Publications by authors named "Daniel Schulz"

26 Publications

  • Page 1 of 1

Cutaneous and systemic hyperinflammation drives maculopapular drug exanthema in severely ill COVID-19 patients.

Allergy 2021 Jun 22. Epub 2021 Jun 22.

Department of Dermatology, University Hospital Zurich, Zurich, Switzerland.

Background: Coronavirus disease-2019 (COVID-19) has been associated with cutaneous findings, some being the result of drug hypersensitivity reactions such as maculopapular drug rashes (MDR). The aim of this study was to investigate whether COVID-19 may impact the development of the MDR.

Methods: Blood and skin samples from COVID-19 patients (based on a positive nasopharyngeal PCR) suffering from MDR (COVID-MDR), healthy controls, non-COVID-19-related patients with drug rash with eosinophilia and systemic symptoms (DRESS), and MDR were analyzed. We utilized imaging mass cytometry (IMC) to characterize the cellular infiltrate in skin biopsies. Furthermore, RNA sequencing transcriptome of skin biopsy samples and high-throughput multiplexed proteomic profiling of serum were performed.

Results: IMC revealed by clustering analyses a more prominent, phenotypically shifted cytotoxic CD8 T cell population and highly activated monocyte/macrophage (Mo/Mac) clusters in COVID-MDR. The RNA sequencing transcriptome demonstrated a more robust cytotoxic response in COVID-MDR skin. However, severe acute respiratory syndrome coronavirus 2 was not detected in skin biopsies at the time point of MDR diagnosis. Serum proteomic profiling of COVID-MDR patients revealed upregulation of various inflammatory mediators (IL-4, IL-5, IL-6, TNF, and IFN-γ), eosinophil and Mo/Mac -attracting chemokines (MCP-2, MCP-3, MCP-4 and CCL11). Proteomics analyses demonstrated a massive systemic cytokine storm in COVID-MDR compared with the relatively milder cytokine storm observed in DRESS, while MDR did not exhibit such features.

Conclusion: A systemic cytokine storm may promote activation of Mo/Mac and cytotoxic CD8 T cells in severe COVID-19 patients, which in turn may impact the development of MDR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/all.14983DOI Listing
June 2021

Outcome of Repeat Pulmonary Metastasectomy.

Adv Exp Med Biol 2021 Apr 23. Epub 2021 Apr 23.

Department of Thoracic Surgery, HELIOS University Hospital Wuppertal, Wuppertal, Germany.

Pulmonary metastasectomy is a well-established contribution to the cure of oligometastatic cancers, but its exact effectiveness is poorly understood. Here we report the outcomes of repeat pulmonary metastasectomy from a multicenter trial. This retrospective study included patients who underwent re-do metastasectomies between January 2010 and December 2014. The exclusion criterion was metastasectomy without curative intent. We reviewed medical files of 621 consecutive patients who underwent initial pulmonary metastasectomy. Of those, 64 patients underwent repeat metastasectomies, and these patients were included in the analysis. All the 64 patients underwent a second metastasectomy, later 35 of them underwent a third metastasectomy, 12 underwent a fourth metastasectomy, and 6 underwent a fifth metastasectomy. The total number of re-do metastasectomies was 181. The median overall survival among the patients undergoing re-do metastasectomy was 66.0 ± 3.8 months. Three and 5-year survival rates were 82.3% and 63.3%, respectively. The 5-year survival rates were 63.3% after the first, 50.9% after the second, 74.4% after the third, 83.3% after the fourth, and 60.0% after the fifth metastasectomy. We conclude that at the current stage of knowledge, there is an indication for repeat re-do metastasectomy with curative intent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/5584_2021_635DOI Listing
April 2021

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow.

Nat Commun 2020 05 22;11(1):2570. Epub 2020 May 22.

Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, 10117, Berlin, Germany.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1 stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16464-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244721PMC
May 2020

Hair eruption initiates and commensal skin microbiota aggravate adverse events of anti-EGFR therapy.

Sci Transl Med 2019 12;11(522)

Institute of Cancer Research, Department of Medicine I, Medical University of Vienna and Comprehensive Cancer Center, Vienna 1090, Austria.

Epidermal growth factor receptor (EGFR)-targeted anticancer therapy induces stigmatizing skin toxicities affecting patients' quality of life and therapy adherence. The lack of mechanistic details underlying these adverse events hampers their management. We found that EGFR/ERK signaling is required in LRIG1-positive stem cells during de novo hair eruption to secure barrier integrity and prevent the invasion of commensal microbiota and inflammatory skin disease. EGFR-deficient epidermis is permissive for microbiota outgrowth and displays an atopic-like T2-dominated signature. The opening of the follicular ostia during hair eruption allows invasion of commensal microbiota into the hair follicle, initiating an additional T1 and T17 response culminating in chronic folliculitis. Restoration of epidermal ERK signaling via prophylactic FGF7 treatment or transgenic SOS expression rescues the barrier defect in the absence of EGFR, highlighting a therapeutic anchor point. These data reveal that commensal skin microbiota provoke atopic-like inflammatory skin diseases by invading into the follicular opening of erupting hair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aax2693DOI Listing
December 2019

Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.

