34 results on '"Oliver Keminer"'
Search Results
2. Persister state-directed transitioning and vulnerability in melanoma
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Heike Chauvistré, Batool Shannan, Sheena M. Daignault-Mill, Robert J. Ju, Daniel Picard, Stefanie Egetemaier, Renáta Váraljai, Christine S. Gibhardt, Antonio Sechi, Farnusch Kaschani, Oliver Keminer, Samantha J. Stehbens, Qin Liu, Xiangfan Yin, Kirujan Jeyakumar, Felix C. E. Vogel, Clemens Krepler, Vito W. Rebecca, Linda Kubat, Smiths S. Lueong, Jan Forster, Susanne Horn, Marc Remke, Michael Ehrmann, Annette Paschen, Jürgen C. Becker, Iris Helfrich, Daniel Rauh, Markus Kaiser, Sheraz Gul, Meenhard Herlyn, Ivan Bogeski, José Neptuno Rodríguez-López, Nikolas K. Haass, Dirk Schadendorf, and Alexander Roesch
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Science - Abstract
Slow-cycling melanoma persister cells are characterised by a high, reversible expression of H3K4 demethylase KDM5B. Here, the authors use genetic and chemical methods to enforce a permanent high expression of KDM5B and show that these cells transit to a melanocytic differentiated state and undergo cell cycle arrest.
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- 2022
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3. A SARS-CoV-2 cytopathicity dataset generated by high-content screening of a large drug repurposing collection
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Bernhard Ellinger, Denisa Bojkova, Andrea Zaliani, Jindrich Cinatl, Carsten Claussen, Sandra Westhaus, Oliver Keminer, Jeanette Reinshagen, Maria Kuzikov, Markus Wolf, Gerd Geisslinger, Philip Gribbon, and Sandra Ciesek
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Science - Abstract
Measurement(s) cytotoxicity • killing by virus of host cell Technology Type(s) bright-field microscopy Factor Type(s) concentration • incubation time Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.13562570
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- 2021
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4. Generation of two human isogenic iPSC lines from fetal dermal fibroblasts
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Rashmi Tandon, Björn Brändl, Natalya Baryshnikova, Alexandro Landshammer, Laura Steenpaß, Oliver Keminer, Ole Pless, and Franz-Josef Müller
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Biology (General) ,QH301-705.5 - Abstract
Two isogenic hiPSC lines, ZIPi013-B and ZIPi013-E, were generated by reprogramming fetal dermal fibroblasts with episomal vectors. Previously, the same fetal fibroblasts were reprogrammed multiple times in a study comparing other reprogramming methods. As a consequence, the genomes have been sequenced multiple times. Both new cell lines offer the opportunity to study basic stem cell biology and model human disease. They can be applied as reference cell lines for creating isogenic clones bearing disease mutations on a well-characterized genomic background, as both cell lines have demonstrated excellent differentiation capacity in multiple labs.Resource tableUnlabelled TableUnique stem cell lines identifierZIPi013-BZIPi013-EAlternative names of stem cell linesZIP13K2 (ZIPi013-B)ZIP13K5 (ZIPi013-E)InstitutionZentrum für Integrative Psychiatrie gGmbH, Kiel, GermanyContact information of distributorPD Dr. Franz-Josef Müller, franz-josef.mueller@uksh.deType of cell linesiPSCOriginhumanCell SourceFibroblastsClonalityClonalMethod of reprogrammingTransgene free, episomalMultiline rationaleIsogenic clonesGene modificationNOType of modificationN/AAssociated diseaseN/AGene/locusN/AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateStock date ZIPi013-B 8th December 2017, stock date ZIPi013-E 12th December 2017Cell line repository/bankN/AEthical approvalhttps://www.sciencellonline.com/technical-support/ethical-statement.htmlEthikkommission der medizinischen Fakultät der Christian-Albrechts-Universität zu Kiel, approval number A145/11
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- 2018
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5. A high-content small molecule screen identifies novel inducers of definitive endoderm
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Alexander Korostylev, Pallavi U. Mahaddalkar, Oliver Keminer, Kamyar Hadian, Kenji Schorpp, Philip Gribbon, and Heiko Lickert
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Rock inhibition ,Fasudil ,Pancreatic progenitors ,Anterior definitive endoderm ,Differentiations ,Internal medicine ,RC31-1245 - Abstract
Objectives: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can generate any given cell type in the human body. One challenge for cell-replacement therapy is the efficient differentiation and expansion of large quantities of progenitor cells from pluripotent stem cells produced under good manufacturing practice (GMP). FOXA2 and SOX17 double positive definitive endoderm (DE) progenitor cells can give rise to all endoderm-derived cell types in the thymus, thyroid, lung, pancreas, liver, and gastrointestinal tract. FOXA2 is a pioneer transcription factor in DE differentiation that is also expressed and functionally required during pancreas development and islet cell homeostasis. Current differentiation protocols can successfully generate endoderm; however, generation of mature glucose-sensitive and insulin-secreting β-cells is still a challenge. As a result, it is of utmost importance to screen for small molecules that can improve DE and islet cell differentiation for cell-replacement therapy for diabetic patients. Methods: The aim of this study was to identify and validate small molecules that can induce DE differentiation and further enhance pancreatic progenitor differentiation. Therefore, we developed a large scale, high-content screen for testing a chemical library of 23,406 small molecules to identify compounds that induce FoxA2 in mouse embryonic stem cells (mESCs). Results: Based on our high-content screen algorithm, we selected 84 compounds that directed differentiation of mESCs towards the FoxA2 lineage. Strikingly, we identified ROCK inhibition (ROCKi) as a novel mechanism of endoderm induction in mESCs and hESCs. DE induced by the ROCK inhibitor Fasudil efficiently gives rise to PDX1+ pancreatic progenitors from hESCs. Conclusion: Taken together, DE induction by ROCKi can simplify and improve current endoderm and pancreatic differentiation protocols towards a GMP-grade cell product for β-cell replacement.
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- 2017
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6. Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience
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Paul A. De Sousa, Rachel Steeg, Elisabeth Wachter, Kevin Bruce, Jason King, Marieke Hoeve, Shalinee Khadun, George McConnachie, Julie Holder, Andreas Kurtz, Stefanie Seltmann, Johannes Dewender, Sascha Reimann, Glyn Stacey, Orla O'Shea, Charlotte Chapman, Lyn Healy, Heiko Zimmermann, Bryan Bolton, Trisha Rawat, Isobel Atkin, Anna Veiga, Bernd Kuebler, Blanca Miranda Serano, Tomo Saric, Jürgen Hescheler, Oliver Brüstle, Michael Peitz, Cornelia Thiele, Niels Geijsen, Bjørn Holst, Christian Clausen, Majlinda Lako, Lyle Armstrong, Shailesh K. Gupta, Alexander J. Kvist, Ryan Hicks, Anna Jonebring, Gabriella Brolén, Andreas Ebneth, Alfredo Cabrera-Socorro, Patrik Foerch, Martine Geraerts, Tina C. Stummann, Shawn Harmon, Carol George, Ian Streeter, Laura Clarke, Helen Parkinson, Peter W. Harrison, Adam Faulconbridge, Luca Cherubin, Tony Burdett, Cesar Trigueros, Minal J Patel, Christa Lucas, Barry Hardy, Rok Predan, Joh Dokler, Maja Brajnik, Oliver Keminer, Ole Pless, Philip Gribbon, Carsten Claussen, Annette Ringwald, Beate Kreisel, Aidan Courtney, and Timothy E. Allsopp
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Biology (General) ,QH301-705.5 - Abstract
A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.
