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Additional file 1: Supplementary Figure S1. EDI3 is highly expressed in ER-HER2+ cells and is regulated by HER2. A (left panel), HER2 mRNA expression measured using qRT-PCR. Quantification of A (right panel), HER2 and B, ER protein expression from immunoblotting as represented in Figure 1D. C, Immunofluorescence staining, and D, metabolite levels by NMR in a panel of breast cancer cell lines representing the different breast cancer subtypes. E, HER2 and EDI3 mRNA, and F, protein expression in HCC1954 cells upon HER2 silencing. G, EDI3 and HER2 mRNA expression and H, EDI3, pHER2, and HER2 protein expression in SKBR3 cells after silencing EDI3 with siRNA. I, EDI3 and HER2 mRNA expression and J, EDI3, pHER2, and HER2 protein expression in HCC1954 cells after silencing EDI3 with siRNA. K, Representative Western blots showing EDI3, pHER2 and HER2 protein expression in HCC1954 cells treated with 0.1 µM or 1.0 µM lapatinib for 24, 48, 72 and 96h, with corresponding quantification of protein levels. Data are mean ± SD or mean ± SE for HER2 protein expression in panel A of at least three independent experiments (*, P

Additional file 3: Supplementary Figure S3. Effect of targeting EDI3 on viability, alone or in combination with lapatinib, in ER-HER2+ and ER+HER2+ breast cancer cell lines. A-B, EDI3 mRNA (left panels) and protein expression (right panels) after silencing EDI3 using siRNA in A, BT474 and B, EFM192A cells. C, Effect of inhibiting HER2 using increasing concentrations of lapatinib on viability in SKBR3, HCC1954, BT474 and EFM192A cells. D-G Influence of silencing EDI3 with siRNA (oligo #2) and inhibiting HER2 with lapatinib (0.01 µM, 0.1 µM and 1 µM), as well as the combined inhibition of both EDI3 and HER2 on viability in D, SKBR3, E, HCC1954, F, BT474 and G, EFM192A cells. H, Effect of inhibiting HER2 using different concentrations of trastuzumab on viability in SKBR3, HCC1954, BT474 and EFM192A cells. I-L Influence of inhibiting EDI3 with siRNA (oligos #1 and #2) and HER2 with trastuzumab (1 µg/ml and 10 µg/ml), as well as the combined inhibition of both EDI3 and HER2 on viability in I, SKBR3, J, HCC1954, K, BT474 and L, EFM192A. Data are mean ± SD (A-C and H) or mean ± SE (D-G and I-L) of at least three independent experiments (*, P

Additional file 2: Supplementary Figure S2. GSK3β and mTOR are key proteins in HER2-mediated upregulation of EDI3. A, Quantification of HER2, EDI3 and p-Akt protein expression after inhibiting PI3K with 5 and 10 µM LY294002 for 3 and 7 days in MCF7-NeuT cells. B, Representative Western blot and HER2, EDI3 and p-PKCα/βII protein expression quantification after inhibiting PLCγ with 1 and 3 µM U73122 for 3 and 7 days in MCF7-NeuT cells. C, Representative Western blot and quantification of HER2, EDI3 and p-ERK1/2 protein expression after inhibiting MEK with 5 and 25 µM PD98059 for 3 and 7 days in MCF7-NeuT cells. D, HER2, EDI3 and p-mTOR protein expression after inhibiting mTORC1 with 1 and 3 µM everolimus for 3 and 7 days in MCF7-NeuT cells. E, HER2, EDI3 and p-mTOR protein expression after inhibiting mTORC1 with 1 and 3 µM everolimus for 1, 2 and 3 days in HCC1954 cells. F, HER2, EDI3 and p-mTOR protein expression after inhibiting mTORC1 with 1 and 3 µM everolimus for 1, 2 and 3 days in SKBR3 cells. G, HER2, EDI3 and β-catenin protein expression after inhibiting GSKβ with 1 and 2.5 µM CHIR-99021 for 3 and 7 days in MCF7/NeuT cells. H, HER2, EDI3 and β-catenin protein expression after inhibiting GSKβ with 1 and 2.5 µM CHIR-99021 for 1, 2 and 3 days in HCC1954 cells. I, HER2, EDI3 and β-catenin protein expression after inhibiting GSKβ with 1 and 2.5 µM CHIR-99021 for 1, 2 and 3 days in SKBR3 cells. Graphs D-I are quantification of Western blots exemplified in Fig. 3C-H, respectively. J, EDI3 mRNA expression after inhibiting c-Myc with 5 and 10 µM 10074-G5, K, NFκB with 5 and 10 µM SC75741, or L, KLF5 with 5 and 10 µM SR18662 for 1, 2 and 3 days in SKBR3 cells. Data are mean ± SD of at least three independent experiments (*, P

Nature Communications 14, 3304 (2023). doi:10.1038/s41467-023-38840-8, Published by Nature Publishing Group UK, [London]

Additional file 7: Supplementary Table S2. List of used reagents including A, QuantiTect primer assays, B, siRNA and shRNA oligos, and C, antibodies.

Additional file 6: Supplementary Table S1. EDI3 (GPCPD1) expression in human breast cancer tissue was evaluated in publicly available Affymetrix HG U133 Plus 2.0 gene expression microarray dataset that were downloaded from the Gene Expression Omnibus webportal (GEO) together with available clinicopathological data.

