Publications by authors named "Ralf Mrowka"

59 Publications

Preselector.uni-jena.de: optimize your cloning-a resource for identifying restriction enzymes for preselection reactions.

Nucleic Acids Res 2021 07;49(W1):W541-W543

Experimentelle Nephrologie, Universitätsklinikum Jena KIM III, Am Nonnenplan 4, D-07743 Jena, Germany.

Preselection digests are a common strategy to reduce the background in the ligation step of molecular cloning. However, choosing fitting restriction enzymes by hand is not trivial and may lead to errors, potentially costing a lot of time and work. We therefore created preselector.uni-jena.de (https://preselector.uni-jena.de/), a free online tool to find such restriction enzymes. The tool uses regular expressions to find recognition sites of restriction enzymes in the DNA sequences provided by the user. This new tool compares the sets of restriction sites and reports the enzymes that cut one sequence but not the other sequences to the user. These enzymes are then the ones suitable for a preselection digest. Thus, preselector.uni-jena.de is a fast, reliable, and free-to-use tool to help researchers designing preselection digestion strategies for their cloning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262699PMC
July 2021

From small molecules to dinosaurs - Recent advances in blood pressure research.

Authors:
Ralf Mrowka

Acta Physiol (Oxf) 2021 07 27;232(3):e13677. Epub 2021 May 27.

Klinik für Innere Medizin III, AG Experimentelle Nephrologie, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13677DOI Listing
July 2021

The impact of episporic modification of on virulence and interaction with phagocytes.

Comput Struct Biotechnol J 2021 20;19:880-896. Epub 2021 Jan 20.

Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI), Jena, Germany.

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on and . This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of , one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.csbj.2021.01.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851798PMC
January 2021

Kidney research.

Authors:
Ralf Mrowka

Acta Physiol (Oxf) 2020 12 3;230(4):e13569. Epub 2020 Nov 3.

Klinik für Innere Medizin III, AG Experimentelle Nephrologie, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13569DOI Listing
December 2020

Sex Differences in Diabetes- and TGF-β1-Induced Renal Damage.

Cells 2020 10 3;9(10). Epub 2020 Oct 3.

Department of Internal Medicine III, Jena University Hospital, Am Klinikum 1, D-07747 Jena, Germany.

While females are less affected by non-diabetic kidney diseases compared to males, available data on sex differences in diabetic nephropathy (DN) are controversial. Although there is evidence for an imbalance of sex hormones in diabetes and hormone-dependent mechanisms in transforming growth factor β1 (TGF-β1) signaling, causes and consequences are still incompletely understood. Here we investigated the influence of sex hormones and sex-specific gene signatures in diabetes- and TGF-β1-induced renal damage using various complementary approaches (a diabetes mouse model, ex vivo experiments on murine renal tissue, and experiments with a proximal tubular cell line TKPTS). Our results show that: (i) diabetes affects sex hormone concentrations and renal expression of their receptors in a sex-specific manner; (ii) sex, sex hormones and diabetic conditions influence differences in expression of TGF-β1, its receptor and bone morphogenetic protein 7 (BMP7); (iii) the sex and sex hormones, in combination with variable TGF-β1 doses, determine the net outcome in TGF-β1-induced expression of connective tissue growth factor (CTGF), a profibrotic cytokine. Altogether, these results suggest complex crosstalk between sex hormones, sex-dependent expression pattern and profibrotic signals for the precise course of DN development. Our data may help to better understand previous contradictory findings regarding sex differences in DN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9102236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600610PMC
October 2020

Influence of Macitentan on the Vascular Tone and Recruitment of Finger Capillaries Under Hypobaric Hypoxia in High Altitude.

High Alt Med Biol 2020 Dec 31;21(4):336-345. Epub 2020 Jul 31.

Division of Cardiology, Department of Internal Medicine I, University Hospital Jena, Jena, Germany.

