Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway

BACKGROUND: The anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of the Chelidonium majus L. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell lines in vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry. METHODS: Apoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling. RESULTS: Ukrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-x(L )and expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events. However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis. Instead, the Chelidonium majus L. alkaloids chelidonine, sanguinarine, chelerythrine, protopine and allocryptopine were identified as major components of Ukrain. Apart from sanguinarine and chelerythrine, chelidonine turned out to be a potent inducer of apoptosis triggering cell death at concentrations of 0.001 mM, while protopine and allocryptopine were less effective. Similar to Ukrain, apoptosis signalling of chelidonine involved Bcl-2 controlled mitochondrial alterations and caspase-activation. CONCLUSION: The potent proapoptotic effects of Ukrain are not due to the suggested "Ukrain-molecule" but to the cytotoxic efficacy of Chelidonium majus L. alkaloids including chelidonine

Genomic analyses inform on migration events during the peopling of Eurasia.

High-coverage whole-genome sequence studies have so far focused on a limited number of geographically restricted populations, or been targeted at specific diseases, such as cancer. Nevertheless, the availability of high-resolution genomic data has led to the development of new methodologies for inferring population history and refuelled the debate on the mutation rate in humans. Here we present the Estonian Biocentre Human Genome Diversity Panel (EGDP), a dataset of 483 high-coverage human genomes from 148 populations worldwide, including 379 new genomes from 125 populations, which we group into diversity and selection sets. We analyse this dataset to refine estimates of continent-wide patterns of heterozygosity, long- and short-distance gene flow, archaic admixture, and changes in effective population size through time as well as for signals of positive or balancing selection. We find a genetic signature in present-day Papuans that suggests that at least 2% of their genome originates from an early and largely extinct expansion of anatomically modern humans (AMHs) out of Africa. Together with evidence from the western Asian fossil record, and admixture between AMHs and Neanderthals predating the main Eurasian expansion, our results contribute to the mounting evidence for the presence of AMHs out of Africa earlier than 75,000 years ago.Support was provided by: Estonian Research Infrastructure Roadmap grant no 3.2.0304.11-0312; Australian Research Council Discovery grants (DP110102635 and DP140101405) (D.M.L., M.W. and E.W.); Danish National Research Foundation; the Lundbeck Foundation and KU2016 (E.W.); ERC Starting Investigator grant (FP7 - 261213) (T.K.); Estonian Research Council grant PUT766 (G.C. and M.K.); EU European Regional Development Fund through the Centre of Excellence in Genomics to Estonian Biocentre (R.V.; M.Me. and A.Me.), and Centre of Excellence for Genomics and Translational Medicine Project No. 2014-2020.4.01.15-0012 to EGC of UT (A.Me.) and EBC (M.Me.); Estonian Institutional Research grant IUT24-1 (L.S., M.J., A.K., B.Y., K.T., C.B.M., Le.S., H.Sa., S.L., D.M.B., E.M., R.V., G.H., M.K., G.C., T.K. and M.Me.) and IUT20-60 (A.Me.); French Ministry of Foreign and European Affairs and French ANR grant number ANR-14-CE31-0013-01 (F.-X.R.); Gates Cambridge Trust Funding (E.J.); ICG SB RAS (No. VI.58.1.1) (D.V.L.); Leverhulme Programme grant no. RP2011-R-045 (A.B.M., P.G. and M.G.T.); Ministry of Education and Science of Russia; Project 6.656.2014/K (S.A.F.); NEFREX grant funded by the European Union (People Marie Curie Actions; International Research Staff Exchange Scheme; call FP7-PEOPLE-2012-IRSES-number 318979) (M.Me., G.H. and M.K.); NIH grants 5DP1ES022577 05, 1R01DK104339-01, and 1R01GM113657-01 (S.Tis.); Russian Foundation for Basic Research (grant N 14-06-00180a) (M.G.); Russian Foundation for Basic Research; grant 16-04-00890 (O.B. and E.B); Russian Science Foundation grant 14-14-00827 (O.B.); The Russian Foundation for Basic Research (14-04-00725-a), The Russian Humanitarian Scientific Foundation (13-11-02014) and the Program of the Basic Research of the RAS Presidium “Biological diversity” (E.K.K.); Wellcome Trust and Royal Society grant WT104125AIA & the Bristol Advanced Computing Research Centre (http://www.bris.ac.uk/acrc/) (D.J.L.); Wellcome Trust grant 098051 (Q.A.; C.T.-S. and Y.X.); Wellcome Trust Senior Research Fellowship grant 100719/Z/12/Z (M.G.T.); Young Explorers Grant from the National Geographic Society (8900-11) (C.A.E.); ERC Consolidator Grant 647787 ‘LocalAdaptatio’ (A.Ma.); Program of the RAS Presidium “Basic research for the development of the Russian Arctic” (B.M.); Russian Foundation for Basic Research grant 16-06-00303 (E.B.); a Rutherford Fellowship (RDF-10-MAU-001) from the Royal Society of New Zealand (M.P.C.)