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- 2017
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7. The COVID-19 Ontology.
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Astghik Sargsyan, Alpha Tom Kodamullil, Shounak Baksi, Johannes Darms, Sumit Madan, Stephan Gebel, Oliver Keminer, Geena Mariya Jose, Helena Balabin, Lauren Nicole Delong, Manfred Kohler, Marc Jacobs 0001, and Martin Hofmann-Apitius
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- 2021
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8. Droplet-based vitrification of adherent human induced pluripotent stem cells on alginate microcarrier influenced by adhesion time and matrix elasticity
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Julia C. Neubauer, Julia Majer, Philip Gribbon, Ina Meiser, Gesa Witt, Ole Pless, André Schulz, Katharina Schmidt, Alisa Katsen-Globa, Heiko Zimmermann, Frank Stracke, John Gardner, Eirini Koutsouraki, Oliver Keminer, Carsten Claussen, and Publica
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Cryopreservation ,Cell type ,Alginates ,Chemistry ,Induced Pluripotent Stem Cells ,Cell ,Microcarrier ,General Medicine ,Adhesion ,Matrix (biology) ,Vitrification ,Elasticity ,General Biochemistry, Genetics and Molecular Biology ,medicine.anatomical_structure ,Freezing ,medicine ,Biophysics ,Humans ,Stem cell ,General Agricultural and Biological Sciences - Abstract
The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialised cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35%) versus stiff (0.65%) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.
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- 2021
9. Mapping Chromone-3-Phenylcarboxamide Pharmacophore: Quid Est Veritas?
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Stefano Alcaro, Francesco Mesiti, Annalisa Maruca, Fernanda Borges, Alexandra Gaspar, Sheraz Gul, Oliver Keminer, Sandra Barreiro, Carlos M.C.G. Fernandes, Renata Silva, Roberta Rocca, Eva Gil Martins, Daniel Chavarria, and Publica
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chemistry.chemical_compound ,chemistry ,Stereochemistry ,In vivo ,Drug Discovery ,Chromone ,Molecular Medicine ,Monoamine oxidase B ,Pharmacophore ,Cytotoxicity ,In vitro ,ADME ,Benzopyran - Abstract
Chromone-3-phenylcarboxamides (Crom-1 and Crom-2) were identified as potent, selective, and reversible inhibitors of human monoamine oxidase B (hMAO-B). Since they exhibit some absorption, distribution, metabolism, and excretion (ADME)-toxicity liabilities, new derivatives were synthesized to map the chemical structural features that compose the pharmacophore, a process vital for lead optimization. Structure-activity relationship data, supported by molecular docking studies, provided a rationale for the contribution of the heterocycle's rigidity, the carbonyl group, and the benzopyran heteroatom for hMAO-B inhibitory activity. From the study, N-(3-chlorophenyl)-4H-thiochromone-3-carboxamide (31) (hMAO-B IC50 = 1.52 ± 0.15 nM) emerged as a reversible tight binding inhibitor with an improved pharmacological profile. In in vitro ADME-toxicity studies, compound 31 showed a safe cytotoxicity profile in Caco-2, SH-SY5Y, HUVEC, HEK-293, and MCF-7 cells, did not present cardiotoxic effects, and did not affect P-gp transport activity. Compound 31 also protected SH-SY5Y cells from iron(III)-induced damage. Collectively, these studies highlighted compound 31 as the first-in-class and a suitable candidate for in vivo preclinical investigation.
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- 2021
10. The COVID-19 Ontology
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Martin Hofmann-Apitius, Johannes Darms, Stephan Gebel, Sumit Madan, Oliver Keminer, Lauren Nicole DeLong, Marc Jacobs, Astghik Sargsyan, Helena Balabin, Geena Mariya Jose, Alpha Tom Kodamullil, Manfred Kohler, Shounak Baksi, and Publica
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Statistics and Probability ,AcademicSubjects/SCI01060 ,Computer science ,MEDLINE ,interoperability ,Context (language use) ,text mining ,Ontology (information science) ,Biochemistry ,World Wide Web ,03 medical and health sciences ,Text mining ,Development (topology) ,semantic search ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,semantic integration ,business.industry ,ontology and terminology ,030302 biochemistry & molecular biology ,semantic interoperability ,Computer Science Applications ,Applications Note ,Computational Mathematics ,Computational Theory and Mathematics ,Ontology ,business ,Scope (computer science) - Abstract
Motivation The COVID-19 pandemic has prompted an impressive, worldwide response by the academic community. In order to support text mining approaches as well as data description, linking and harmonization in the context of COVID-19, we have developed an ontology representing major novel coronavirus (SARS-CoV-2) entities. The ontology has a strong scope on chemical entities suited for drug repurposing, as this is a major target of ongoing COVID-19 therapeutic development. Results The ontology comprises 2270 classes of concepts and 38 987 axioms (2622 logical axioms and 2434 declaration axioms). It depicts the roles of molecular and cellular entities in virus-host interactions and in the virus life cycle, as well as a wide spectrum of medical and epidemiological concepts linked to COVID-19. The performance of the ontology has been tested on Medline and the COVID-19 corpus provided by the Allen Institute. Availabilityand implementation COVID-19 Ontology is released under a Creative Commons 4.0 License and shared via https://github.com/covid-19-ontology/covid-19. The ontology is also deposited in BioPortal at https://bioportal.bioontology.org/ontologies/COVID-19. Supplementary information Supplementary data are available at Bioinformatics online.