Additional file 8: Supplementary Table S3. Association of EDI3 expression for the three probesets on the Affymetrix HG U133 Plus 2.0 array and available clinicopathological parameters shown for A, the six datasets combined, as well as separately for B, GSE16446, C, GSE19615, D, GSE28844, E, GSE32646, F, GSE6532, and G, GSE9195. P value from the Mann Whitney or Kruskal-Wallis U test.

Additional file 5: Supplemental Figure S5. Inducibly silencing EDI3 inhibits colony formation and cell viability. A, Representative images (top) and corresponding quantification (bottom) of colony number (left) and size (right) formed by HCC1954 shNEG and HCC1954 shEDI3 (oligos shEDI3 #1, #2, #3) cells treated with 0 or 0.1 µg/ml doxycycline. B, Viability (RFU) after treating HCC1954 shNEG and HCC1954 shEDI3 (oligos shEDI3 #1, #2) with 0.1 or 1 µg/ml doxycycline. All in vitro data are mean ± SD of three independent experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). RFU, relative fluorescence units.

Immunity 56(7), 1578-1595, e1-e8 (2023). doi:10.1016/j.immuni.2023.05.017, Published by Elsevier, New York, NY

Additional file 4: Supplementary Figure S4. Inhibiting EDI3 with dipyridamole, alone or in combination with HER2-targeting therapy, in ER-HER2+ tumors in mice. A-D EDI3 activity in percent after treatment with dipyridamole at different concentrations in A, HCC1954 B, SKBR3, C, BT474 and D, EFM192 cells. E, Cell viability in percent of vehicle control after treatment with different concentrations of dipyridamole in SKBR3, HCC1954, BT474 and EFM192A cells (upper panel) and EC50-values with corresponding 95% confidence intervals (CI) (lower panel). F, Concentration of dipyridamole in plasma after a single oral dose of 120 mg/kg dipyridamole in mice over time. G, Mouse weight (grams) after treatment with dipyridamole, lapatinib and the combination for up to 4 weeks. H, Images of CD1 nude mice with tumors encircled, after 4-week treatment of vehicle, dipyridamole, lapatinib and the combination of both. I, Plasma and tumor concentrations (µM) of dipyridamole (upper panel) and lapatinib (lower panel) after treatment with each compound alone or in combination (D+L). Data (A-E) are mean ± SD of three independent experiments; mean ± SD of three CD1 mice (F); mean ± SD 5 to 6 mice (G); and representative images of 4 mice per condition H. D+L (combined dipyridamole and lapatinib treatment); BQL (below quantification limit); EC50 (half maximal effective concentration).

Funder: Children’s Liver Disease Foundation; Id: http://dx.doi.org/10.13039/501100000290, Funder: European Society for Paediatric Research; Id: http://dx.doi.org/10.13039/501100008873, Funder: Virtutis Opus Foundation, Funder: European Association for the Study of the Liver; Id: http://dx.doi.org/10.13039/501100009253, Funder: For Wishdom Foundation, Funder: Italian Ministry of Health, Funder: Van den Broek Lohman Foundation, Genome-wide association studies in adults have identified variants in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) and mitochondrial amidoxime reducing component 1 (MTARC1) as protective against nonalcoholic fatty liver disease (NAFLD). We aimed to test their association with pediatric NAFLD liver histology and investigate their function using metabolomics. A total of 1450 children (729 with NAFLD, 399 with liver histology) were genotyped for rs72613567T>TA in HSD17B13, rs2642438G>A in MTARC1, and rs738409C>G in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Genotype-histology associations were tested using ordinal regression. Untargeted hepatic proteomics and plasma lipidomics were performed in a subset of children. We found rs72613567T>TA in HSD17B13 to be associated with lower odds of NAFLD diagnosis (odds ratio, 0.7; 95% confidence interval, 0.6-0.9) and a lower grade of portal inflammation (p < 0.001). rs2642438G>A in MTARC1 was associated with a lower grade of hepatic steatosis (p = 0.02). Proteomics found reduced expression of HSD17B13 in carriers of the protective -TA allele. MTARC1 levels were unaffected by genotype. Both variants were associated with down-regulation of fibrogenic pathways. HSD17B13 perturbs plasma phosphatidylcholines and triglycerides. In silico modeling suggested p.Ala165Thr disrupts the stability and metal binding of MTARC1. Conclusion: Both HSD17B13 and MTARC1 variants are associated with less severe pediatric NAFLD. These results provide further evidence for shared genetic mechanisms between pediatric and adult NAFLD.

