum Thema Rachengonorrhoe.

Experimental inoculations of the pharynx with gonococci were undertaken. In seven volunteers gonococci could be cultivated and identified from 16 hours to 8 weeks following infection. In three cases, it was tried to infect the female genital tract using positive pharyngeal smears. This was successful in one instance and the diagnosis was again confirmed by culture and sugar fermentation.