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- 2020
11. Mapping Chromone-3-Phenylcarboxamide Pharmacophore
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Francesco, Mesiti, Alexandra, Gaspar, Daniel, Chavarria, Annalisa, Maruca, Roberta, Rocca, Eva, Gil Martins, Sandra, Barreiro, Renata, Silva, Carlos, Fernandes, Sheraz, Gul, Oliver, Keminer, Stefano, Alcaro, and Fernanda, Borges
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Structure-Activity Relationship ,Monoamine Oxidase Inhibitors ,Dose-Response Relationship, Drug ,Molecular Structure ,Chromones ,Humans ,Monoamine Oxidase ,Cell Line - Abstract
Chromone-3-phenylcarboxamides (
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- 2021
12. Generation of two human isogenic iPSC lines from fetal dermal fibroblasts
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Oliver Keminer, Rashmi Tandon, Alexandro Landshammer, Laura Steenpaß, Franz-Josef Müller, Ole Pless, Natalya Baryshnikova, Björn Brändl, and Publica
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0301 basic medicine ,Fetus ,Transgene ,Induced Pluripotent Stem Cells ,Medizin ,Cell Differentiation ,Locus (genetics) ,Cell Biology ,General Medicine ,Fibroblasts ,Biology ,Molecular biology ,Genome ,03 medical and health sciences ,030104 developmental biology ,lcsh:Biology (General) ,Cell culture ,Humans ,Female ,Gene ,Reprogramming ,Stem cell biology ,lcsh:QH301-705.5 ,Developmental Biology - Abstract
Two isogenic hiPSC lines, ZIPi013-B and ZIPi013-E, were generated by reprogramming fetal dermal fibroblasts with episomal vectors. Previously, the same fetal fibroblasts were reprogrammed multiple times in a study comparing other reprogramming methods. As a consequence, the genomes have been sequenced multiple times. Both new cell lines offer the opportunity to study basic stem cell biology and model human disease. They can be applied as reference cell lines for creating isogenic clones bearing disease mutations on a well-characterized genomic background, as both cell lines have demonstrated excellent differentiation capacity in multiple labs. Resource table Unique stem cell lines identifier ZIPi013-B ZIPi013-E Alternative names of stem cell lines ZIP13K2 (ZIPi013-B) ZIP13K5 (ZIPi013-E) Institution Zentrum fur Integrative Psychiatrie gGmbH, Kiel, Germany Contact information of distributor PD Dr. Franz-Josef Muller, franz-josef.mueller@uksh.de Type of cell lines iPSC Origin human Cell Source Fibroblasts Clonality Clonal Method of reprogramming Transgene free, episomal Multiline rationale Isogenic clones Gene modification NO Type of modification N/A Associated disease N/A Gene/locus N/A Method of modification N/A Name of transgene or resistance N/A Inducible/constitutive system N/A Date archived/stock date Stock date ZIPi013-B 8th December 2017, stock date ZIPi013-E 12th December 2017 Cell line repository/bank N/A Ethical approval https://www.sciencellonline.com/technical-support/ethical-statement.html Ethikkommission der medizinischen Fakultat der Christian-Albrechts-Universitat zu Kiel, approval number A145/11
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- 2018
13. A SARS-CoV-2 cytopathicity dataset generated by high-content screening of a large drug repurposing collection
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Gerd Geisslinger, Jindrich Cinatl, Oliver Keminer, Sandra Westhaus, Maria Kuzikov, Bernhard Ellinger, Sandra Ciesek, Jeanette Reinshagen, Carsten Claussen, Andrea Zaliani, Denisa Bojkova, Markus Wolf, Philip Gribbon, and Publica
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Statistics and Probability ,Camostat ,Drug ,Data Descriptor ,Science ,media_common.quotation_subject ,Drug Evaluation, Preclinical ,Cetylpyridinium ,Library and Information Sciences ,medicine.disease_cause ,Antiviral Agents ,Guanidines ,Lopinavir ,Education ,03 medical and health sciences ,chemistry.chemical_compound ,Papaverine ,medicine ,Humans ,030304 developmental biology ,Coronavirus ,media_common ,0303 health sciences ,SARS-CoV-2 ,030306 microbiology ,Mefloquine ,business.industry ,Small molecules ,Drug Repositioning ,virus diseases ,COVID-19 ,Esters ,Virology ,Benzamidines ,Computer Science Applications ,Nafamostat ,Drug repositioning ,Drug screening ,chemistry ,Viral infection ,High-content screening ,Caco-2 Cells ,Statistics, Probability and Uncertainty ,business ,Information Systems ,medicine.drug - Abstract
SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies., Measurement(s) cytotoxicity • killing by virus of host cell Technology Type(s) bright-field microscopy Factor Type(s) concentration • incubation time Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13562570
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- 2021
14. An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds
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Gesa Witt, Isabelle Sébastien, Rashmi Tandon, Ina Meiser, Philip Gribbon, Julia C. Neubauer, Jennifer Leu, Oliver Keminer, Ole Pless, Björn Brändl, Heiko Zimmermann, Franz-Josef Müller, Anne Willing, Jana-Christin Abt, Manuel A. Friese, Ingo Winschel, Carsten Claussen, and Publica
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0301 basic medicine ,Pluripotent Stem Cells ,Embryonic stem cells ,Computer science ,Cell Survival ,Health, Toxicology and Mutagenesis ,High-throughput screening ,Induced Pluripotent Stem Cells ,Developmental toxicity ,Embryonic Development ,High-throughput ,Computational biology ,010501 environmental sciences ,Toxicology ,01 natural sciences ,Small Molecule Libraries ,03 medical and health sciences ,Automation ,Mice ,Adenosine Triphosphate ,In vivo ,Toxicity Tests ,Animals ,Humans ,Myocytes, Cardiac ,Flow cytometry ,Induced pluripotent stem cell ,Embryoid Bodies ,0105 earth and related environmental sciences ,Cell Death ,Reproduction ,Mouse Embryonic Stem Cells ,Cell Biology ,Fibroblasts ,Small molecule ,Embryonic stem cell ,High-Throughput Screening Assays ,030104 developmental biology ,Workflow ,NIH 3T3 Cells ,Original Article ,Biological Assay ,Reproductive toxicity - Abstract
The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract•The embryonic stem cell test to predict teratogenicity was made automation-compatible.•Several key improvements to the assay procedure have been introduced to increase performance.•The workflow was adapted to human iPS cells and isogenic fibroblast donor cells. Electronic supplementary material The online version of this article (10.1007/s10565-020-09538-0) contains supplementary material, which is available to authorized users.
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- 2021
15. Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform
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Flavio Di Pisa, Elisa Uliassi, Lucio H. Freitas-Junior, Maria Laura Bolognesi, Antonio Quotadamo, Maria Paola Costi, Claudia Danielli Pereira Bertolacini, Joachim Clos, Joanna Panecka-Hofman, Chiara Borsari, María Jesús Corral, G. Landi, Maria Kuzikov, Deepa Karunakaran, Pasquale Linciano, Wolfgang Müller, Birte Behrens, Kyriakos C. Prousis, Carolina B. Moraes, Lucia Dello Iacono, Markus Wolf, Oliver Keminer, Cecilia Pozzi, Rebecca C. Wade, Bruno dos Santos Pascoalino, José María Alunda, Stefania Ferrari, Martina Fenske, Alberto Venturelli, Sheraz Gul, Jeanette Reinshagen, Nuno Santarém, Matteo Santucci, Jennifer Leu, Stephen Wrigley, Ina Pöhner, Bethlehem Kebede, Theodora Calogeropoulou, Bernhard Ellinger, Stefano Mangani, Anabela Cordeiro-da-Silva, Gesa Witt, Carolina B. Moraes, Gesa Witt, Maria Kuzikov, Bernhard Ellinger, Theodora Calogeropoulou, Kyriakos C. Prousis, Stefano Mangani, Flavio Di Pisa, Giacomo Landi, Lucia Dello Iacono, Cecilia Pozzi, Lucio H. Freitas-Junior, Bruno dos Santos Pascoalino, Claudia P. Bertolacini, Birte Behrens, Oliver Keminer, Jennifer Leu, Markus Wolf, Jeanette Reinshagen, Anabela Cordeiro-da-Silva, Nuno Santarem, Alberto Venturelli, Stephen Wrigley, Deepa Karunakaran, Bethlehem Kebede, Ina Pöhner, Wolfgang Müller, Joanna Panecka-Hofman, Rebecca C. Wade, Martina Fenske, Joachim Clos, José María Alunda, María Jesús Corral, Elisa Uliassi, Maria Laura Bolognesi, Pasquale Linciano, Antonio Quotadamo, Stefania Ferrari, Matteo Santucci, Chiara Borsari, Maria Paola Costi, Sheraz Gul, and Publica
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Chagas disease ,cell-based assays ,Antiparasitic ,medicine.drug_class ,cell-based assay ,Computational biology ,liquid handling ,Trypanosoma brucei ,01 natural sciences ,Biochemistry ,Article ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,Trypanosomiasis ,anti-infective drugs ,Drug Discovery ,parasitic diseases ,medicine ,Humans ,compound repositories ,enzyme assays or enzyme kinetics ,Biotechnology ,Molecular Medicine ,030304 developmental biology ,0303 health sciences ,Biological Products ,Natural product ,biology ,Drug discovery ,Leishmaniasis ,biology.organism_classification ,medicine.disease ,Trypanocidal Agents ,anti-infective drug ,0104 chemical sciences ,3. Good health ,010404 medicinal & biomolecular chemistry ,chemistry ,compound repositorie ,Neglected tropical diseases ,enzyme assays or enzyme kinetic - Abstract
According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.