Upon different types of liver injury, there are distinct patterns of hepatic fibrosis developed, such as ECM septa (fibrotic walls) connecting pericentral (CV) areas due to toxic injury or septa connecting portal (PV) compartments due to cholestatic injury. Since liver fibrosis is a multi-cellular process and difficult to capture for biological in vivo and in vitro models, computational models may shed light on exploring the mechanisms behind particular pattern formation. In this work, we present a novel computational liver model that permits to assess the potential role of biomechanics in the formation of fibrotic walls. It for the first time studies the orchestration of cell types during fibrosis development and the interaction of cell populations with the ECM network mechanics in a liver lobule. Our model results are quantitatively confronted with experimental findings. The determination of the pattern-characterizing parameters in this study e.g. the density of hepatic stellate cells (HSC) and macrophages (MC) were obtained through image analysis of 2D and 3D images from mouse experiments. Together with a model of ECM networks, these non-parenchymal cells and their intercellular signaling were integrated as new elements into computational model of basic liver micro-architecture that included hepatocytes (the main parenchymal liver cell), sinusoids, CV and PV. The new model is applied to test possible mechanisms of how the fibrotic wall may form during liver fibrosis in space and time.

The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades. Bisphenol F (BPF) and S (BPS) have been suggested as “safer” alternatives. In the present study we used hepatocyte-like cells (HLCs) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system and were exposed to BPA, BPF and BPS for 8 days during their transition to HLCs. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC50) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as downregulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity., EXCLI Journal; 19:Doc1459; ISSN 1611-2156

Partial hepatectomy (PHx) is a surgical intervention where a part of the liver is removed. Due to its extraordinary capacity to regenerate, the liver is able to regenerate about two-thirds of its mass within a few weeks. Nevertheless, in some patients regeneration fails. Understanding the principles and limitations underlying regeneration may permit to control this process and prospectively improve the regeneration. Here, we established a simulation model to mimic the process of regeneration in the liver lobe of a mouse. This model represents each hepatocyte individually and builds upon a previous computational model of regeneration of drug induced damage in a single liver lobule. The present study simulates entire liver lobes that consist of hundreds to thousands of lobules. It accounts for biomechanical control of cell cycle progression (Biomechanical Growth Control), which has not been considered in that previous work. The model reproduced the available experimental observations only if BGC was taken into account. Interestingly, the model predicted that BGC minimizes the number of proliferating neighbor cells of a proliferating cell resulting in a checkerboard-like proliferation pattern. Moreover, the model predicted different cell proliferation patterns in pigs and mice that corresponded to data obtained from regenerating tissue of the two species. In conclusion, the here established model suggest that biomechanical control mechanisms may play a significant role in liver regeneration after PHx.

Proceedings of the National Academy of Sciences of the United States of America 117(1), 454-463 (2020). doi:10.1073/pnas.1909145117, Published by National Acad. of Sciences, Washington, DC

Thousands of transcriptome data sets are available, but approaches for their use in dynamic cell response modelling are few, especially for processes affected simultaneously by two orthogonal influencing variables. We approached this problem for neuroepithelial development of human pluripotent stem cells (differentiation variable), in the presence or absence of valproic acid (signaling variable). Using few basic assumptions (sequential differentiation states of cells; discrete on/off states for individual genes in these states), and time-resolved transcriptome data, a comprehensive model of spontaneous and perturbed gene expression dynamics was developed. The model made reliable predictions (average correlation of 0.85 between predicted and subsequently tested expression values). Even regulations predicted to be non-monotonic were successfully validated by PCR in new sets of experiments. Transient patterns of gene regulation were identified from model predictions. They pointed towards activation of Wnt signaling as a candidate pathway leading to a redirection of differentiation away from neuroepithelial cells towards neural crest. Intervention experiments, using a Wnt/beta-catenin antagonist, led to a phenotypic rescue of this disturbed differentiation. Thus, our broadly applicable model allows the analysis of transcriptome changes in complex time/perturbation matrices. published

Since its foundation in 2002, Experimental and Clinical Sciences (EXCLI Journal) published more than 760 original articles and reviews, particularly in the field of cancer research (Abbastabar et al., 2018; Nojadeh et al., 2018; Karimian et al., 2018), cell biology (Li et al., 2017; Niknami et al., 2017; Ahmadi et al., 2017), toxicology (Randjelovic et al., 2017; Hassani et al., 2018; Nakhaee and Mehrpour, 2018), neurosciences (Farajdokht et al., 2017; Ebrahimi et al., 2017), drug discovery (Li et al., 2018; Khedher et al., 2017) and immunology (Fahimi et al., 2018; Sarvari et al., 2018). However, the editors are keen to keep a broad view of science and technology and also welcome manuscripts from other fields of life sciences and interdisciplinary studies. Our main criterion during the review process is the scientific quality of the study. Editors and reviewers focus particularly on whether the methods are sufficiently described, results are presented in a transparent way, have been sufficiently reproduced in independent experiments and justify the main conclusions. If this is the case, also confirmatory studies or studies reporting negative results may be published. Each manuscript should include a statement on the aim of the study and convincingly explain why the experiments are relevant. While a high degree of novelty is welcome, it is not our most important criterion in selecting manuscripts for publication., EXCLI Journal;Vol. 18 2019

Small-molecule flux in tissue-microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods applicable to live animals. We developed a methodology based on dynamic and correlative imaging for quantitative intravital flux analysis. Application to the liver, challenged the prevailing ‘mechano-osmotic’ theory of canalicular bile flow. After active transport across hepatocyte membranes bile salts are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts, diffusion is augmented by regulatable advection. We corroborate these observations with in silico simulations and pan-species comparisons of lobule size. This study demonstrates a flux mechanism, where the energy invested in transmembrane transport entropically dissipates in a sub-micron scale vessel network. One Sentence Summary Bile flux proceeds by diffusion in canaliculi, augmented by advection in ducts.

