Systems microscopy to unravel cellular stress response signalling in drug induced liver injury

Toxicological insults are met by cellular adaptive stress response pathway activation. We find that activation of adaptive stress responses occur well before the typical ultimate outcome of chemical cell injury. To increase our understanding of chemically-induced adaptive stress response pathway activation and its contribution to safety assessment we believe that a time-resolved, sensitive and multiplex readout of chemical-induced toxicological relevant cellular stress responses will be essential. For that purpose, we developed a platform containing a panel of distinct adaptive stress response reporter cell lines. These are used for automated high content live cell imaging and quantitative multi-parameter image analysis to elucidate critical adaptive stress response pathway activation that can contribute to human chemical safety assessment. To conserve the endogenous gene regulatory programs, we tag selected reporter target genes with GFP using BAC-transgenomics approaches. In this thesis we demonstrate the functionality of individual BAC-GFP pathway in toxicity reporter cell lines. The application of these reporters in chemical safety assessment in relation to drug-induced liver injury is discussed in detail. We anticipate that ultimately a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses assisting in the safety assessment of chemicals.UBL - phd migration 201

Mobilising Q methodology within a realist evaluation – lessons from an empirical study

Realist evaluation and Q Methodology are established approaches in social science. However, integration of Q methodology within a realist evaluation is scarce. This paper attempts to illustrate (through a recent evaluation) how Q methodology can support a realist evaluation. The paper attempts to capture the philosophical compatibility of the two approaches creating an argument for Q’s integration within realist evaluation. Through the case study selected (a realist evaluation of an evaluation capacity building framework) the iterative methodological process is presented, capturing a snapshot of the findings from the evaluation. This illuminates how Q met the philosophical aims of a realist evaluation to make sense of how and why and under what circumstances a programme or intervention works. It is argued that Q methodology is entirely suitable for capturing the three key stages in a realist evaluation of developing, testing, and refining programme theory. It is intended that this paper can contribute to inspire other realist evaluators and methodologists to make use of Q as a tool in their evaluation

Effects of cold winters and roost site stability on population development of non-native Asian ring-necked parakeets (Alexandrinus manillensis) in temperate Central Europe – Results of a 16-year census

Asian ring-necked parakeets (Alexandrinus manillensis, formerly Psittacula krameri, hereafter RNP) first bred in Germany in 1969. Since then, RNP numbers increased in all three major German subpopulations (Rhineland, Rhine-Main, Rhine-Neckar) over the period 2003–2018. In the Rhine-Neckar region, the population increased to more than fivefold within only 15 years. Interestingly, there was no significant breeding range expansion of  RNP in the period 2010–2018. In 2018, the total number of RNP in Germany amounted to >16,200 birds. Differences in RNP censuses between years were evident. Surprisingly, cold winters (extreme value, −13.7 °C) and cold weather conditions in the breeding season (coldest month average, −1.36 °C) were not able to explain between-year variation. This finding suggests that in general winter mortality is low – with exceptions for winters 2008/2009 and 2009/2010, and a population-relevant loss of broods is low in our study population. Surprisingly, the social behaviour in terms of spatio-temporal stability of roost sites could well explain positive and negative population trends. Years of spatially stable and regularly used roost sites seem to correlate with increasing population sizes. In contrast, known shifts of RNP among different roost sites or the formations of new roost sites by split are related to population stagnation or a decrease in numbers. Climate change may lead to further range expansion as cities not suitable yet for RNP may become so in the near future.

Investigation of Nrf2, AhR and ATF4 Activation in Toxicogenomic Databases

Toxicological responses to chemical insult are largely regulated by transcriptionally activated pathways that may be independent, correlated and partially or fully overlapping. Investigating the dynamics of the interactions between stress responsive transcription factors from toxicogenomic data and defining the signature of each of them is an additional step toward a system level understanding of perturbation driven mechanisms. To this end, we investigated the segregation of the genes belonging to the three following transcriptionally regulated pathways: the AhR pathway, the Nrf2 pathway and the ATF4 pathway. Toxicogenomic datasets from three projects (carcinoGENOMICS, Predict-IV and TG-GATEs) obtained in various experimental conditions (in human and rat in vitro liver and kidney models and rat in vivo, with bolus administration and with repeated doses) were combined and consolidated where overlaps between datasets existed. A bioinformatic analysis was performed to refine pathways' signatures and to create chemical activation capacity scores to classify chemicals by their potency and selectivity of activation of each pathway. With some refinement such an approach may improve chemical safety classification and allow biological read across on a pathway level

Uncovering the Signaling Landscape Controlling Breast Cancer Cell Migration Identifies Novel Metastasis Driver Genes

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug developmen

Uncovering the signaling landscape controlling breast cancer cell migration identifies novel metastasis driver genes

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development

Neuronal Nitric Oxide Synthase-Rescue of Dystrophin/Utrophin Double Knockout Mice does not Require nNOS Localization to the Cell Membrane

Survival of dystrophin/utrophin double-knockout (dko) mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS) transgene. Dko mice expressing the transgene (nNOS TG+/dko) experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide)-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Alternative signaling network activation through different insulin receptor family members caused by pro-mitogenic antidiabetic insulin analogues in human mammary epithelial cells

INTRODUCTION: Insulin analogues are designed to have improved pharmacokinetic parameters compared to regular human insulin. This provides a sustained control of blood glucose levels in diabetic patients. All novel insulin analogues are tested for their mitogenic side effects, however these assays do not take into account the molecular mode of action of different insulin analogues. Insulin analogues can bind the insulin receptor and the insulin-like growth factor 1 receptor with different affinities and consequently will activate different downstream signaling pathways. METHODS: Here we used a panel of MCF7 human breast cancer cell lines that selectively express either one of the isoforms of the INSR or the IGF1R. We applied a transcriptomics approach to assess the differential transcriptional programs activated in these cells by either insulin, IGF1 or X10 treatment. RESULTS: Based on the differentially expressed genes between insulin versus IGF1 and X10 treatment, we retrieved a mitogenic classifier gene set. Validation by RT-qPCR confirmed the robustness of this gene set. The translational potential of these mitogenic classifier genes was examined in primary human mammary cells and in mammary gland tissue of mice in an in vivo model. The predictive power of the classifier genes was evaluated by testing all commercial insulin analogues in the in vitro model and defined X10 and glargine as the most potent mitogenic insulin analogues. CONCLUSIONS: We propose that these mitogenic classifier genes can be used to test the mitogenic potential of novel insulin analogues as well as other alternative molecules with an anticipated affinity for the IGF1R. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13058-015-0600-5) contains supplementary material, which is available to authorized users