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- 2019
16. Persister state-directed transitioning and vulnerability in melanoma
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Marc Remke, Stefanie Egetemaier, Nikolas K. Haass, Smiths S Lueong, Markus Kaiser, Heike Chauvistré, José Neptuno Rodríguez-López, Linda Kubat, Clemens Krepler, Antonio Sechi, Batool Shannan, Qin Liu, Robert J. Ju, Farnusch Kaschani, Alexander Roesch, Sheraz Gul, Jürgen C. Becker, Vito W. Rebecca, Jan Forster, Iris Helfrich, Susanne Horn, Annette Paschen, Daniel Rauh, S. M. Daignault, Kirujan Jeyakumar, Meenhard Herlyn, Xiangfan Yin, Dirk Schadendorf, Daniel Picard, Felix C. E. Vogel, Michael Ehrmann, Renáta Váraljai, Oliver Keminer, Samantha J. Stehbens, and Publica
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Cell ,Medizin ,General Physics and Astronomy ,Context (language use) ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,medicine ,Humans ,Melanoma ,Gene ,Cell Proliferation ,030304 developmental biology ,Phenocopy ,0303 health sciences ,Multidisciplinary ,biology ,Monophenol Monooxygenase ,Cell growth ,General Chemistry ,medicine.disease ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,Melanocytes ,Demethylase ,JARID1B - Abstract
Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. For example, melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a new dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor growth and plasticity and primes melanoma cells towards lineage-specific elimination.
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- 2020
17. A tiered high-throughput screening approach for evaluation of estrogen and androgen receptor modulation by environmentally relevant bisphenol A substitutes
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Andrea Wenzel, Matthias Teigeler, Björn Windshügel, Elke Eilebrecht, Oliver Keminer, Franziska Kaßner, Jürgen Arning, Manfred Kohler, and Publica
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endocrine system ,Bisphenol A ,Environmental Engineering ,010504 meteorology & atmospheric sciences ,medicine.drug_class ,High-throughput screening ,Estrogen receptor ,010501 environmental sciences ,Endocrine Disruptors ,01 natural sciences ,chemistry.chemical_compound ,Phenols ,substitution candidate ,androgen receptor ,medicine ,Environmental Chemistry ,Endocrine system ,Benzhydryl Compounds ,Waste Management and Disposal ,screening cascade ,0105 earth and related environmental sciences ,Retrospective Studies ,urogenital system ,endocrine disrupting potential ,Pollution ,Androgen receptor ,chemistry ,Biochemistry ,Nuclear receptor ,Estrogen ,Docking (molecular) ,Receptors, Androgen ,hormones, hormone substitutes, and hormone antagonists ,estrogen receptor - Abstract
Bisphenol A (BPA) is a high production volume chemical with a broad application spectrum. As an endocrine disrupting chemical, mainly by modulation of nuclear receptors (NRs), BPA has an adverse impact on organisms and is identified as a substance of very high concern under the European REACH regulation. Various BPA substitution candidates have been developed in recent years, however, information concerning the endocrine disrupting potential of these substances is still incomplete or missing. In this study, we intended to investigate the endocrine potential of BPA substitution candidates used in environmentally relevant applications such as thermal paper or epoxy resins. Based on an extensive literature and patent search, 33 environmentally relevant BPA substitution candidates were identified. In order to evaluate the endocrine potential of the BPA replacements, a screening cascade consisting of biochemical and cell-based assays was employed to investigate substance binding to the NRs estrogen receptor α and β, as well as androgen receptor, co-activator recruitment and NR-mediated reporter gene activation. In addition, a computational docking approach for retrospective prediction of receptor binding was carried out. Our results show that some BPA substitution candidates, for which so far no or only very few data were available, possess a substantial endocrine disrupting potential (TDP, BPZ), while several substances (BPS, D-8, DD70, DMP-OH, TBSA, D4, CBDO, ISO, VITC, DPA, and DOPO) did not reveal any NR binding.
- Published
- 2020
18. Mutations in PMPCB Encoding the Catalytic Subunit of the Mitochondrial Presequence Protease Cause Neurodegeneration in Early Childhood
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F.-Nora Vögtle, Carolyn F. Delto, Hiltrud Muhle, Nils Burger, Marisa W. Friederich, Johanna A. Jähn, Ingo Helbig, Audrey Burnett, Maie Walsh, Naomichi Matsumoto, Cansu Kücükköse, Björn Brändl, Franz-Josef Müller, Hung-Chun Yu, Andreas van Baalen, Deepali N. Shinde, Austin Larson, Susan M. White, Alice Basinger, Noriko Miyake, Oliver Keminer, Ulrich Stephani, Manuela Pendziwiat, Dirk Mossmann, Lisa Myketin, Katherine L. Helbig, Mark A. Lovell, Johan L.K. Van Hove, and Publica
- Subjects
Iron-Sulfur Proteins ,Male ,0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Mitochondrial disease ,Induced Pluripotent Stem Cells ,Respiratory chain ,Saccharomyces cerevisiae ,Mitochondrion ,medicine.disease_cause ,Proto-Oncogene Mas ,Article ,Electron Transport ,03 medical and health sciences ,Catalytic Domain ,Genetics ,medicine ,Humans ,Child ,PMPCB ,Genetics (clinical) ,Mutation ,biology ,Neurodegeneration ,Metalloendopeptidases ,Dermis ,Fibroblasts ,medicine.disease ,Magnetic Resonance Imaging ,Mitochondria ,Pedigree ,Cell biology ,030104 developmental biology ,Child, Preschool ,Nerve Degeneration ,Frataxin ,biology.protein ,Female ,Biogenesis - Abstract
Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.
- Published
- 2018
19. Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience
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Oliver Brüstle, Annette Ringwald, Christian Clausen, Charlotte Chapman, Glyn Stacey, Alexander J. Kvist, Marieke A. Hoeve, Shailesh Kumar Gupta, Blanca Miranda Serano, Minal Patel, Gabriella Brolén, Tomo Saric, George McConnachie, Cornelia Thiele, Julie Holder, Trisha Rawat, Tina C. Stummann, Joh Dokler, Maja Brajnik, Bryan Bolton, Lyle Armstrong, Bernd Kuebler, Alfredo Cabrera-Socorro, Isobel Atkin, Adam Faulconbridge, Christa Lucas, Jason A. King, Johannes Dewender, Ian Streeter, Rok Predan, Majlinda Lako, Lyn Healy, Bjørn Holst, Martine Geraerts, Luca Cherubin, Carsten Claussen, Heiko Zimmermann, Timothy E Allsopp, Orla O'Shea, Peter W. Harrison, K. Bruce, Niels Geijsen, Michael Peitz, Tony Burdett, Paul A. De Sousa, Ole Pless, Elisabeth Wachter, Anna Veiga, A. Courtney, Laura Clarke, Helen Parkinson, Cesar Trigueros, Anna Jonebring, Shalinee Khadun, Rachel Steeg, Patrik Foerch, Sascha Reimann, Ryan Hicks, Carol George, Andreas Ebneth, Oliver Keminer, Philip Gribbon, Barry Hardy, Stefanie Seltmann, Andreas Kurtz, Shawn Harmon, Jürgen Hescheler, Beate Kreisel, Hubrecht Institute for Developmental Biology and Stem Cell Research, and Publica
- Subjects
0301 basic medicine ,QH301-705.5 ,Process (engineering) ,media_common.quotation_subject ,Induced Pluripotent Stem Cells ,Biology ,Tissue procurement ,Cell Line ,03 medical and health sciences ,Resource (project management) ,Journal Article ,Humans ,Quality (business) ,Biology (General) ,Induced pluripotent stem cell ,lcsh:QH301-705.5 ,Biological Specimen Banks ,media_common ,Cryopreservation ,International research ,Medicine(all) ,Hot start ,General Medicine ,Cell Biology ,Europe ,Engineering management ,030104 developmental biology ,lcsh:Biology (General) ,Work flow ,Developmental Biology - Abstract
A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community.eTOCThe report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.