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2–12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival.

The effects of engineered nanomaterials on human health are still intensively studied in order to facilitate their safe application. However, relatively little is known how mechanical strain as induced in alveolar epithelial cells by breathing movements modifies biological responses to nanoparticles (NPs). In this study, A549 cells as a model for alveolar epithelial cells were exposed to 25 nm amorphous colloidal silica NPs under dynamic and static culture conditions. Gene array data, qPCR, and ELISA revealed an amplified effect of NPs when cells were mechanically stretched in order to model the physiological mechanical deformation during breathing. In contrast, treatment of cells with either strain or NPs alone only led to minor changes in gene expression or interleukin-8 (IL-8) secretion. Confocal microscopy revealed that stretching does not lead to an increased internalization of NPs, indicating that elevated intracellular NP accumulation is not responsible for the observed effect. Gene expression alterations induced by combined exposure to NPs and mechanical strain showed a high similarity to those known to be induced by TNF-α. This study suggests that the inclusion of mechanical strain into in vitro models of the human lung may have a strong influence on the test results.

The present debate on chemicals with Hormonal activity, often termed 'endocrine disruptors', is highly controversial and includes challenges of the present paradigms used in toxicology and in hazard identification and risk characterization. In our opinion, chemicals with hormonal activity can be subjected to the well-evaluated health risk characterization approach used for many years including adverse outcome pathways. Many of the points arguing for a specific approach for risk characterization of chemicals with hormonal activity are based on highly speculative conclusions. These conclusions are not well supported when evaluating the available information. The present debate on chemicals with Hormonal activity, often termed 'endocrine disruptors', is highly controversial and includes challenges of the present paradigms used in toxicology and in hazard identification and risk characterization. In our opinion, chemicals with hormonal activity can be subjected to the well-evaluated health risk characterization approach used for many years including adverse outcome pathways. Many of the points arguing for a specific approach for risk characterization of chemicals with hormonal activity are based on highly speculative conclusions. These conclusions are not well supported when evaluating the available information.

Non-small cell lung cancer (NSCLC) represents a genomically unstable cancer type with extensive copy number aberrations. The relationship of gene copy number alterations and subsequent mRNA levels has only fragmentarily been described. The aim of this study was to conduct a genome-wide analysis of gene copy number gains and corresponding gene expression levels in a clinically well annotated NSCLC patient cohort (n = 190) and their association with survival. While more than half of all analyzed gene copy number-gene expression pairs showed statistically significant correlations (10,296 of 18,756 genes), high correlations, with a correlation coefficient >0.7, were obtained only in a subset of 301 genes (1.6%), including KRAS, EGFR and MDM2. Higher correlation coefficients were associated with higher copy number and expression levels. Strong correlations were frequently based on few tumors with high copy number gains and correspondingly increased mRNA expression. Among the highly correlating genes, GO groups associated with posttranslational protein modifications were particularly frequent, including ubiquitination and neddylation. In a meta-analysis including 1,779 patients we found that survival associated genes were overrepresented among highly correlating genes (61 of the 301 highly correlating genes, FDR adjusted p

Candidate gene and genome-wide association studies (GWAS) have identified 15 independent genomic regions associated with bladder cancer risk. In search for additional susceptibility variants, we followed up on four promising single-nucleotide polymorphisms (SNPs) that had not achieved genome-wide significance in 6911 cases and 11 814 controls (rs6104690, rs4510656, rs5003154 and rs4907479, P < 1 × 10(-6)), using additional data from existing GWAS datasets and targeted genotyping for studies that did not have GWAS data. In a combined analysis, which included data on up to 15 058 cases and 286 270 controls, two SNPs achieved genome-wide statistical significance: rs6104690 in a gene desert at 20p12.2 (P = 2.19 × 10(-11)) and rs4907479 within the MCF2L gene at 13q34 (P = 3.3 × 10(-10)). Imputation and fine-mapping analyses were performed in these two regions for a subset of 5551 bladder cancer cases and 10 242 controls. Analyses at the 13q34 region suggest a single signal marked by rs4907479. In contrast, we detected two signals in the 20p12.2 region-the first signal is marked by rs6104690, and the second signal is marked by two moderately correlated SNPs (r(2) = 0.53), rs6108803 and the previously reported rs62185668. The second 20p12.2 signal is more strongly associated with the risk of muscle-invasive (T2-T4 stage) compared with non-muscle-invasive (Ta, T1 stage) bladder cancer (case-case P ≤ 0.02 for both rs62185668 and rs6108803). Functional analyses are needed to explore the biological mechanisms underlying these novel genetic associations with risk for bladder cancer.

