DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BACKGROUND: Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

METHODS: HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity Pathway Analysis.

RESULTS: Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

CONCLUSION: This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.

Street Medicine – Assessment Work Strategies of Paramedics on the Front lines of Emergency Health Services

This article is based on an institutional ethnographic inquiry into the work of paramedics and the institutional setting that organizes and coordinates their work processes in a major City in Canada. Drawing on over 200 hours of observations and over 100 interviews with paramedics (average length of 18 minutes) and other emergency medical personnel, this article explores the standard and not so standard work of paramedics as they assess and care for their patients on the front lines of emergency health services. The multiplicity of interfacing social, demographic, locational, and situational factors that shape and organize the work of paramedics are analyzed. In doing so, this article provides insights into the complex work of an understudied yet ever-important profession in healthcare.

The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF-mutant colorectal cancer cell lines.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor Vemurafenib (PLX4032) has shown significant increases in response rates and overall survival, only minor responses to Vemurafenib treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MEK inhibition in BRAFMT CRC. Methods: Paired BRAFMT/WT RKO and VACO432 CRC cells and non-isogenic BRAFMT LIM2405, WiDR, HT-29 and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interactions between MEK1/2 and JAK1/2 or c-MET inhibition were assessed using the MTT cell viability assays and Flow Cytometry. Apoptosis was measured using Western Blotting for PARP, cleaved caspase 3, 8 and 9, and caspase 3/7 and 8 activity assays. Results: Treatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in synergy and significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or trametinib in BRAFMT CRC cells. The RTK c-MET is activated upstream of STAT3 following MEK1/2 inhibition. Inhibition of c-MET and MEK1/2, using pharmacological inhibitors (crizotinib and AZD6244), results in synergy and increased cell death in BRAFMT CRC cells. Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition in vitro. Combinations of JAK/MEKi or MET/MEKi can be a potential novel treatment strategy for poor prognostic BRAFMT advanced CRC patients.

The effects of radiative cascades on the x-ray diagnostic lines of Fe16+

We present complete collisional-radiative modelling results for the soft x-ray emission lines of Fe16+ in the 15 Å–17 Å range. These lines have been the subject of much controversy in the astrophysical and laboratory plasma community. Radiative transition rates are generated from fully relativistic atomic structure calculations. Electron-impact excitation cross sections are determined using a fully relativistic R-matrix method employing 139 coupled atomic levels through n = 5. We find that, in all cases, using a simple ratio of the collisional rate coefficient times a radiative branching factor is not sufficient to model the widely used diagnostic line ratios. One has to include the effects of collisional-radiative cascades in a population model to achieve accurate line ratios. Our line ratio results agree well with several previous calculations and reasonably well with tokamak experimental measurements, assuming a Maxwellian electron-energy distribution. Our modelling results for four EBIT line ratios, assuming a narrow Gaussian electron-energy distribution, are in generally poor agreement with all four NIST measurements but are in better agreement with the two LLNL measurements. These results suggest the need for an investigation of the theoretical polarization calculations that are required to interpret the EBIT line ratio measurements.

Ultraviolet emission lines of Si II in cool star and solar spectra

Recent atomic physics calculations for Si II are employed within the CLOUDY modelling code to analyse Hubble Space Telescope (HST) STIS ultraviolet spectra of three cool stars, β Geminorum, α Centauri A and B, as well as previously published HST/GHRS observations of α Tau, plus solar quiet Sun data from the High Resolution Telescope and Spectrograph. Discrepancies found previously between theory and observation for line intensity ratios involving the 3s²3p ²PJ-3s3p^{2 4}P^J' intercombination multiplet of Si II at ~ 2335 Å are significantly reduced, as are those for ratios containing the 3s²3p ²PJ-3s3p^{2 2}DJ ~ transitions at ~1816 Å. This is primarily due to the effect of the new Si II transition probabilities. However, these atomic data are not only very different from previous calculations, but also show large disagreements with measurements, specifically those of Calamai et al. for the intercombination lines. New measurements of transition probabilities for Si II are hence urgently required to confirm (or otherwise) the accuracy of the recently calculated values. If the new calculations are confirmed, then a long-standing discrepancy between theory and observation will have finally been resolved. However, if the older measurements are found to be correct, then the agreement between theory and observation is simply a coincidence and the existing discrepancies remain.

Passive Intermodulation of Analog and Digital Signals on Transmission Lines with Distributed Nonlinearities: Modelling and Characterization

Passive intermodulation (PIM) often limits the performance of communication systems with analog and digitally-modulated signals and especially of systems supporting multiple carriers. Since the origins of the apparently multiple physical sources of nonlinearity causing PIM are not fully understood, the behavioral models are frequently used to describe the process of PIM generation. In this paper a polynomial model of memoryless nonlinearity is deduced from PIM measurements of a microstrip line with distributed nonlinearity with two-tone CW signals. The analytical model of nonlinearity is incorporated in Keysight Technology’s ADS simulator to evaluate the metrics of signal fidelity in the receive band for analog and digitally-modulated signals. PIM-induced distortion and cross-band interference with modulated signals are compared to those with two-tone CW signals. It is shown that conventional metrics can be applied to quantify the effect of distributed nonlinearities on signal fidelity. It is found that the two-tone CW test provides a worst-case estimate of cross-band interference for two-carrier modulated signals whereas with a three-carrier signal PIM interference in the receive band is noticeably overestimated. The simulated constellation diagrams for QPSK signals demonstrate that PIM interference exhibits the distinctive signatures of correlated distortion and this indicates that there are opportunities for mitigating PIM interference and that PIM interference cannot be treated as noise. One of the interesting results is that PIM distortion on a transmission line results in asymmetrical regrowth of output PIM interference for modulated signals.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <or=-5.60 and <or=-8.00, respectively. Using this assay, it was found that GL-HCl permeates through human skin with a flux 1.497+/-0.42 microg cm-2 h-1, a permeability coefficient of 5.66+/-1.6x10(-6) cm h-1 and with a lag time of 10.9+/-4.6 h.