- Published
- 2017
20. Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3
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Serene M. L. Lee, Björn Windshügel, Tobias S. Schiergens, Frank Essmann, Oliver Burk, Matthias Schwab, and Oliver Keminer
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0301 basic medicine ,Agonist ,Gene isoform ,Models, Molecular ,medicine.drug_class ,Health, Toxicology and Mutagenesis ,High-throughput screening ,Receptors, Cytoplasmic and Nuclear ,010501 environmental sciences ,Molecular Dynamics Simulation ,Toxicology ,01 natural sciences ,Proof of Concept Study ,03 medical and health sciences ,Constitutive androstane receptor ,medicine ,Humans ,Protein Isoforms ,Constitutive Androstane Receptor ,0105 earth and related environmental sciences ,chemistry.chemical_classification ,Cell Nucleus ,In vitro toxicology ,General Medicine ,Amino acid ,Clopidogrel ,High-Throughput Screening Assays ,Molecular Docking Simulation ,Cytochrome P-450 CYP2B6 ,Protein Transport ,030104 developmental biology ,HEK293 Cells ,Retinoid X Receptors ,Nuclear receptor ,chemistry ,Biochemistry ,Gene Expression Regulation ,Hepatocytes ,Immortalised cell line - Abstract
Prediction of drug interactions, based on the induction of drug disposition, calls for the identification of chemicals, which activate xenosensing nuclear receptors. Constitutive androstane receptor (CAR) is one of the major human xenosensors; however, the constitutive activity of its reference variant CAR1 in immortalized cell lines complicates the identification of agonists. The exclusively ligand-dependent isoform CAR3 represents an obvious alternative for screening of CAR agonists. As CAR3 is even more abundant in human liver than CAR1, identification of its agonists is also of pharmacological value in its own right. We here established a cellular high-throughput screening assay for CAR3 to identify ligands of this isoform and to analyse its suitability for identifying CAR ligands in general. Proof-of-concept screening of 2054 drug-like compounds at 10 µM resulted in the identification of novel CAR3 agonists. The CAR3 assay proved to detect the previously described CAR1 ligands in the screened libraries. However, we failed to detect CAR3-selective compounds, as the four novel agonists, which were selected for further investigations, all proved to activate CAR1 in different cellular and in vitro assays. In primary human hepatocytes, the compounds preferentially induced the expression of the prototypical CAR target gene CYP2B6. Failure to identify CAR3-selective compounds was investigated by molecular modelling, which showed that the isoform-specific insertion of five amino acids did not impact on the ligand binding pocket but only on heterodimerization with retinoid X receptor. In conclusion, we demonstrate here the usability of CAR3 for screening compound libraries for the presence of CAR agonists.
- Published
- 2019
21. Effects of exercise on Irisin, BDNF and IL-6 serum levels in patients with progressive multiple sclerosis
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Stefan Patra, Ole Pless, Gesche Ketels, Sven Briken, Oliver Keminer, Stefan M. Gold, Karl-Heinz Schulz, Christoph Heesen, Rainer Hellweg, and Sina C. Rosenkranz
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Adult ,Male ,0301 basic medicine ,Oncology ,medicine.medical_specialty ,Multiple Sclerosis ,Immunology ,Pilot Projects ,Cohort Studies ,03 medical and health sciences ,0302 clinical medicine ,Physical medicine and rehabilitation ,Degenerative disease ,Endurance training ,Neurotrophic factors ,Internal medicine ,Myokine ,medicine ,Humans ,Immunology and Allergy ,Aerobic exercise ,Dementia ,Interleukin 6 ,Exercise ,biology ,Interleukin-6 ,business.industry ,Brain-Derived Neurotrophic Factor ,Multiple sclerosis ,Middle Aged ,medicine.disease ,Fibronectins ,030104 developmental biology ,Neurology ,Exercise Test ,biology.protein ,Female ,Neurology (clinical) ,business ,Biomarkers ,030217 neurology & neurosurgery - Abstract
Clinical studies have suggested beneficial effects of exercise on cognitive function in ageing adults and neurodegenerative diseases such as dementia. Recent work indicates the same for progressive multiple sclerosis (MS), an inflammatory and degenerative disease of the central nervous system (CNS). The biological pathways associated with these effects are however not well understood.In this randomized controlled study, we explored serum levels of the myokine Irisin, the neurotrophin brain-derived neurotrophic factor (BDNF) and Interleukin-6 (IL-6) during acute endurance exercise and over the course of a 9-weeks endurance exercise training period in n=42 patients with progressive MS.We detected a significant increase of BDNF levels in progressive MS patients after 30min of bicycling (p0.001). However, there were no significant changes for baseline levels after 22 sessions of training. No significant effects of acute or prolonged exercise could be found for Irisin or Interleukin-6.Our results indicate that BDNF is strongly induced during acute exercise even in patients with progressive MS and advanced physical disability. Long-term effects of exercise programs on biological parameters (Irisin, BDNF, IL-6) were much less pronounced. Given the hypothesis-driven selection of a limited set of biological markers in this pilot study, future studies should use unbiased approaches in larger samples to obtain a comprehensive picture of the networks involved in exercise effects on neurological diseases.
- Published
- 2016
22. Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs
- Author
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Paloma Tejera Nevado, Anabela Cordeiro-da-Silva, María Jesús Corral, Gesa Witt, Catarina Baptista, Rebecca C. Wade, G. Landi, José Mª Alunda, Juan J. Torrado, Bernhard Ellinger, Maria Kuzikov, Maria Paola Costi, Julia Eick, Stefano Mangani, Jeanette Reinshagen, Eugenia Bifeld, Nuno Santarém, Sheraz Gul, Manfred Kohler, Stefania Ferrari, Joachim Clos, María Dolores Jiménez-Antón, Lucia Dello Iacono, Birte Behrens, Philip Gribbon, Cecilia Pozzi, Oliver Keminer, Markus Wolf, Luca Costantino, Stefan Henrich, Matteo Trande, Rosaria Luciani, Ina Poehner, Flavio Di Pisa, Annalisa Tait, Federica Pellati, Chiara Borsari, and Publica
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Models, Molecular ,0301 basic medicine ,Flavonols ,Stereochemistry ,Phenotypic screening ,Trypanosoma brucei brucei ,Trypanosoma brucei ,01 natural sciences ,Flavonol Derivatives, Drug Development, Antitrypanosomatidic Drugs, enzyme inhibition, x-ray crystallography, drug delivery ,Cell Line ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,Parasitic Sensitivity Tests ,Drug Discovery ,Animals ,Humans ,Structure–activity relationship ,EC50 ,chemistry.chemical_classification ,Biological Products ,Mice, Inbred BALB C ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Macrophages ,Drug Discovery3003 Pharmaceutical Science ,biology.organism_classification ,Trypanocidal Agents ,In vitro ,0104 chemical sciences ,3. Good health ,010404 medicinal & biomolecular chemistry ,030104 developmental biology ,chemistry ,Biochemistry ,Molecular Medicine ,Docking (molecular) ,Toxicity - Abstract
Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.