Candidate gene and genome-wide association studies (GWAS) have identified 15 independent genomic regions associated with bladder cancer risk. In search for additional susceptibility variants, we followed up on four promising single-nucleotide polymorphisms (SNPs) that had not achieved genome-wide significance in 6911 cases and 11 814 controls (rs6104690, rs4510656, rs5003154 and rs4907479, P < 1x 10(-6)), using additional data from existing GWAS datasets and targeted genotyping for studies that did not have GWAS data. In a combined analysis, which included data on up to 15 058 cases and 286 270 controls, two SNPs achieved genome-wide statistical significance: rs6104690 in a gene desert at 20p12.2 (P = 2.19 x 10(-11)) and rs4907479 within the MCF2L gene at 13q34 (P = 3.3 x 10(-10)). Imputation and fine-mapping analyses were performed in these two regions for a subset of 5551 bladder cancer cases and 10 242 controls. Analyses at the 13q34 region suggest a single signal marked by rs4907479. In contrast, we detected two signals in the 20p12.2 region-the first signal is marked by rs6104690, and the second signal is marked by two moderately correlated SNPs (r(2) = 0.53), rs6108803 and the previously reported rs62185668. The second 20p12.2 signal is more strongly associated with the risk of muscle-invasive (T2-T4 stage) compared with non-muscle-invasive (Ta, T1 stage) bladder cancer (case-case P

Despite an abundance of studies on chromatin states and dynamics, there is an astonishing dearth of information on the expression of genes responsible for regulating histone and DNA modifications. We used here a set of 156 defined epigenetic modifier genes (EMG) and profiled their expression pattern in cells of different lineages. As reference value, expression data from human embryonic stem cells (hESC) were used. Hepatocyte-like cells were generated from hESC, and their EMG expression was compared to primary human liver cells. In parallel, we generated postmitotic human neurons (Lu d6), and compared their relative EMG expression to human cortex (Ctx). Clustering analysis of all cell types showed that neuronal lineage samples grouped together (94 similarly regulated EMG), as did liver cells (61 similarly-regulated), while the two lineages were clearly distinct. The general classification was followed by detailed comparison of the major EMG groups; genes that were higher expressed in differentiated cells than in hESC included the acetyltransferase KAT2B and the methyltransferase SETD7. Neuro-specific EMGs were the histone deacetylases HDAC5 and HDAC7, and the arginine-methyltransferase PRMT8. Comparison of young (Lu d6) and more aged (Ctx) neuronal samples suggested a maturation-dependent switch in the expression of functionally homologous proteins. For instance, the ratio of the histone H3 K27 methyltransfereases, EZH1 to EZH2, was high in Ctx and low in Lu d6. The same was observed for the polycomb repressive complex 1 (PRC1) subunits CBX7 and CBX8. A large proportion of EMGs in differentiated cells was very differently expressed than in hESC, and absolute levels were significantly higher in neuronal samples than in hepatic cells. Thus, there seem to be distinct qualitative and quantitative differences in EMG expression between cell lineages.

Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining.

EXCLI Journal ; Vol. 8, 2009

We conducted a multi-stage, genome-wide association study of bladder cancer with a primary scan of 591,637 SNPs in 3,532 affected individuals (cases) and 5,120 controls of European descent from five studies followed by a replication strategy, which included 8,382 cases and 48,275 controls from 16 studies. In a combined analysis, we identified three new regions associated with bladder cancer on chromosomes 22q13.1, 19q12 and 2q37.1: rs1014971, (P = 8 x 10(-12)) maps to a non-genic region of chromosome 22q13.1, rs8102137 (P = 2 x 10(-11)) on 19q12 maps to CCNE1 and rs11892031 (P = 1 x 10(-7)) maps to the UGT1A cluster on 2q37.1. We confirmed four previously identified genome-wide associations on chromosomes 3q28, 4p16.3, 8q24.21 and 8q24.3, validated previous candidate associations for the GSTM1 deletion (P = 4 x 10(-11)) and a tag SNP for NAT2 acetylation status (P = 4 x 10(-11)), and found interactions with smoking in both regions. Our findings on common variants associated with bladder cancer risk should provide new insights into the mechanisms of carcinogenesis.

The increased relevance of liver surgery and transplantation as a therapeutic modality over the last two decades mandates the development of novel strategies to improve liver regeneration. Here we studied whether erythropoietin (EPO) improves liver regeneration after hepatectomy in pigs. Eighteen female pigs underwent laparoscopic left lateral liver resection and were allocated randomly into three groups. No EPO was administered to the control group (group 1, n=6). Group 2 (n=6) received EPO topically to the liver resection surface in a fibrin sealant. Group 3 (n=6) received EPO topically and systemically. Pigs were sacrificed 14 days after hepatectomy. The fraction of proliferating hepatocytes was determined by ki-67 immunostaining. Liver volume was determined by the principle of Archimedes, Liver weight and volume were significantly increased in group 3 (1249 ± 223 g, 1073 ± 190 ml) compared to group 2 (1027 ± 167 g, 894 ± 105 ml) and group 1 (923 ± 186 g, 813 ± 165 ml). Ki-67 immunostaining of liver tissue close to the resection surface demonstrated a significantly increased percentage of proliferating hepatocytes in group 3 (4.3 ± 1.96 %) and in group 2 (3.5% ± 0.98 %) as compared to group 1 (1.15 ± 1.2 %) 14 days after hepatectomy. Our results indicate for the first time that EPO supports liver regeneration after hepatectomy., EXCLI Journal ; Vol. 7, 2008

This report covers recent advances in Regenerative Medicine with a special focus on (i) imaging of regeneration, (ii) nanotechnology and tissue engineering, (iii) immunological cell tolerance, (iv) cell therapies in cardiovascular, neurodegenerative, and liver diseases and in spinal regeneration., EXCLI Journal ; Vol. 5, 2006