Betge, Stefan, Stefan Drinda, Thomas Neumann, Laura Bäz, Alexander Pfeil, Christian Schulze, Ralf Mrowka, Christian Jung, and Marcus Franz. Influence of macitentan on the vascular tone and recruitment of finger capillaries under hypobaric hypoxia in high altitude. . 21:336-345, 2020. Acute normobaric (NH) and hypobaric hypoxia (HH) has effects on the vascular tone of larger arteries and may have effects on the microcirculation. These effects may be noninvasively detectable by automated devices. A part of these effects may be mediated by endothelin (ET) and should be influenced by macitentan (MAC), a dual endothelin receptor antagonist (ERA). We used photoplethysmographic sensors, fingertip volume sensors, nailfold capillaroscopy, and laser Doppler probes at rest and after a 5-minute forearm ischemia in healthy study subjects under NH, under HH, and under HH plus a single dose of MAC. NH at simulated 4000 m led to increased heart rates (HR) and pulse wave velocities (PWV) and reduced augmentation index (AIX). The values for the AIX showed a high SD and differed between the used devices. At simulated 5500 m, only baseline mean value (BMV; EndoPAT) showed a further change, indicating less filled capillaries of the fingertips. HH (2978 m) increased HR, blood pressure values, and PWV. Focusing on the microcirculation of the fingertips, HH reduced the BMV and the nailfold capillary density and the postischemic capillary recruitment. MAC had no effect on the BMV, but antagonized the effects of HH on the nailfold capillaries and led to a strongly increased postischemic diameter of the arterial limbs. Concordantly, the postischemic blood flow velocity increment, measured through ultrasound Doppler, was increased at ALT+MAC. The BMV may be a parameter for changes of the microcirculation of the finger tips. A single dose of MAC blocked hypoxia-induced capillary rarefaction and enhanced postischemic hyperemia of the fingertips. These results indicate the importance of ET-1 for the regulation of the microcirculation under hypoxia. The German Registry of Clinical Studies (DRKS) ID: 00005459.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ham.2019.0120DOI Listing
December 2020

Decipher the complexity of cis-regulatory regions by a modified Cas9.

PLoS One 2020 2;15(7):e0235530. Epub 2020 Jul 2.

Experimental Nephrology Group, KIM III, Universitätsklinikum Jena, Jena, Germany.

Background: Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity of renin transcription in its native genomic context.

Methods: With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models.

Results: SAM complexes induced activation of renin in our cells after renin specific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data.

Conclusion: By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235530PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332081PMC
September 2020

How Simulations May Help Us to Understand the Dynamics of COVID-19 Spread. - Visualizing Non-Intuitive Behaviours of a Pandemic (pansim.uni-jena.de).

Acta Physiol (Oxf) 2020 08 21;229(4):e13520. Epub 2020 Jun 21.

Experimentelle Nephrologie, Universitätsklinikum Jena, KIM III, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300557PMC
August 2020

Recent advances in blood pressure research.

Authors:
Ralf Mrowka

Acta Physiol (Oxf) 2020 01 26;228(1):e13412. Epub 2019 Nov 26.

AG Experimentelle Nephrologie, Klinik für Innere Medizin III, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13412DOI Listing
January 2020

Recent advances in hypertension research.

Authors:
Ralf Mrowka

Acta Physiol (Oxf) 2019 08 5;226(4):e13295. Epub 2019 Jun 5.

Klinik für Innere Medizin III, AG Experimentelle Nephrologie, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13295DOI Listing
August 2019

Inflammation-Dysregulated inflammatory response and strategies for treatment.

Acta Physiol (Oxf) 2019 07 16;226(3):e13284. Epub 2019 May 16.

Klinik für Innere Medizin III, AG Experimentelle Nephrologie, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13284DOI Listing
July 2019

Cancer - An ongoing fight searching for reasons and therapies.

Acta Physiol (Oxf) 2019 05 30;226(1):e13275. Epub 2019 Mar 30.

Klinik für Innere Medizin III, AG Experimentelle Nephrologie, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13275DOI Listing
May 2019

Imidazopyridines as Potent KDM5 Demethylase Inhibitors Promoting Reprogramming Efficiency of Human iPSCs.

iScience 2019 Feb 11;12:168-181. Epub 2019 Jan 11.

Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, ImNeuenheimer Feld 364, 69120 Heidelberg, Germany. Electronic address:

Pioneering human induced pluripotent stem cell (iPSC)-based pre-clinical studies have raised safety concerns and pinpointed the need for safer and more efficient approaches to generate and maintain patient-specific iPSCs. One approach is searching for compounds that influence pluripotent stem cell reprogramming using functional screens of known drugs. Our high-throughput screening of drug-like hits showed that imidazopyridines-analogs of zolpidem, a sedative-hypnotic drug-are able to improve reprogramming efficiency and facilitate reprogramming of resistant human primary fibroblasts. The lead compound (O4I3) showed a remarkable OCT4 induction, which at least in part is due to the inhibition of H3K4 demethylase (KDM5, also known as JARID1). Experiments demonstrated that KDM5A, but not its homolog KDM5B, serves as a reprogramming barrier by interfering with the enrichment of H3K4Me3 at the OCT4 promoter. Thus our results introduce a new class of KDM5 chemical inhibitors and provide further insight into the pluripotency-related properties of KDM5 family members.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2019.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354736PMC
February 2019

Obesity, adipocytes and insulin resistance-Friends for life?

Acta Physiol (Oxf) 2019 03;225(3):e13258

Experimental Nephrology, KIM III, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13258DOI Listing
March 2019

Literature optimized integration of gene expression for organ-specific evaluation of toxicogenomics datasets.

PLoS One 2019 14;14(1):e0210467. Epub 2019 Jan 14.

Faculty of Biology, Biozentrum I, Mainz, Germany.

The study of drug toxicity in human organs is complicated by their complex inter-relations and by the obvious difficulty to testing drug effects on biologically relevant material. Animal models and human cell cultures offer alternatives for systematic and large-scale profiling of drug effects on gene expression level, as typically found in the so-called toxicogenomics datasets. However, the complexity of these data, which includes variable drug doses, time points, and experimental setups, makes it difficult to choose and integrate the data, and to evaluate the appropriateness of one or another model system to study drug toxicity (of particular drugs) of particular human organs. Here, we define a protocol to integrate drug-wise rankings of gene expression changes in toxicogenomics data, which we apply to the TG-GATEs dataset, to prioritize genes for association to drug toxicity in liver or kidney. Contrast of the results with sets of known human genes associated to drug toxicity in the literature allows to compare different rank aggregation approaches for the task at hand. Collectively, ranks from multiple models point to genes not previously associated to toxicity, notably, the PCNA clamp associated factor (PCLAF), and genes regulated by the master regulator of the antioxidant response NFE2L2, such as NQO1 and SRXN1. In addition, comparing gene ranks from different models allowed us to evaluate striking differences in terms of toxicity-associated genes between human and rat hepatocytes or between rat liver and rat hepatocytes. We interpret these results to point to the different molecular functions associated to organ toxicity that are best described by each model. We conclude that the expected production of toxicogenomics panels with larger numbers of drugs and models, in combination with the ongoing increase of the experimental literature in organ toxicity, will lead to increasingly better associations of genes for organism toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210467PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331104PMC
October 2019

New insights into the astonishing diversity of hormone functions.

Acta Physiol (Oxf) 2018 12 22;224(4):e13188. Epub 2018 Oct 22.

Klinik für Innere Medizin III, AG Experimentelle Nephrologie, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13188DOI Listing
December 2018

Modifiers of hypertension.

Authors:
Ralf Mrowka

Acta Physiol (Oxf) 2018 11 25;224(3):e13184. Epub 2018 Sep 25.

Experimentelle Nephrologie, Universitätsklinikum Jena, KIM III, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13184DOI Listing
November 2018

Nephropathy: New aspects of mechanisms, diagnosis and therapy.

Acta Physiol (Oxf) 2018 09 25;224(1):e13162. Epub 2018 Jul 25.