- Published
- 2016
23. Abstract 2999: Identification of Src's role in governing chemotherapy resistance in oesophageal adenocarcinoma through functional genomic and high-throughput drug screening approaches
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Niamh H McCabe, Richard D. Kennedy, Jaine K. Blayney, Enya Scanlon, Leanne Stevenson, Bjoern Windshuegel, Oliver Keminer, and Richard C. Turkington
- Subjects
Cisplatin ,Cancer Research ,Gene knockdown ,Candidate gene ,business.industry ,Cancer ,medicine.disease ,Dasatinib ,Oncology ,Cancer research ,medicine ,Gene silencing ,Viability assay ,business ,medicine.drug ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Introduction: Five-year survival rates for oesophageal adenocarcinoma (OAC) remain poor at just 15%. The development of drug-resistance limits the effectiveness of current chemotherapies, so the discovery of underlying resistance mechanisms and novel agents to target these pathways is crucial. Materials and Methods: 273 pre-treatment OAC biopsies were transcriptionally profiled using the Almac Diagnostics Xcel array™. The Broad Institute's Gene Set Enrichment Analysis (GSEA) software was used to identify pathways enriched in non-responders to chemotherapy compared to responders. A focused siRNA screen of 80 target genes identified from the GSEA results was then devised and completed, highlighting the role of Src in chemotherapy non-responders. An Enzo compound screen of FDA-approved drugs was carried out in OE33 and FLO-1 cell lines in combination with cisplatin, and cell viability was measured by CellTiter Glo. Src inhibition using dasatinib and saracatinib was assessed with MTT viability assays, western blotting and flow cytometry to investigate the mechanisms of action. CI values were generated to assess synergy. Results: GSEA identified pathways associated with resistance to chemotherapy in OAC. Candidate genes were selected according to predefined criteria and 80 genes were taken forward in a siRNA screen to study the effects on cell viability of gene silencing alone or in combination with cisplatin or 5-FU. Twelve genes were found to have an additive effect with chemotherapy to improve chemo-efficacy across an OAC cell line panel, including Src. A high-throughput drug screen highlighted a number of novel compounds that displayed additive effects when combined with cisplatin in OAC cell lines including dasatinib, a Src inhibitor. Src was then validated in an in-vitro OAC setting, investigating the effects of Src inhibition using siRNA, dasatinib and saracatinib. In-vitro combination experiments showed that combining Src inhibitors with traditional chemotherapies provides synergistic effects. Conclusions: Using a functional genomic approach Src was identified as a novel target associated with reduced cell viability following siRNA-mediated knockdown. By the use of small molecule inhibitors, the results of the siRNA screen were reinforced, verifying the clinical potential and synergistic effects of targeting Src alongside traditional chemotherapy treatments. Citation Format: Niamh Helen McCabe, Leanne Stevenson, Enya Scanlon, Oliver Keminer, Richard D. Kennedy, Bjoern Windshuegel, Jaine K. Blayney, Richard C. Turkington. Identification of Src's role in governing chemotherapy resistance in oesophageal adenocarcinoma through functional genomic and high-throughput drug screening approaches [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2999.
- Published
- 2020
24. Identification of approved drugs as potent inhibitors of pregnane X receptor activation with differential receptor interaction profiles
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Carsten Wrenger, Oliver Keminer, Maria Kuzikov, Oliver Burk, Matthias Schwab, Thales Kronenberger, Wolfgang E. Thasler, Judith Jeske, Björn Windshügel, and Publica
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0301 basic medicine ,Drug ,Indazoles ,Health, Toxicology and Mutagenesis ,media_common.quotation_subject ,In silico ,Pharmacology ,Toxicology ,digestive system ,Tacrolimus ,Cell Line ,Pazopanib ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Drug Approval ,media_common ,Pregnane X receptor ,Sulfonamides ,Chemistry ,In vitro toxicology ,Pregnane X Receptor ,General Medicine ,Hep G2 Cells ,digestive system diseases ,In vitro ,3. Good health ,Molecular Docking Simulation ,030104 developmental biology ,Pyrimidines ,Nuclear receptor ,CÉLULAS KUPFFER ,030220 oncology & carcinogenesis ,Hepatocytes ,Efflux ,medicine.drug ,Protein Binding - Abstract
Activation of pregnane X receptor (PXR) results in the induction of first-pass metabolism and drug efflux. Hereby, PXR may cause adverse drug reactions or therapeutic failure of drugs. PXR inhibition is thus an attractive option to minimise adverse effects or to improve therapeutic efficiencies; however, only a limited number of antagonists have been identified so far. We performed a cell-based high-throughput screen to identify PXR antagonists, using a library of approved and investigational drugs. Two approved drugs, pimecrolimus and pazopanib, emerged as novel potent antagonists of PXR activation, with IC50 values of 1.2 and 4.1 µM, respectively. We further characterised these with respect to receptor specificity, assembly of the PXR ligand-binding domain (LBD) and interactions with co-factors. In vitro and in silico assays were carried out to identify the site(s) of interaction with the PXR LBD. Primary human hepatocytes were used to investigate antagonism of the induction of endogenous PXR target genes. Pimecrolimus and pazopanib did not affect the transcriptional activity of other nuclear receptors. Both induced the release of co-repressor from PXR and likewise interfered with agonist-induced recruitment of co-activator. Cumulative evidence from cellular and in vitro assays, as well as molecular docking, suggested additional or exclusive binding outside the PXR ligand-binding pocket for both. The compounds differentially antagonised the induction of PXR-regulated genes by rifampicin in primary human hepatocytes. In conclusion, we here have identified two approved drugs as novel potent PXR inhibitors with differential receptor interaction profiles and gene selectivity in primary human hepatocytes.
- Published
- 2018
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25. Arc/Arg3.1 governs inflammatory dendritic cell migration from the skin and thereby controls T cell activation
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Pablo Vargas, Nina Kursawe, Joseph Tintelnot, Dietmar Kuhl, Friederike Ufer, Ole Pless, Ora Ohana, Gabriela Salinas-Riester, Oliver Keminer, Hana Winkler, Benjamin Schattling, Anne Willing, Manuel A. Friese, Simone Bauer, Jan Broder Engler, and Publica
- Subjects
0301 basic medicine ,Arc (protein) ,Follicular dendritic cells ,T cell ,Immunology ,Experimental autoimmune encephalomyelitis ,Inflammation ,General Medicine ,Biology ,medicine.disease ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Immune system ,medicine.anatomical_structure ,Cancer research ,medicine ,medicine.symptom ,Dendritic cell migration ,Cytoskeleton ,030217 neurology & neurosurgery - Abstract
Skin-migratory dendritic cells (migDCs) are pivotal antigen-presenting cells that continuously transport antigens to draining lymph nodes and regulate immune responses. However, identification of migDCs is complicated by the lack of distinguishing markers, and it remains unclear which molecules determine their migratory capacity during inflammation. We show that, in the skin, the neuronal plasticity molecule activity-regulated cytoskeleton-associated protein/activity-regulated gene 3.1 (Arc/Arg3.1) was strictly confined to migDCs. Mechanistically, Arc/Arg3.1 was required for accelerated DC migration during inflammation because it regulated actin dynamics through nonmuscle myosin II. Accordingly, Arc/Arg3.1-dependent DC migration was critical for mounting T cell responses in experimental autoimmune encephalomyelitis and allergic contact dermatitis. Thus, Arc/Arg3.1 was restricted to migDCs in the skin and drove fast DC migration by exclusively coordinating cytoskeletal changes in response to inflammatory challenges. These findings commend Arc/Arg3.1 as a universal switch in migDCs that may be exploited to selectively modify immune responses.