Eur J Immunol 2019 09 7;49(9):1372-1379. Epub 2019 Jun 7.

Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201848053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771914PMC
September 2019

In-Depth Characterization of Monocyte-Derived Macrophages using a Mass Cytometry-Based Phagocytosis Assay.

Sci Rep 2019 02 13;9(1):1925. Epub 2019 Feb 13.

Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, 8057, Zürich, Switzerland.

Phagocytosis is a process in which target cells or particles are engulfed and taken up by other cells, typically professional phagocytes; this process is crucial in many physiological processes and disease states. The detection of targets for phagocytosis is directed by a complex repertoire of cell surface receptors. Pattern recognition receptors directly detect targets for binding and uptake, while opsonic and complement receptors detect objects coated by soluble factors. However, the importance of single and combinatorial surface marker expression across different phenotypes of professional phagocytes is not known. Here we developed a novel mass cytometry-based phagocytosis assay that enables the simultaneous detection of phagocytic events in combination with up to 40 other protein markers. We applied this assay to distinct monocyte derived macrophage (MDM) populations and found that prototypic M2-like MDMs phagocytose more E. coli than M1-like MDMs. Surface markers such as CD14, CD206, and CD163 rendered macrophages phagocytosis competent, but only CD209 directly correlated with the amount of particle uptake. Similarly, M2-like MDMs also phagocytosed more cancer cells than M1-like MDMs but, unlike M1-like MDMs, were insensitive to anti-CD47 opsonization. Our approach facilitates the simultaneous study of single-cell phenotypes, phagocytic activity, signaling and transcriptional events in complex cell mixtures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-38127-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374473PMC
February 2019

Bringing together what belongs together: Optimizing murine infection models by using mouse-adapted Staphylococcus aureus strains.

Int J Med Microbiol 2019 Jan 22;309(1):26-38. Epub 2018 Oct 22.

Department of Immunology, University Medicine Greifswald, Greifswald, Germany. Electronic address:

Staphylococcus (S.) aureus is a leading cause of bacterial infection world-wide, and currently no vaccine is available for humans. Vaccine development relies heavily on clinically relevant infection models. However, the suitability of mice for S. aureus infection models has often been questioned, because experimental infection of mice with human-adapted S. aureus requires very high infection doses. Moreover, mice were not considered to be natural hosts of S. aureus. The latter has been disproven by our recent findings, showing that both laboratory mice, as well as wild small mammals including mice, voles, and shrews, are naturally colonized with S. aureus. Here, we investigated whether mouse-and vole-derived S. aureus strains show an enhanced virulence in mice as compared to the human-adapted strain Newman. Using a step-wise approach based on the bacterial genotype and in vitro assays for host adaptation, we selected the most promising candidates for murine infection models out of a total of 254 S. aureus isolates from laboratory mice as well as wild rodents and shrews. Four strains representing the clonal complexes (CC) 8, 49, and 88 (n = 2) were selected and compared to the human-adapted S. aureus strain Newman (CC8) in murine pneumonia and bacteremia models. Notably, a bank vole-derived CC49 strain, named DIP, was highly virulent in BALB/c mice in pneumonia and bacteremia models, whereas the other murine and vole strains showed virulence similar to or lower than that of Newman. At one tenth of the standard infection dose DIP induced disease severity, bacterial load and host cytokine and chemokine responses in the murine bacteremia model similar to that of Newman. In the pneumonia model, DIP was also more virulent than Newman but the effect was less pronounced. Whole genome sequencing data analysis identified a pore-forming toxin gene, lukF-PV(P83)/lukM, in DIP but not in the other tested S. aureus isolates. To conclude, the mouse-adapted S. aureus strain DIP allows a significant reduction of the inoculation dose in mice and is hence a promising tool to develop clinically more relevant infection models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijmm.2018.10.007DOI Listing
January 2019

Inactivation of the Mouse L-Proline Transporter PROT Alters Glutamatergic Synapse Biochemistry and Perturbs Behaviors Required to Respond to Environmental Changes.

Front Mol Neurosci 2018 20;11:279. Epub 2018 Aug 20.

Institute for Pharmaceutical Biology, University of Bonn Bonn, Germany.

