UNIVERSALIZABILITY, IMPARTIALITY AND THE EXPANDING CIRCLE

This paper deals with the question of whether the universalizability of moral judgment, and impartiality in ethics, would require us to expand our scope of moral consideration to cover all sentient beings, human or non-human, as de Lazari-Radek and Singer wish to establish in their new volume on Sidgwick and contemporary ethics. I will argue that the former two concepts, universalizability and impartiality, may not entail the third concept, the expanding circle. De Lazari-Radek and Singer’s arguments may presuppose their moral intuitions that are not entirely self-evident, and the philosophical foundation of ethics Sidgwick presented does not necessarily lead us to their version of utilitarianism

How the Atacama Skeleton Might Advance Discussion of Responsible Conduct of Research Responsibilities

Controversies resulting from genetic testing on skeletal remains of disputed stewardship raise important questions about obligations inherent on genetic researchers to assure ethical chain of custody. In this paper, we analyze and evaluate several proposed positions on whether such research should be published. Following jurisprudential standards for legitimate regulatory systems, we argue that responsible conduct of research requires reasonable attention to chain of custody, but cannot require guarantees, particularly in cases of ancient remains

Multi-kinase inhibitor C1 triggers mitotic catastrophe of glioma stem cells mainly through MELK kinase inhibition.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors

Globalization and the Logic of Participation: Unions and the Politics of Coalition Building

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors

C1 treatment accumulates GSCs in G2/M and triggers subsequent mitotic catastrophe.

<p>A, Proliferation assays on two glioblastoma cell lines (U87 and U251). U87 and U251 cells were treated with 5.7 μM C1 or DMSO. Cells were trypsinized and estimated by counting, in duplicate, after 72 h of treatment. Two different experiments were conducted with similar results. B, Time-Lapse on mitotic U251 cells stably expressing GFP was performed in the absence (DMSO) or in the presence of C1 (at 1 μM). The compound was added to the cell culture just before imaging and then cells were continuously imaged. Three independent experiments were conducted and 10 to 15 fields were followed in each. None of the followed mitotic cells divided in two daughter cells. Representative field is imaged, DNA is in blue and the merge shows GFP and DNA. Several polyploid cells were present in the image. Arrows and arrowheads in upper panels of DMSO and C1- treated cell indicate the same cells through time-lapse. The elapse times are indicated on each photo, in some assays, a zoom of one cell is shown (the red bar represents 5 μm) this cell is present on the former field and labelled with an arrow). Images in bottom panels show DNA only (left) and DNA overlap with GFP (right) after 72-hour treatment with DMSO or C1 (the red bars on each panel represent 20 μm). Arrows in the bottom panel of C1-treated cells indicate mitotic catastrophe by C1 treatment. C, Pictures demonstrate pre-mitotic phase (left panel), mitotic (mid panel) process by full karyokinesis and cytokinesis and after cell division (right panel). MELK expression was determined with immunocytochemistry of GBM1600 cells with anti-MELK antibody (red), chromatin staining with Hoechst stain (blue). Picture of pre-mitosis shows GBM1600 cells highly expressed MELK at pre-mitosis phase (400× magnification). Then GBM1600 cells were treated with 5 μM C1 or control and were subjected to immunocytochemistry 3 days later with anti-MELK and chromatin staining (640× magnification). Data were confirmed by three independent experiments. C1 treated cells are micronucleated at metaphase and followed multinuclear chromatin condensation (mid panel). Right panel show multinuclear asymmetric divided chromatin of C1 treated cell compared with DMSO treated cell. D, Flow cytometric analysis of C1- and DMSO-treated GBM1600 cells with Propidium Iodide at 3 days after treatment shows 62.7% of C1-treated cells resulted in the G2/M arrest, whereas the control cells have 19.3% of the G2/M arrested cells.E, Graph indicating the proportions of live, early apoptotic, and late apoptotic U251 cells with varying doses of C1 or DMSO.</p

EZH2 Protects Glioma Stem Cells from Radiation-Induced Cell Death in a MELK/FOXM1-Dependent Manner

Glioblastoma (GBM)-derived tumorigenic stem-like cells (GSCs) may play a key role in therapy resistance. Previously, we reported that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 in GSCs. Here, we demonstrate that the catalytic subunit of Polycomb repressive complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are coexpressed in GBM and significantly induced in postirradiation recurrent tumors whose expression is inversely correlated with patient prognosis. Through a gain-and loss-of-function study, we show that MELK or FOXM1 contributes to GSC radioresistance by regulation of EZH2. We further demonstrate that the MELK-EZH2 axis is evolutionarily conserved in Caenorhabditis elegans. Collectively, these data suggest that the MELK-FOXM1-EZH2 signaling axis is essential for GSC radioresistance and therefore raise the possibility that MELK-FOXM1-driven EZH2 signaling can serve as a therapeutic target in irradiation-resistant GBM tumors

C1 treatment inhibits GSC proliferation <i>in vitro</i> and <i>in vivo</i>.

<p>A, Graph of neurosphere forming assay indicating the relative neurosphere numbers of C1-treated patient-derived GBM samples (GBM146, GBM157, and GBM206) and normal neural progenitors (16wf). B, CD133(+) and (−) cells, separated from GBM157-derived sphere cultures, were treated with 1 μM C1 or DMSO (control) under the identical serum-free conditions for 48 hours. The effect on CD133(+) cells was assessed by the neurosphere number per well, and the effect on CD133(−) cells was assessed by the % change of the total cell number in comparison to the control sample. C, Schematic showing organotypic slice cultures explanted GBM tissues and treated with C1 or DMSO (control) for 16 hours and evaluated with H&E, Ki67, and Nestin staining. D, Immunohistochemistry of C1- or DMSO-treated GBM slice cultures with anti-Ki-67 monoclonal antibody (Original magnification, ×200). E, Graph indicating the numbers of neurospheres (left) or total cells (right) in serum-free medium derived from C1- or DMSO-treated slice cultures for 16 hours. F, Schematic drawing of the effect of C1 treatment for the mouse intracranial GBM models derived from GSCs. Cells from GBM157 spheres were injected intracranially into immunocompromised mice (C1 mice: n = 4, control mice: n = 12). At day 7 post transplantation, C1 was injected intratumorally at quantities of 2.5 pmol (n = 3), 25 pmol (n = 4), or 250 pmol (n = 5). G, Representative images for immunohistochemistry with Ki-67 staining of GBM slice cultures treated with 25 pmol C1 or DMSO at day 10 of treatment. Ki-67 positive cells in each group were analyzed automated digital image analysis (Original magnification, ×200). H, Representative images for immunohistochemistry with human-specific Nestin antibody using GBM157-derived mouse intracranial tumors treated with varying doses of C1 or DMSO intratumoral injection (bar: 1 mm). I, Graph indicates tumor sizes in each group as determined by Nestin staining intensities analyzed using automated digital image analysis.</p