Klinik für Innere Medizin III, AG Experimentelle Nephrologie, Universitätsklinikum Jena, Jena, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apha.13162DOI Listing
September 2018

Human serum alters cell culture behavior and improves spheroid formation in comparison to fetal bovine serum.

Exp Cell Res 2018 04 21;365(1):57-65. Epub 2018 Feb 21.

Placenta-Lab, Department of Obstetrics, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany.

Background: The use of fetal bovine serum (FBS) as growth supplement for human cell and tissue culture is widely spread in basic research as well as in clinical approaches, although several limitations must be considered, such as unstable composition and availability, biosafety and ethical aspects. Regarding interspecies differences, xenogeneic growth factors may evoke incompatibilities and non-desired interactions with human cells resulting in imprecise outcome of human-relevant data.

Methods: In this study the functionality of human serum (HS) has been investigated in comparison to FBS by assessing proliferation, migration and invasion of the human cervical cancer cell lines SiHa and HeLa. The effects of both sera on spheroid formation were analyzed microscopically.

Results: Both, FBS and HS, stimulate cell proliferation and migration similarly, whereas HS significantly enhanced cell invasion. The spheroid formation assay revealed remarkable differences between both sera, especially for SiHa cells. While in FBS supplemented medium cells only formed loose aggregates, HS induced regularly shaped spheroids under all tested conditions.

Conclusion: We were able to demonstrate that HS and FBS differently influence behavior of cells in culture which may have an impact on experimental results, especially in 3D cultures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2018.02.017DOI Listing
April 2018

Liver-Kidney-on-Chip To Study Toxicity of Drug Metabolites.

ACS Biomater Sci Eng 2018 Jan 4;4(1):78-89. Epub 2017 Dec 4.

Institute of Pharmacy and Molecular Biotechnology, Pharmaceutical Biology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.

Advances in organ-on-chip technologies for the application in in vitro drug development provide an attractive alternative approach to replace ethically controversial animal testing and to establish a basis for accelerated drug development. In recent years, various chip-based tissue culture systems have been developed, which are mostly optimized for cultivation of one single cell type or organoid structure and lack the representation of multi organ interactions. Here we present an optimized microfluidic chip design consisting of interconnected compartments, which provides the possibility to mimic the exchange between different organ specific cell types and enables to study interdependent cellular responses between organs and demonstrate that such tandem system can greatly improve the reproducibility and efficiency of toxicity studies. In a simplified liver-kidney-on-chip model, we showed that hepatic cells that grow in microfluidic conditions abundantly and stably expressed metabolism-related biomarkers. Moreover, we applied this system for investigating the biotransformation and toxicity of Aflatoxin B1 (AFB1) and Benzoalphapyrene (BαP), as well as the interaction with other chemicals. The results clearly demonstrate that the toxicity and metabolic response to drugs can be evaluated in a flow-dependent manner within our system, supporting the importance of advanced interconnected multiorgans in microfluidic devices for application in in vitro toxicity testing and as optimized tissue culture systems for in vitro drug screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsbiomaterials.7b00417DOI Listing
January 2018

Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation.

Arch Toxicol 2018 Mar 5;92(3):1133-1149. Epub 2017 Dec 5.

Institute of Pharmacy and Molecular Biotechnology, Pharmaceutical Biology, Heidelberg University, Im Neuenheimer Feld 364, 69120, Heidelberg, Germany.

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00204-017-2128-1DOI Listing
March 2018

Evaluation of in vivo and in vitro models of toxicity by comparison of toxicogenomics data with the literature.

Methods 2018 01 14;132:57-65. Epub 2017 Jul 14.