Emission of volatile organic substances (VOC) from articles inside a car may lead to adverse health effects in exposed drivers. Presently, no general concept to derive maximum exposure levels inside cars has been published. Therefore, we recommend techniques for three types of maximum exposure levels inside cars, namely for (i) chronic exposure to non-genotoxic substances (ELIA, chronic), (ii) short term exposure inside automotive vehicles (STELIA) and (iii) genotoxic substances acting by threshold mechanisms (ELIA, cm). For derivation of the ELIA, chronic, we recommend to start with a Lowest Observed Adverse Effect Level (LOEL) or a Benchmark Dose 10 (BMD10) and use a procedure including four steps: a. estimation of the No Observed Effect Level (NOEL), b. extrapolation from laboratory animal to man, c. extrapolation to the general population due to interindividual differences and d. extrapolation to continuous exposure. To derive STELIAs a three-step-procedure is recommended, starting with a LOEL and a. estimating the NOEL, b. extrapolating from animal to man and c. extrapolating to the general population. Derivation of ELIA, cm, the maximum exposure level for carcinogens acting by a threshold mechanism, is certainly the most problematic procedure. We recommend to start with the lower 95% confidence limit for the most sensitive tumor type known in animals (BMD05) and a. extrapolate from animal to man and b. use a safety factor of 1/50 000 unless specific research succeeded in demonstrating specific levels of thresholds. It must be considered that a general concept for maximum exposure levels can not replace an intelligent toxicological approach considering the mechanism of action of individual substances. However, the strategy suggested here offers a practical technique for identification of individual problematic exposures that require an intensive toxicological evaluation., EXCLI Journal ; Vol. 3, 2004

Tumor incidence for renal cell carcinoma is two-fold higher in males than in females. Members of the protein kinase C (PKC) gene family have been shown to be relevant for carcinogenesis. However, little is known about a possible gender specific role of PKC in renal cell carcinoma (RCC). In this study, we quantified expression of eleven PKC-isoforms in clear cell RCCs (ccRCC) and in the corresponding normal renal tissue. A possible association of PKC-isoforms with gender of the patients was examined. Tissue specimens of 27 patients, 14 males and 13 females, with ccRCC and of the corresponding normal renal tissue were examined. Expression of PKC-isoforms were detected by Western blot analysis and quantified by computer-aided integration. In ccRCCs, as well as in the corresponding normal renal tissue, all PKC-isoforms except PKCy and 0 were detectable. Clear associations with gender of the patients were observed: (i) PKCd was reduced in tumor tissue of female patients (p = 0.023), but not of male patients (p = 0.198). (ii) A 3.6-fold enhanced expression of the oncogene PKC? was found in tumor tissue of female compared to male patients (p = 0.049). Gender specific differences in PKCd as well as PKCn expression suggest that different molecular mechanisms are relevant for carcinogenesis of ccRCC in male and female patients. This may become important for classification and treatment of ccRCC., EXCLI Journal ; Vol. 2, 2003

EXCLI Journal ; Vol. 2, 2003

The human microsomal epoxide hydrolase, a single enzyme, has to detoxify a broad range of structurally diverse, potentially genotoxic epoxides that are formed in the course of xenobiotic metabolism. The enzyme has developed a unique strategy to combine a broad substrate specificity with a high detoxification efficacy, by immediately trapping the reactive compounds as covalent intermediates and by being expressed at high levels for high trapping capacity. Computer simulation and experimental data as well as existing epidemiologic studies reveal this detoxification strategy as the mechanistic basis for a threshold in the tumorigenesis of mutagenic carcinogens., EXCLI Journal ; Vol. 2, 2003

EXCLI Journal ; Vol. 2, 2003

Cells 8(12), 1556 (2019). doi:10.3390/cells8121556, Published by MDPI, Basel

Stable reconstitution of vascular endothelial beds upon transplantation of progenitor cells represents an important challenge due to the paucity and generally limited integration/expansion potential of most identified vascular related cell subsets. We previously showed that mouse fetal liver (FL) hemato/vascular cells from day 12 of gestation (E12), expressing the Stem Cell Leukaemia (SCL) gene enhancer transgene (SCL-PLAP+ cells), had robust endothelial engraftment potential when transferred to the blood stream of newborns or adult conditioned recipients, compared to the scarce vascular contribution of adult bone marrow cells. However, the specific SCL-PLAP+ hematopoietic or endothelial cell subset responsible for the long-term reconstituting endothelial cell (LTR-EC) activity and its confinement to FL developmental stages remained unknown. Using a busulfan-treated newborn transplantation model, we show that LTR-EC activity is restricted to the SCL-PLAP+ VE-cadherin+ CD45- cell population, devoid of hematopoietic reconstitution activity and largely composed by Lyve1+ endothelial-committed cells. SCL-PLAP+ Ve-cadherin+ CD45- cells contributed to the liver sinusoidal endothelium and also to the heart, kidney and lung microvasculature. LTR-EC activity was detected at different stages of FL development, yet marginal activity was identified in the adult liver, revealing unknown functional differences between fetal and adult liver endothelial/endothelial progenitors. Importantly, the observations that expanding donor-derived vascular grafts colocalize with proliferating hepatocyte-like cells and participate in the systemic circulation, support their functional integration into young livers. These findings offer new insights into the engraftment, phonotypical, and developmental characterization of a novel endothelial/endothelial progenitor cell subtype with multiorgan LTR-EC activity, potentially instrumental for the treatment/genetic correction of vascular diseases. Stem Cells 2017;35:507-521.