The endogenous neutral amino acid L-proline exhibits a variety of physiological and behavioral actions in the nervous system, highlighting the importance of accurately regulating its extracellular abundance. The L-proline transporter PROT () is believed to control the spatial and temporal distribution of L-proline at glutamatergic synapses by rapid uptake of this amino acid into presynaptic terminals. Despite the importance of members of the transporter family regulating neurotransmitter signaling and homeostasis in brain, evidence that PROT dysfunction supports risk for mental illness is lacking. Here we report the disruption of the PROT gene by homologous recombination. Mice defective in PROT displayed altered expression of glutamate transmission-related synaptic proteins in cortex and thalamus. PROT deficiency perturbed mouse behavior, such as reduced locomotor activity, decreased approach motivation and impaired memory extinction. Thus, our study demonstrates that PROT regulates behaviors that are needed to respond to environmental changes and suggests that PROT dysfunctions might contribute to mental disorders showing altered response choice following task contingency changes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2018.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110171PMC
August 2018

Simultaneous Multiplexed Imaging of mRNA and Proteins with Subcellular Resolution in Breast Cancer Tissue Samples by Mass Cytometry.

Cell Syst 2018 Jan 27;6(1):25-36.e5. Epub 2017 Dec 27.

Insitute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland. Electronic address:

To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cels.2017.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791659PMC
January 2018

Global spread of mouse-adapted Staphylococcus aureus lineages CC1, CC15, and CC88 among mouse breeding facilities.

Int J Med Microbiol 2018 Aug 20;308(6):598-606. Epub 2017 Nov 20.

Department of Immunology, University Medicine Greifswald, Germany. Electronic address:

We previously reported that laboratory mice from all global vendors are frequently colonized with Staphylococcus aureus (S. aureus). Genotyping of a snap sample of murine S. aureus isolates from Charles River, US, showed that mice were predominantly colonized with methicillin-sensitive CC88 strains. Here, we expanded our view and investigated whether laboratory mice from other global animal facilities are colonized with similar strains or novel S. aureus lineages, and whether the murine S. aureus isolates show features of host adaptation. In total, we genotyped 230 S. aureus isolates from various vendor facilities of laboratory mice around the globe (Charles River facilities in the USA, Canada, France, and Germany; another US facility) and university- or company-associated breeding facilities in Germany, China and New Zealand. Spa typing was performed to analyse the clonal relationship of the isolates. Moreover, multiplex PCRs were performed for human-specific virulence factors, the immune-evasion cluster (IEC) and superantigen genes (SAg). We found a total of 58 different spa types that clustered into 15 clonal complexes (CCs). Three of these S. aureus lineages had spread globally among laboratory mice and accounted for three quarters of the isolates: CC1 (13.5%), CC15 (14.3%), and CC88 (47.0%). Compared to human colonizing isolates of the same lineages, the murine isolates frequently lacked IEC genes and SAg genes on mobile genetic elements, implying long-term adaptation to the murine host. In conclusion, laboratory mice from various vendors are colonized with host-adapted S. aureus-strains of a few lineages, predominantly the CC88 lineage. S. aureus researchers must be cautioned that S. aureus colonization might be a relevant confounder in infection and vaccination studies and are therefore advised to screen their mice before experimentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijmm.2017.11.006DOI Listing
August 2018

Wild rodents and shrews are natural hosts of Staphylococcus aureus.

Int J Med Microbiol 2018 Aug 22;308(6):590-597. Epub 2017 Sep 22.

Department of Immunology, University Medicine Greifswald, Sauerbruchstraße DZ7, 17475 Greifswald, Germany. Electronic address:

Laboratory mice are the most commonly used animal model for Staphylococcus aureus infection studies. We have previously shown that laboratory mice from global vendors are frequently colonized with S. aureus. Laboratory mice originate from wild house mice. Hence, we investigated whether wild rodents, including house mice, as well as shrews are naturally colonized with S. aureus and whether S. aureus adapts to the wild animal host. 295 animals of ten different species were caught in different locations over four years (2012-2015) in Germany, France and the Czech Republic. 45 animals were positive for S. aureus (15.3%). Three animals were co-colonized with two different isolates, resulting in 48 S. aureus isolates in total. Positive animals were found in Germany and the Czech Republic in each studied year. The S. aureus isolates belonged to ten different spa types, which grouped into six lineages (clonal complex (CC) 49, CC88, CC130, CC1956, sequence type (ST) 890, ST3033). CC49 isolates were most abundant (17/48, 35.4%), followed by CC1956 (14/48, 29.2%) and ST890 (9/48, 18.8%). The wild animal isolates lacked certain properties that are common among human isolates, e.g., a phage-encoded immune evasion cluster, superantigen genes on mobile genetic elements and antibiotic resistance genes, which suggests long-term adaptation to the wild animal host. One CC130 isolate contained the mecC gene, implying wild rodents might be both reservoir and vector for methicillin-resistant . In conclusion, we demonstrated that wild rodents and shrews are naturally colonized with S. aureus, and that those S. aureus isolates show signs of host adaptation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijmm.2017.09.014DOI Listing
August 2018

histoCAT: analysis of cell phenotypes and interactions in multiplex image cytometry data.