Nitrate reduction in geologically heterogeneous catchments:a framework for assessing the scale of predictive capability of hydrological models

International audienceIn order to fulfil the requirements of the EU Water Framework Directive nitrate load from agricultural areas to surface water in Denmark needs to be reduced by about 40%. The regulations imposed until now have been uniform, i.e. the same restrictions for all areas independent of the subsurface conditions. Studies have shown that on a national basis about 2/3 of the nitrate leaching from the root zone is reduced naturally, through denitrification, in the subsurface before reaching the streams. Therefore, it is more cost-effective to identify robust areas, where nitrate leaching through the root zone is reduced in the saturated zone before reaching the streams, and vulnerable areas, where no subsurface reduction takes place, and then only impose regulations/restrictions on the vulnerable areas. Distributed hydrological models can make predictions at grid scale, i.e. at much smaller scale than the entire catchment. However, as distributed models often do not include local scale hydrogeological heterogeneities, they are typically not able to make accurate predictions at scales smaller than they are calibrated. We present a framework for assessing nitrate reduction in the subsurface and for assessing at which spatial scales modelling tools have predictive capabilities. A new instrument has been developed for airborne geophysical measurements, Mini-SkyTEM, dedicated to identifying geological structures and heterogeneities with horizontal and lateral resolutions of 30-50 m and 2 in, respectively, in the upper 30 m. The geological heterogeneity and uncertainty are further analysed by use of the geostatistical software TProGS by generating stochastic geological realisations that are soft conditioned against the geophysical data. Finally, the flow paths within the catchment are simulated by use of the MIKE SHE hydrological modelling system for each of the geological models generated by TProGS and the prediction uncertainty is characterised by the variance between the predictions of the different models. (C) 2013 Elsevier B.V. All rights reserved

“It was a lot Tougher than I Thought It would be”. A Qualitative Study on the Changing Nature of Being a Hemophilia Carrier

Studies on carriers of genetic disorders mainly focus on the process of genetic testing and reproductive choices, and less on how psychosocial aspects of being a carrier change over time. Our study sought to understand more about the psychosocial aspects of hemophilia carrier status, and thereby improve counseling aiming to advance carriers' quality of life and well-being. We analyzed 16 in-depth interviews from women who were carriers of hemophilia and had a son with hemophilia. Three themes emerged: Guilt and sorrow across generations; the choices and future consequences of genetic testing; and preparing to have a child with hemophilia. Experience with being a hemophilia carrier is a process that changes over time while feelings of guilt and sorrow run across generations. The carrier status may create "mothers-in-waiting" living at risk of having a sick child or not. The women think they are prepared to have a son with hemophilia, but experience more sadness than they expect when a son is diagnosed. Our findings suggest that health professionals, especially clinical geneticists and genetic counselors, carriers, families and patient organizations need to be aware that women's experiences of being a carrier of hemophilia changes during the biographical life course. The women may benefit from several rounds of genetic counseling at different stages of life