- Published
- 2016
26. IL-17 production by CSF lymphocytes as a biomarker for cerebral vasculitis
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Christian Gerloff, Götz Thomalla, Markus Glatzel, Vivien Thom, Romy Hackbusch, Manuela Kolster, Mathias Gelderblom, Christian Bernreuther, Sabrina Schmid, Simon Schuster, Tim Magnus, Ole Pleß, Oliver Keminer, Eva Tolosa, Karl Wegscheider, and Publica
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Bioinformatics ,Gastroenterology ,Article ,Flow cytometry ,Proinflammatory cytokine ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Internal medicine ,Medicine ,Stroke ,medicine.diagnostic_test ,business.industry ,medicine.disease ,3. Good health ,030104 developmental biology ,Neurology ,Biomarker (medicine) ,Neurology (clinical) ,Interleukin 17 ,business ,Vasculitis ,030217 neurology & neurosurgery ,Cerebral vasculitis - Abstract
Objective: To explore the possibility of using interleukin-17 (IL-17) production by CD4+ T cells in the CSF as a potential biomarker for cerebral vasculitis in stroke patients. Methods: In this consecutive case study, we performed prospective analysis of CSF and blood in patients admitted to a university medical center with symptoms of stroke and suspected cerebral vasculitis. Flow cytometry was performed for intracellular detection of inflammatory cytokines in peripheral blood lymphocytes and expanded T cells from CSF. Results: CSF CD4+ lymphocytes from patients with cerebral vasculitis showed significantly higher levels of the proinflammatory cytokine IL-17 compared to patients with stroke not due to vasculitis or with other, noninflammatory neurologic diseases. There was no difference in the production of interferon-γ in the CSF and no overall differences in the relative frequencies of peripheral immune cells. Conclusions: Intracellular IL-17 in CSF cells is potentially useful in discriminating cerebral vasculitis as a rare cause in patients presenting with ischemic stroke. Classification of evidence: This study provides Class II evidence that an increased proportion of IL-17-producing CD4+ cells in CSF of patients presenting with stroke symptoms is indicative of cerebral vasculitis (sensitivity 73%, 95% confidence interval [CI] 39–94%; specificity 100%, 95% CI 74%–100%).
- Published
- 2016
27. Utilizing Regulatory Networks for Pluripotency Assessment in Stem Cells
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Lena Böhnke, Lea A. I. Vaas, Franz-Josef Müller, Rainer Fischer, Bernhard Schuldt, Ole Pless, Oliver Keminer, Björn Brändl, and Publica
- Subjects
0301 basic medicine ,Genetics ,Cellular differentiation ,Rex1 ,Cell Biology ,Embryoid body ,Biology ,Embryonic stem cell ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Stem cell ,Induced pluripotent stem cell ,Molecular Biology ,Cell potency ,Reprogramming ,Developmental Biology - Abstract
Pluripotency is a term in cell biology describing a unique state present in distinct stem cell lines, which were either established from the inner cell mass of the mammalian embryo or derived from somatic cells that have been reprogrammed to induced pluripotent stem cells. Pluripotent stem cells are continuously self-renewing, and their differentiation capacity enables them to develop into all derivatives of the three germ layers of a gastrulating embryo (endoderm, ectoderm, mesoderm). Both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) are virtually indistinguishable, at least based on their global RNA expression patterns. Yet, after these in vitro cell cultures have been generated, the cell lines’ pluripotent properties may change considerably on the genetic and/or epigenetic level as a consequence of long-term propagation. Among other unphysiological changes, cell lines might acquire aneuploidies, loose physiological imprinting marks, or develop differentiation biases favoring one cell lineage over the other. As a result, stem cell researchers have to continuously monitor each stem cell line’s integrity, transcriptional profile, and functional properties. Regulatory transcription factors, protein-protein interactions, and signaling networks govern the pluripotent state. As a consequence, emerging small- and large-scale perturbations to these gene regulatory networks mediate the outlined unfavorable changes to the pluripotent phenotype. Here, we describe a reliable bioinformatic framework called PluriTest for confirmation and assessment of pluripotency as an animal-free, fast, and inexpensive way based on genome-wide transcriptional RNA profiles from microarrays. Additionally, we discuss future developments using RNA expression profiling for pluripotency assessment.
- Published
- 2016
28. Nuclear Receptor Modulators — Current Approaches and Future Perspectives
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BjörnWindshügel, Carsten Wrenger, Thales Kronenberger, and Oliver Keminer
- Subjects
Nuclear receptor ,Chemistry ,Current (fluid) ,Neuroscience - Published
- 2015
29. Treatment non-response to boswellic acids is associated with MIR-302B expression in CD4+ T cells of relapsing–remitting multiple sclerosis patients in the Saba proof-of-concept trial
- Author
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Benjamin Otto, Christoph Heesen, Klarissa Hanja Stürner, Oliver Keminer, and Ole Pless
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Neurology ,Expression (architecture) ,Relapsing remitting ,business.industry ,Multiple sclerosis ,Immunology ,Immunology and Allergy ,Medicine ,Neurology (clinical) ,business ,medicine.disease - Published
- 2014
30. Novel MK2 inhibitors by fragment screening
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Dirk Winkler, Jan Kahmann, Sabine Schaertl, Joachim Kraemer, Oliver Keminer, Osamu Ichihara, Dirk Ullmann, Ina Sternberger, Christoph Scheich, Thomas Hesterkamp, Mark Whittaker, and Joern Jungmann
- Subjects
Stereochemistry ,Chemistry ,Tumor Necrosis Factor-alpha ,Organic Chemistry ,Intracellular Signaling Peptides and Proteins ,General Medicine ,Nuclear magnetic resonance spectroscopy ,Protein Serine-Threonine Kinases ,Peptide Fragments ,Computer Science Applications ,Fragment (logic) ,Biochemistry ,Drug Discovery ,Humans ,Biological Assay ,MAPKAP Kinase 2 ,Signal transduction ,Enzyme Inhibitors ,Signal Transduction - Abstract
Inhibitors of MAPKAP kinase 2 (MK2) are expected to attenuate the p38alpha signal transduction pathway in macrophages in a similar way to p38alpha inhibitors and to have a lower propensity for toxic side effects that have slowed the clinical development of the latter. Therefore, novel MK2 inhibitors may find therapeutic application in acute and chronic, TNFalpha-mediated inflammatory conditions like rheumatoid arthritis and others. Herein we have applied fragment screening, using physiologically relevant bioassays and fragment binding mode mapping by protein-observed NMR spectroscopy to the discovery of novel efficient chemical starting points for MK2.