Johannes-Gutenberg University of Mainz, Gresemundweg 2, 55128 Mainz, Germany; Institute of Molecular Biology gGmbH, Ackermannweg 4, 55128 Mainz, Germany. Electronic address:

Toxicity affecting humans is studied by observing the effects of chemical substances in animal organisms (in vivo) or in animal and human cultivated cell lines (in vitro). Toxicogenomics studies collect gene expression profiles and histopathology assessment data for hundreds of drugs and pollutants in standardized experimental designs using different model systems. These data are an invaluable source for analyzing genome-wide drug response in biological systems. However, a problem remains that is how to evaluate the suitability of heterogeneous in vitro and in vivo systems to model the many different aspects of human toxicity. We propose here that a given model system (cell type or animal organ) is supported to appropriately describe a particular aspect of human toxicity if the set of compounds associated in the literature with that aspect of toxicity causes a change in expression of genes with a particular function in the tested model system. This approach provides candidate genes to explain the toxicity effect (the differentially expressed genes) and the compounds whose effect could be modeled (the ones producing both the change of expression in the model system and that are associated with the human phenotype in the literature). Here we present an application of this approach using a computational pipeline that integrates compound-induced gene expression profiles (from the Open TG-GATEs database) and biomedical literature annotations (from the PubMed database) to evaluate the suitability of (human and rat) in vitro systems as well as rat in vivo systems to model human toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2017.07.010DOI Listing
January 2018

Human microRNA-299-3p decreases invasive behavior of cancer cells by downregulation of Oct4 expression and causes apoptosis.

PLoS One 2017 20;12(4):e0174912. Epub 2017 Apr 20.

Experimentelle Nephrologie, Klinik für Innere Medizin III, Universitätsklinikum Jena, Jena, Germany.

Purpose: Oct4 was reported to be one of the most important pluripotency transcription factors in the biology of stem cells including cancer stem cells, and progressed malignant cells. Here we report the investigation of gene expression control of Oct4 by selected human microRNAs and the physiological effect of Oct4 silencing in invasive cancer cells.

Methods And Results: High throughput luciferase activity assay revealed the microRNA-299-3p to be the most effective in reducing gene expression of Oct4, which was confirmed by Western blot analysis and Oct4 promoter activity in a target luciferase assay. Furthermore, it could be demonstrated that downregulation of Oct4 by microRNAs-299-3p in breast cancer and fibrosarcoma cells lead to a decreased invasiveness in a microfluidic chip assay. Additionally, microRNA-299-3p causes apoptosis in cancer cells. Comparison with Oct4 specific siRNA transfection confirmed that this effect is primary due to the blockade of Oct4 expression.

Conclusion: The results suggest that microRNA-299-3p is an interesting target for potential clinical use. It may be able to decrease invasive behaviour of carcinoma cells; or even kill these cells by causing apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398498PMC
September 2017

Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols.

J Mammary Gland Biol Neoplasia 2016 12 12;21(3-4):89-98. Epub 2016 Aug 12.

Department of Obstetrics, Placenta-Lab, Bachstraße 18, 07743, Jena, Germany.

Multicellular tumor spheroids are widely used models in tumor research. Because of their three dimensional organization they can simulate avascular tumor areas comprising proliferative and necrotic cells. Nonetheless, protocols for spheroid generation are still inconsistent. Therefore, in this study the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3 have been used to compare different spheroid generation models including hanging drop, liquid overlay and suspension culture techniques, each under several conditions. Experimental approaches differed in cell numbers (400-10,000), media and additives (25 % methocel, 25 % methocel plus 1 % Matrigel, 3.5 % Matrigel). In total, 42 different experimental setups have been tested. Generation of spheroids was evaluated by light microscopy and the structural composition was assessed immunohistochemically by means of Ki-67, cleaved poly (ADP-ribose) polymerase (cPARP) and mucin-1 (MUC-1) expression. Although the tested cell lines diverged widely in their capacity of forming spheroids we recommend hanging drops supplemented with 25 % methocel as the most reliable and efficient method with regard to success of generation of uniform spheroids, costs, experimental complexity and time expenditure in the different cell lines. MCF-7 cells formed spheroids under almost all analyzed conditions, and MDA-MB-231 cells under only one protocol (liquid overlay technique, 3.5 % Matrigel), while SK-BR-3 did not under neither condition. Therefore, we outline specific methods and recommend the use of adapted and standardized spheroid generation protocols for each cell line.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10911-016-9359-2DOI Listing
December 2016

Vitamin D3 Partly Antagonizes Advanced-Glycation Endproducts-Induced NFκB Activation in Mouse Podocytes.

