Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability.

Porous graphitic carbon (PGC) is an important tool in a chromatographer's armory that retains polar compounds with mass spectrometry (MS)-compatible solvents. However, its applicability is severely limited by an unpredictable loss of retention, which can be attributed to contamination. The solutions offered fail to restore the original retention and our observations of retention time shifts of gemcitabine/metabolites on PGC are not consistent with contamination. The mobile phase affects the ionization state of analytes and the polarizable PGC surface that influences the strength of dispersive forces governing retention on the stationary phase. We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC. Herein, we optimize the choice of mobile phase solvent in a gradient program with three parts: a preparatory phase, which allows binding of analytes to column; an elution phase, which gives the required separation/peak shape; and a maintenance phase, to preserve the required retention capacity. Via liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolites extracted from tumor tissue, we demonstrate reproducible chromatography on three PGC columns of different ages. This approach simplifies use of the PGC to the same level as that of a C-18 column, removes the need for column regeneration, and minimizes run times, thus allowing PGC columns to be used to their full potential.This work was funded by the Cancer Research UK Cambridge Institute (Grant NO. C14303/A17197).This is the final version of the article. It first appeared from American Chemical Society via https://doi.org/ 10.1021/acs.analchem.6b0116

Anti-tumour effects of a specific anti-ADAM17 antibody in an ovarian cancer model in vivo.

ADAM 17 (TNF-α converting enzyme, TACE) is a potential target for cancer therapy, but the small molecule inhibitors reported to date are not specific to this ADAM family member. This membrane-bound metalloproteinase is responsible for ectodomain shedding of pathologically significant substrates including TNF-α and EGFR ligands. The aim of this study was to evaluate the pharmacokinetics, pharmacodynamics and anti-tumour efficacy of the first specific inhibitor, an anti-human ADAM17 IgG antibody, clone D1(A12). We used intraperitoneal xenografts of the human ovarian cancer cell line IGROV1-Luc in Balb/c nude mice, chosen because it was previously reported that growth of these xenografts is inhibited by knock-down of TNF-α. In vitro, 200 nM D1(A12) inhibited shedding of ADAM17 substrates TNF-α, TNFR1-α, TGF-α, amphiregulin (AREG), HB-EGF and IL-6Rα, from IGROV1-Luc cells, (4.7 nM IC(50) for TNF-α shedding). In IGROV1-Luc xenografts in vivo, D1(A12) IgG showed pharmacokinetic properties suitable for efficacy studies, with a single i.p. dose of 10 mg/kg D1(A12) sufficient to maintain IgG plasma and ascites fluid concentrations above 100 nM for more than 7 days. The plasma half life was 8.6 days. Next, an efficacy study was performed, dosing D1(A12) or anti-human TNF-α antibody infliximab at 10 mg/kg q7d, quantifying IGROV1-Luc tumour burden by bioluminescence. D1(A12) IgG showed a significant reduction in tumour growth (p = 0.005), 56% of vehicle control. Surprisingly, D1(A12) did not reduce the concentration of circulating human TNF-α, suggesting that another enzyme may compensate for inhibition of ADAM17 in vivo (but not in vitro). However, D1(A12) did show clear pharmacodynamic effects in the mice, with significant inhibition of shedding from tumour of ADAM17 substrates TNFR1-α, AREG, and TGF-α (4-15-fold reductions, p<0.0001 for all three). Thus, D1(A12) has anti-ADAM17 activity in vivo, inhibits shedding of EGFR ligands and has potential for use in EGF ligand-dependent tumours

Exploring Gothic Tourism: A new form of urban tourism?

Purpose: The concept of ‘Gothic tourism’ has recently been proposed within the discipline of English Literature. Such tourism is claimed to be a distinct form of special interest tourism grounded in familiarity with the Gothic, distinctive aesthetics, and experiences of frights and scares. It is increasingly common in towns and cities around the world. This paper examines and critiques the concept of Gothic tourism, and considers its similarities with existing forms of urban tourism. Design/Methodology/Approach: This is a conceptual paper and no empirical data is presented. Findings: Gothic tourism is not as clearly differentiated from other forms of tourism as has been claimed. In particular Gothic tourism can be conceptualised as a particular form of ‘lighter’ dark tourism, but it can also be considered as a form of literary tourism. A conceptual model is presented which places Gothic tourism as the nexus of dark and literary tourism. Research limitations: This study is a conceptual exploration of Gothic tourism. Further empirical research is required to test the ideas presented in this paper at established Gothic tourism attractions. Originality: This study examines the recently-proposed (but little-researched) concept of Gothic tourism and considers its relationships with other forms of special interest tourism. It also illustrates the broader issue of how typologies of special interest tourism do not necessarily correspond with the motives and experiences of tourists themselves, or of the providers of tourist experiences

Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes

Centrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics

CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.