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)- derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (\20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

Primary human hepatocytes (PHH) are important for clinical therapy as well as for studies in pharmacology and toxicology. Hepatocyte-like cells (HLC) derived from pluripotent stem cells offer the perspective of an unlimited supply of PHH, however, genome- and proteome-wide analyses demonstrated that HLC still show major differences compared to PHH. More recently it was shown that, HLC express hepatocyte- and non-hepatocyte-associated genes within the same cells, indicating that HLC reside in a hybrid state that can be targeted by bioinformatics-guided intervention [1]. In this context, it remains to be clarified whether cell line or differentiation protocol-specific differences lead to comparable hybrid states and if the reported hybrid state is a common feature among HLC. In this thesis, HLC obtained by two different protocols from three different induced pluripotent stem cell lines (iPSC) were compared using genome-wide transcriptomics. Furthermore, it was demonstrated that interventions to improve HLC differentiation by targeting the FXR gene regulatory network (GRN) increased the expression of hepatocyte-associated genes and suppressed undesired non-liver genes in HLC, thereby increasing their similarity to PHH. However, this has yet only been shown on the transcriptomic level. Here, functional assays of bile acid secretion and lipid droplet formation were performed to confirm that an FXR targeting intervention strategy does indeed increase the similarity of HLC to PHH.

Metastasis remains a major problem for tumor therapy. In endometrial and ovarian cancer, metastasis and worse survival was found to be associated with elevated EDI3 (GPCPD1; GDE5; GDPD6) expression in primary tumors. EDI3 is a glycerophosphodiesterase which cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P) and is therefore considered one of the key enzymes involved in choline metabolism. Altered choline metabolism is a recognized metabolic hallmark of cancer and was reported in breast, ovarian, and prostate cancers. Previously, in vitro studies revealed that silencing EDI3 transiently in various breast cancer cell lines resulted in altered choline metabolism and impaired cellular migration, attachment, and spreading. However, stable constitutive EDI3 knockdown led to compensation of metabolite levels over time, which was accompanied by a loss of the migration phenotype. Therefore, in the present work a doxycycline (Dox) inducible EDI3 knockdown system was established in luciferase-expressing ER-HER2+ breast cancer cells, which reduces compensatory effects and allows to investigate EDI3 in tumor growth and metastasis in vivo. To create cell lines in which EDI3 is inducibly silenced, stable luciferase-expressing HCC1954 cells were generated and subsequently transduced with lentiviral particles, which resulted in three different Dox-inducible EDI3 knockdown cell lines containing independent EDI3-targeting shRNA oligos. Dox treatment led to a time and dose dependent decrease in EDI3 RNA and protein expression. Mass spectrometry analyses revealed that induced EDI3 knockdown also led to dose dependent alterations in endogenous choline metabolites and phospholipid levels. Using various in vitro assays, it could be shown that EDI3 knockdown resulted in significant reduction in colony formation and proliferation, processes which are relevant in the formation of metastasis. Furthermore, EDI3 silencing rendered cells more susceptible towards anoikis. However, Dox-induced EDI3 knockdown had only little effect on adhesion and no effect on migration. To investigate EDI3’s role in tumor growth and metastasis in vivo, different tumor models were established in immunodeficient mice. The subcutaneous tumor model showed no significant effect on primary tumor growth. However, in a mouse model for peritoneal metastasis, luminescence imaging revealed lower signals indicative of less metastasis formation in the EDI3 knockdown condition. Furthermore, it could be shown that silencing EDI3 was associated with reduced tumor burden, less ascites fluid and longer survival time. Altogether, this thesis provides, for the first time, in vivo evidence that supports a role for EDI3 in metastasis formation, which further emphasizes the importance of choline and glycerophospholipid metabolism in this process.