Nephron 2016 10;134(2):105-116. Epub 2016 Aug 10.

Department of Internal Medicine III, University Hospital Jena, Jena, Germany.

Background/aims: We have previously shown that advanced glycation-endproducts (AGEs) induced NFκB activation in differentiated mouse podocytes. This NFκB activation may contribute to the progression of renal disease and mediation of fibrosis by various mechanisms. This study was undertaken to test whether this detrimental response may be reversed by vitamin D3 or its analogue paricalcitol.

Methods: Differentiated mouse podocytes were challenged with glycated bovine serum albumin (AGE-BSA), or non-glycated control BSA (in the presence or absence of various concentrations of vitamin D3 (decostriol, 1α,25-dihydroxyvitamin D3)) or its active analog paricalcitol. Quantitative mRNA expressions were measured by real-time PCR, whereas protein expressions were determined by Western blotting followed by densitometry. Cytoplasmic and nuclear protein expression of the NFκB subunit p65 (Rel A) were determined by Western blotting. Furthermore, the ratio of phosphorylated to non-phosphorylated IκB-α was measured using specific antibodies. Electrophoretic mobility shift assays and a capture ELISA assay were used to assess NFκB transactivation in vitro. In addition, NFκB transactivation was also monitored in HEK-NFκBIA reporter cells using live cell luminometry.

Results: Podocytes expressed the receptor for vitamin D. The vitamins did not suppress receptor for AGEs (RAGE) expression; instead, they rather upregulated RAGE. Although vitamin D3 and paricalcitol partly and differentially modified some of the studied parameters, both hormones inhibited AGE-BSA-induced NFκB transactivation, presumably by various mechanisms including the upregulation of IκB-α protein, keeping NFκB sequestered in an inactive state in the cytoplasm.

Conclusion: Vitamin D3 or its analog paricalcitol partly prevented AGE-mediated NFκB activation, an important feature of diabetic nephropathy (DN). Whether this in vitro finding is of clinical relevance to prevent/treat DN requires further studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000448106DOI Listing
July 2017

Ethyl 2-((4-Chlorophenyl)amino)thiazole-4-carboxylate and Derivatives Are Potent Inducers of Oct3/4.

J Med Chem 2015 Aug 23;58(15):5742-50. Epub 2015 Jul 23.

†Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.

The octamer-binding transcription factor 4 (Oct3/4) is a master gene in the transcriptional regulatory network of pluripotent cells. Repression of Oct3/4 in embryonic stem cells (ESCs) is associated with cell differentiation and loss of pluripotency, whereas forced overexpression in cooperation with other transcriptional factors, such as Nanog, Sox2, and Lin28, can reprogram somatic cells back into pluripotent cells, termed induced pluripotent stem cells (iPSCs). However, random integration and potential tumorigenic transformation caused by viral transduction limit the clinical application of iPSCs. By performing a cell-based high throughput screening (HTS) campaign, we identified several potential small molecules as inducers of Oct3/4 expression. Here we report a lead structure ethyl 2-((4-chlorophenyl)amino)-thiazole-4-carboxylate, termed O4I2, showing high activity in enforcing Oct3/4 expression. On the basis of chemical expansion, we further identified derivatives having increased activities toward Oct3/4 induction. Thus, O4I2 and its derivatives should provide a new class of small molecules suitable for iPSC generation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b00226DOI Listing
August 2015

Identification of 2-[4-[(4-Methoxyphenyl)methoxy]-phenyl]acetonitrile and Derivatives as Potent Oct3/4 Inducers.

J Med Chem 2015 Jun 7;58(12):4976-83. Epub 2015 May 7.

†Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.