Combining cell-cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G2-M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biologic analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G2-M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S-G2 duration was extended in treated cells, leading to a definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a "foot-in-the-door" mechanism for drug synergy where cells were destroyed not by frank G2-M phase abrogation but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis.This study was funded by Cancer Research UK via Institute Senior Group Leader funding (C14303/A17197) to DI Jodrell, and by Sentinel Oncology through an award from Innovate UK.This is the author accepted manuscript. The final version is available from American Association for Cancer Research via http://dx.doi.org/10.1158/0008-5472.CAN-14-334

AGN Populations in Large Volume X-ray Surveys: Photometric Redshifts and Population Types found in the Stripe 82X Survey

Multi-wavelength surveys covering large sky volumes are necessary to obtain an accurate census of rare objects such as high luminosity and/or high redshift active galactic nuclei (AGN). Stripe 82X is a 31.3 deg$^2$ X-ray survey with $Chandra$ and $XMM$-Newton observations overlapping the legacy Sloan Digital Sky Survey (SDSS) Stripe 82 field, which has a rich investment of multi-wavelength coverage from the ultraviolet to the radio. The wide-area nature of this survey presents new challenges for photometric redshifts for AGN compared to previous work on narrow-deep fields because it probes different populations of objects that need to be identified and represented in the library of templates. Here we present an updated X-ray plus multi-wavelength matched catalog, including $Spitzer$ counterparts, and estimated photometric redshifts for 5961 (96% of a total of 6181) X-ray sources, which have a normalized median absolute deviation, $\sigma_{\rm nmad}$ = 0.06 and an outlier fraction, $\eta$ = 13.7%. The populations found in this survey, and the template libraries used for photometric redshifts, provide important guiding principles for upcoming large-area surveys such as $eROSITA$ and 3$XMM$ (in X-ray) and the Large Synoptic Survey Telescope (LSST; optical).Comment: Accepted for publication by The Astrophysical Journal (33 pages, 20 figures, 13 tables). Final catalog of counterparts and photo-z supplementing the paper available here: http://stripe82x.com/docs/stripe82x-photometric-redshifts-and-multiwavelength-data-catalog

Combenefit: an interactive platform for the analysis and visualization of drug combinations.

MOTIVATION: Many drug combinations are routinely assessed to identify synergistic interactions in the attempt to develop novel treatment strategies. Appropriate software is required to analyze the results of these studies. RESULTS: We present Combenefit, new free software tool that enables the visualization, analysis and quantification of drug combination effects in terms of synergy and/or antagonism. Data from combinations assays can be processed using classical Synergy models (Loewe, Bliss, HSA), as single experiments or in batch for High Throughput Screens. This user-friendly tool provides laboratory scientists with an easy and systematic way to analyze their data. The companion package provides bioinformaticians with critical implementations of routines enabling the processing of combination data. AVAILABILITY AND IMPLEMENTATION: Combenefit is provided as a Matlab package but also as standalone software for Windows (http://sourceforge.net/projects/combenefit/). CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.This work has been supported by the Cancer Research UK grant C14303/A17197This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/bioinformatics/btw23

Using the Responsible Suicide Reporting Model to increase adherence to global media reporting guidelines

Numerous guidelines on responsible reporting of suicide are available to journalists globally, offering advice on best practice regarding approaches and suitability of content. Whilst their advice is compelling and legitimate, their use is uneven at best. With a suicide death every 40 seconds worldwide, it is imperative journalists understand and recognise the best ethical practices in order to report suicide responsibly. To address these shortcomings, the authors present a model for responsible suicide reporting (RSR) that is grounded in news-work and embeds media reporting guidelines within journalistic storytelling practices. The RSR model consists of a typology of suicide narratives and ‘othering’, ethical rules and a standard of moderation. Methodologically, these typologies emerged from analysis of 159 suicide news stories published in 2018–19, with particular focus on adherence and non-adherence to global media reporting guidelines. We posit through the process of producing stories using the RSR model, journalists should interact more effectively with critical risk factors for example, stigmatisation, copycat effects, harmful speculation, highlighted by media reporting guidelines