Worldwide, approximately two million deaths per year are caused by different liver diseases. The understanding of the underlying mechanisms causing liver diseases remain incomplete. In the last two decades, the involvement of different immune cells during liver disease progression, regeneration and homeostasis have been closely investigated and offered new therapeutic strategies and targets. Recently, natural killer (NK) cells have been observed to contribute to the progression of drug induced liver injury (DILI). In vitro results of primary human hepatocytes (PHH) and human hepatocyte cell lines pretreated with valproic acid, ketoconazole, promethazine and isoniazid showed enhanced expression of activating NK cell ligands. Moreover, enhanced NK cell activity was observed which was characterized by the higher interferon gamma (IFN) production and increased cytotoxicity against the pretreated hepatocytes. In order to validate the in vivo relevance of these findings and investigate the DILI mechanism, the first aim was to reproduce the enhanced NK cell activity ligand expression in drug pretreated primary mouse hepatocytes (PMH). The data of drug pretreated PMH revealed higher expression levels of activating NK cell ligands which were more pronounced in combination with tumor necrosis factor alpha (TNF). In the next step, the aim was to establish a drug induced liver injury mouse model with the most promising drug valproic acid. The attempts to establish a valproic acid induced liver injury mouse model were not successful, since the treatments did not cause increased NK cell numbers in the liver, higher expression of activating NK cell ligands on hepatocytes nor liver damage. During the last decade, another immune cell type was identified which seem to be involved in liver damage, regenaration and homeostasis: innate lymphoid cell 1 (ILC1). Until today, there is not much known about ILC1s, however they have been described to possess a memory potential. Therefore, another aim of this thesis was to identify and characterize the memory potential of ILC1s after multiple applications of the hepatotoxic compound carbon tetrachloride (CCl4). After multiple doses of CCl4 injected in a 30 days interval, memory like ILC1s were discovered which were able to produce more interferon gamma (IFN). Further experiments revealed a transient effect of ILC1s in the liver, since higher cell numbers were observed on day one but until day three, the ILC1 cell numbers were again comparable to control cell numbers. Additionally, the memory effect of ILC1s is long-lasting. Nevertheless, the localization and function of memory like ILC1s in the liver need to be further investigated to evaluate the possible contribution to liver disease progression, regeneration and homeostasis.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries with an increasing prevalence of approximately 25 %. NAFLD comprises several stages, starting as benign steatosis and progressing to non-alcoholic steatohepatitis (NASH), and in some cases to liver cirrhosis and hepatocellular carcinoma (HCC). Although several emerging therapies are currently in clinical trials, so far there are no approved drugs for treatment of NASH. The overarching goal of this thesis was to investigate the mechanisms of NAFLD stage transitions, and to establish preventive measures for the progression from benign steatosis to NASH. First, a mouse model of NAFLD progression was established by long-term feeding of male C57Bl/6N mice with western-style diet (WD) up to 54 weeks. The disease progression was evaluated time-dependently by biochemical, histopathological, and immunohistochemical analyses as well as by intravital two-photon-based imaging. This analyses revealed six stages in NAFLD progression: (1) benign steatosis, (2) macrophage crown-like structure formation, (3) macropinocytosis of bile, (4) ductular reaction, (5) dedifferentiation and functional shutdown, and (6) tumor nodule formation. The novel finding of this thesis was the identification of stage 3, where a retrograde vesicular uptake of bile from bile canaliculi to hepatocytes led to toxic accumulation of bile acids in the liver tissue, providing a link between NAFLD and cholestasis. The phenomenon was further identified as macropinocytosis by treating the mice with a macropinocytosis-specific inhibitor imipramine. As a result, a single application of imipramine efficiently blocked macropinocytosis in WD-fed mice. Interestingly, a long-term application of imipramine for 8 weeks decreased the bile acid concentrations in the liver tissue and led to significant NAFLD amelioration. Moreover, bile macropinocytosis was also found relevant in human NAFLD patients as detected by the presence of fragments of bile canaliculi within steatotic hepatocytes. In conclusion, an NAFLD mouse model recapitulating the different stages of human NAFLD progression to NASH and eventually to HCC was successfully established. Moreover, a novel mechanism possibly driving NALFD progression was identified as macropinocytosis of bile from bile canaliculi back to hepatocytes.

Drug-induced liver injury (DILI) is a major concern due to its poor predictability. Recently, we have developed an in vitro/in silico test system for the prediction of human DILI in relation to oral doses and blood concentrations. Additionally, two indices, the toxicity separation index (TSI) and the toxicity estimation index (TEI) were introduced for the quantitative evaluation of a test system and its input parameters. In this PhD-thesis, I studied whether extending the in vitro test battery of the test system, so far consisting of a cytotoxicity test in primary human hepatocytes, by additional functional readouts would lead to improved performance and thus allow a more accurate prediction. In total, three different approaches that address putative DILI-relevant mechanisms were explored. In the first approach, the influence of a bile acid mix on the cytotoxicity of in total 18 compounds in cultivated primary human hepatocytes was investigated. In summary, increased and decreased susceptibility to both hepatotoxic and non-hepatotoxic substances was observed with the addition of bile acids, which did not improve the TSI (0.79 -> 0.77) nor the TEI (0.73 -> 0.69) compared to cytotoxicity without additional bile acids. In the second approach, an assay was developed and evaluated that measures the inhibition of bile acid export carriers in primary human hepatocytes. In total 36 compounds were tested with the transport inhibtion assay and the cytotoxicity assay. In conclusion, the assay is able to detect bile acid export carrier inhibition and integration into the in vitro test battery improved TSI (0.77 -> 0.89) and TEI (0.69 -> 0.83) compared to cytotoxicity. In a third approach, intracellular lipid accumulation in HepG2 cells was investigated for a total of 60 compounds. Addition of lipid droplet accumulation to cytotoxicity improved TSI (0.74 -> 0.80) and TEI (0.67 -> 0.81). In summary, three assays were developed for the in vitro test battery of a test system to predict drug-induced liver injury. Quantitative analysis revealed that two of the three assays lead to improved separation of hepatotoxic and non-hepatotoxic compounds, as well as improved estimation of in vivo relevant blood concentrations. These improvements allow more accurate prediction of DILI by the test system.