Reprogramming somatic cells into induced-pluripotent cells (iPSCs) provides new access to all somatic cell types for clinical application without any ethical controversy arising from the use of embryonic stem cells (ESCs). Established protocols for iPSCs generation based on viral transduction with defined factors are limited by low efficiency and the risk of genetic abnormality. Several small molecules have been reported as replacements for defined transcriptional factors, but a chemical able to replace Oct3/4 allowing the generation of human iPSCs is still unavailable. Using a cell-based High Throughput Screening (HTS) campaign, we identified that 2-[4-[(4-methoxyphenyl)methoxy]phenyl]acetonitrile (1), termed O4I1, enhanced Oct3/4 expression. Structural verification and modification by chemical synthesis showed that O4I1 and its derivatives not only promoted expression and stabilization of Oct3/4 but also enhanced its transcriptional activity in diverse human somatic cells, implying the possible benefit from using this class of compounds in regenerative medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b00144DOI Listing
June 2015

Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Nucleic Acids Res 2015 Mar 8;43(6):3219-36. Epub 2015 Mar 8.

Charité - Universitätsmedizin Berlin, Institut für Vegetative Physiologie, Charitéplatz 1, D-10117 Berlin, Germany

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381074PMC
March 2015

ICAM1 depletion reduces spinal metastasis formation in vivo and improves neurological outcome.

Eur Spine J 2015 Oct 25;24(10):2173-81. Epub 2015 Feb 25.

Department of Neurosurgery, Universitätsmedizin Charite, Augustenburger Platz 1, 13353, Berlin, Germany.

Introduction: Clinical treatment of spinal metastasis is gaining in complexity while the underlying biology remains unknown. Insufficient biological understanding is due to a lack of suitable experimental animal models. Intercellular adhesion molecule-1 (ICAM1) has been implicated in metastasis formation. Its role in spinal metastasis remains unclear. It was the aim to generate a reliable spinal metastasis model in mice and to investigate metastasis formation under ICAM1 depletion.

Material And Methods: B16 melanoma cells were infected with a lentivirus containing firefly luciferase (B16-luc). Stable cell clones (B16-luc) were injected retrogradely into the distal aortic arch. Spinal metastasis formation was monitored using in vivo bioluminescence imaging/MRI. Neurological deficits were monitored daily. In vivo selected, metastasized tumor cells were isolated (mB16-luc) and reinjected intraarterially. mB16-luc cells were injected intraarterially in ICAM1 KO mice. Metastasis distribution was analyzed using organ-specific fluorescence analysis.

Results: Intraarterial injection of B16-luc and metastatic mB16-luc reliably induced spinal metastasis formation with neurological deficits (B16-luc:26.5, mB16-luc:21 days, p<0.05). In vivo selection increased the metastatic aggressiveness and led to a bone specific homing phenotype. Thus, mB16-luc cells demonstrated higher number (B16-luc: 1.2±0.447, mB16-luc:3.2±1.643) and increased total metastasis volume (B16-luc:2.87±2.453 mm3, mB16-luc:11.19±3.898 mm3, p<0.05) in the spine. ICAM1 depletion leads to a significantly reduced number of spinal metastasis (mB16-luc:1.2±0.84) with improved neurological outcome (29 days). General metastatic burden was significantly reduced under ICAM1 depletion (control: 3.47×10(7)±1.66×10(7); ICAM-1-/-: 5.20×10(4)±4.44×10(4), p<0.05 vs. control)

Conclusion: Applying a reliable animal model for spinal metastasis, ICAM1 depletion reduces spinal metastasis formation due to an organ-unspecific reduction of metastasis development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00586-015-3811-7DOI Listing
October 2015

Shutdown of achaete-scute homolog-1 expression by heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 in hypoxia.

J Biol Chem 2014 Sep 14;289(39):26973-26988. Epub 2014 Aug 14.

Institut für Vegetative Physiologie, Charité-Universitätsmedizin Berlin, D-10117 Berlin,. Electronic address:

The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, whereas additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pulldown experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.579391DOI Listing
September 2014
-->