- Published
- 2008
31. Optical recording of signal-mediated protein transport through single nuclear pore complexes
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Katja Zerf, Oliver Keminer, Jan Peter Siebrasse, and Reiner Peters
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Time Factors ,Nuclear Envelope ,Xenopus ,Protein Sorting Signals ,Models, Biological ,Permeability ,Animals ,Nuclear pore ,Nuclear export signal ,Ovum ,Cell Nucleus ,Multidisciplinary ,biology ,Nuclear Proteins ,Biological Transport ,Intracellular Membranes ,Membrane transport ,Biological Sciences ,biology.organism_classification ,Recombinant Proteins ,Transport protein ,Cell biology ,Cytosol ,Kinetics ,Microscopy, Fluorescence ,Nucleocytoplasmic Transport ,Nuclear localization sequence - Abstract
Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of Xenopus oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate in vivo conditions as closely as possible, a Xenopus egg extract was applied to the cytosolic side and a Xenopus oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, “patched” NPCs depended on signal sequences, extracts, and metabolic energy, as in vivo . All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were ≈2 dimers/s for the import protein and ≈4 dimers/s for the export protein (≈15 μM substrate concentration, 22–24°C), in good agreement with in vivo rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms.
- Published
- 1999
32. Permeability of single nuclear pores
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Reiner Peters and Oliver Keminer
- Subjects
Cell Membrane Permeability ,Photolysis ,Passive transport ,Nuclear Envelope ,Xenopus ,Analytical chemistry ,Biophysics ,Biological Transport ,Dextrans ,Membranes, Artificial ,Permeation ,Transport Pathway ,Fluorescence ,Diffusion ,Homologous series ,chemistry.chemical_compound ,Kinetics ,Membrane ,chemistry ,Permeability (electromagnetism) ,Restricted Diffusion ,Oocytes ,Animals ,Nuclear pore ,Research Article - Abstract
In this first application of optical single transporter recording (OSTR), a recently established technique for optically monitoring the activity of single transporters in membrane patches (Tschodrich-Rotter and Peters. 1998. J. Microsc. 192:114-125), the passive permeability of the nuclear pore complex (NPC) was measured for a homologous series of hydrophilic probe molecules. Nuclei were isolated from Xenopus oocytes and firmly attached to filters containing small cylindrical pores. Transport through membrane patches spanning filter pores was measured by scanning microphotolysis. Thus the permeability coefficients of single NPCs were determined for fluorescently labeled dextrans of approximately 4, 10, and 20 kDa. Dextrans of >/=40 kDa could not permeate the NPC. The data were consistent with a model in which the NPC contains a single diffusion channel. By application of established theories for the restricted diffusion through small pores, the diffusion channel was approximated as a cylinder with a radius of 4.4-6.1 nm (mean 5. 35 nm). Because the transport rate constant of the single NPC was known, the equivalent length of the channel could be also determined and was found to be 40-50 nm (mean 44.5 nm). The symmetry of the NPC implies that a singular component such as the diffusion channel is located at the center of the NPC. Therefore a common transport pathway apparently mediates both passive and signal-dependent transport. To test this hypothesis, measurements of signal-dependent transport and of the mutual effects signal-dependent and passive transport may exert on each other are in progress.
- Published
- 1999
33. Detection of respiratory syncytial virus RNA in blood of neonates by polymerase chain reaction
- Author
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Angela Rohwedder, Johannes Forster, Elisabeth Schneider, Oliver Keminer, Hermann Werchau, and Katja Schneider
- Subjects
Paramyxoviridae ,Molecular Sequence Data ,Viremia ,Respiratory Syncytial Virus Infections ,Biology ,Polymerase Chain Reaction ,Virology ,medicine ,Humans ,Amino Acid Sequence ,Respiratory system ,Sequence Homology, Amino Acid ,Respiratory disease ,Infant, Newborn ,Pneumovirus ,respiratory system ,medicine.disease ,biology.organism_classification ,Infectious Disease Transmission, Vertical ,Respiratory Syncytial Viruses ,Pneumonia ,Infectious Diseases ,Upper respiratory tract infection ,Bronchiolitis ,Immunology ,RNA, Viral ,Sequence Alignment - Abstract
During the winter season of 1994/1995, nasopharyngeal aspirates and blood samples of neonates who were admitted to the Neonatal Intensive Care Unit (NICU) (group 1) and infants with respiratory tract disease (group 2) were examined prospectively for the presence of respiratory syncytial virus (RSV). Examination of nasal washes were done by antigen detection and blood samples were tested by nested reverse transcription and polymerase chain reaction (RT-PCR). The results of the 41 neonates studied were as follows: 14/41 were positive for RSV antigen in nasal washes and for RSV-RNA in blood, 5/41 were only RSV antigen positive, 13/41 neonates had negative nasal washes; 6 had positive RT-PCR results in blood. In 9/41 cases only blood samples were available. Five of these were positive by RT-PCR testing. Group 2 included 20 infants hospitalized with respiratory tract disease, e.g., pneumonia, bronchiolitis, or Upper Respiratory Tract Infection (URTI). Eleven out of twenty were positive for RSV antigen in nasal washes and 6/20 were also positive for RSV-RNA in blood. The conclusion is that viremia may be a frequent occurrence in neonates and young children.
- Published
- 1998
34. Detection of HPV DNA in trichilemmomas by polymerase chain reaction
- Author
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Jörg Schaller, Carlo Hendricks, Oliver Keminer, and Angela Rohwedder
- Subjects
Skin Neoplasms ,Sequence analysis ,Molecular Sequence Data ,Biology ,Polymerase Chain Reaction ,Virus ,law.invention ,chemistry.chemical_compound ,law ,Virology ,medicine ,Humans ,Amino Acid Sequence ,Papillomaviridae ,Polymerase chain reaction ,Neoplasms, Basal Cell ,Skin ,Cloning ,Trichilemmoma ,Papillomavirus Infections ,virus diseases ,Epidermodysplasia verruciformis ,Sequence Analysis, DNA ,Amplicon ,medicine.disease ,female genital diseases and pregnancy complications ,Tumor Virus Infections ,Infectious Diseases ,chemistry ,DNA, Viral ,Hair Follicle ,DNA - Abstract
Paraffin sections of 11 formalin-fixed trichilemmomas were investigated for the presence of human papillomavirus (HPV) DNA by the polymerase chain reaction (PCR) with the degenerated consensus primer pairs. PCRs were conducted with different annealing temperatures. When the annealing temperature was reduced from 55°C to 50°C, amplification products of the expected size were obtained for all 11 cases investigated. Determination of the HPV type was performed by cloning and sequencing of the amplification products. The sequence analysis of the eleven cloned amplicons gave the following data: based on sequence comparison with published amino acid sequences, the best homology was found to epidermodysplasia verruciformis (EV)-associated HPVs (supergroup B). In four specimens an HPV type 23 related type was found; five specimens contained HPV sequences which did not match with one of the known HPV types, but had the closest homology to HPV types 15, 17, and 37. Three of the HPV variants which had not been characterised, displayed identical sequences. Two additional HPV amplification fragments displayed 100% homology to HPV-6b. These results demonstrate, for the first time, the presence of HPV DNA in trichilemmomas. The sequence data suggest that HPV variants or types in trichilemmoma are members of the EV-associated HPV supergroup B. J Med Virol 51:119–125, 1997. © 1997 Wiley-Liss, Inc.
- Published
- 1997
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