Nat Methods 2017 Sep 7;14(9):873-876. Epub 2017 Aug 7.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Single-cell, spatially resolved omics analysis of tissues is poised to transform biomedical research and clinical practice. We have developed an open-source, computational histology topography cytometry analysis toolbox (histoCAT) to enable interactive, quantitative, and comprehensive exploration of individual cell phenotypes, cell-cell interactions, microenvironments, and morphological structures within intact tissues. We highlight the unique abilities of histoCAT through analysis of highly multiplexed mass cytometry images of human breast cancer tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617107PMC
September 2017

Laboratory Mice Are Frequently Colonized with and Mount a Systemic Immune Response-Note of Caution for Infection Experiments.

Front Cell Infect Microbiol 2017 2;7:152. Epub 2017 May 2.

Department of Immunology, University Medicine GreifswaldGreifswald, Germany.

Whether mice are an appropriate model for infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of . We previously identified a mouse-adapted strain, which caused infections in laboratory mice. This raised the question whether laboratory mice are commonly colonized with and whether this might impact on infection experiments. Publicly available health reports from commercial vendors revealed that colonization is rather frequent, with rates as high as 21% among specific-pathogen-free mice. In animal facilities, was readily transmitted from parents to offspring, which became persistently colonized. Among 99 murine isolates from Charles River Laboratories half belonged to the lineage CC88 (54.5%), followed by CC15, CC5, CC188, and CC8. A comparison of human and murine isolates revealed features of host adaptation. In detail, murine strains lacked -converting phages and superantigen-encoding mobile genetic elements, and were frequently ampicillin-sensitive. Moreover, murine CC88 isolates coagulated mouse plasma faster than human CC88 isolates. Importantly, colonization clearly primed the murine immune system, inducing a systemic IgG response specific for numerous proteins, including several vaccine candidates. Phospholipase C emerged as a promising test antigen for monitoring colonization in laboratory mice. In conclusion, laboratory mice are natural hosts of and therefore, could provide better infection models than previously assumed. Pre-exposure to the bacteria is a possible confounder in infection and vaccination studies and should be monitored.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcimb.2017.00152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411432PMC
December 2017

An Immune Atlas of Clear Cell Renal Cell Carcinoma.

Cell 2017 05;169(4):736-749.e18

Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. Electronic address:

Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of samples from 73 clear cell renal cell carcinoma (ccRCC) patients and five healthy controls. In 3.5 million measured cells, we identified 17 tumor-associated macrophage phenotypes, 22 T cell phenotypes, and a distinct immune composition correlated with progression-free survival, thereby presenting an in-depth human atlas of the immune tumor microenvironment in this disease. This study revealed potential biomarkers and targets for immunotherapy development and validated tools that can be used for immune profiling of other tumor types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2017.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422211PMC
May 2017

GenoGAM: genome-wide generalized additive models for ChIP-Seq analysis.

Bioinformatics 2017 Aug;33(15):2258-2265

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, 80333 Munich, Germany.

Motivation: Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a widely used approach to study protein-DNA interactions. Often, the quantities of interest are the differential occupancies relative to controls, between genetic backgrounds, treatments, or combinations thereof. Current methods for differential occupancy of ChIP-Seq data rely however on binning or sliding window techniques, for which the choice of the window and bin sizes are subjective.

Results: Here, we present GenoGAM (Genome-wide Generalized Additive Model), which brings the well-established and flexible generalized additive models framework to genomic applications using a data parallelism strategy. We model ChIP-Seq read count frequencies as products of smooth functions along chromosomes. Smoothing parameters are objectively estimated from the data by cross-validation, eliminating ad hoc binning and windowing needed by current approaches. GenoGAM provides base-level and region-level significance testing for full factorial designs. Application to a ChIP-Seq dataset in yeast showed increased sensitivity over existing differential occupancy methods while controlling for type I error rate. By analyzing a set of DNA methylation data and illustrating an extension to a peak caller, we further demonstrate the potential of GenoGAM as a generic statistical modeling tool for genome-wide assays.

Availability And Implementation: Software is available from Bioconductor: https://www.bioconductor.org/packages/release/bioc/html/GenoGAM.html .

Contact: [email protected]

Supplementary Information: Supplementary information is available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btx150DOI Listing
August 2017

Disentangling Puzzles of Spatial Scales and Participation in Environmental Governance-The Case of Governance Re-scaling Through the European Water Framework Directive.

Environ Manage 2016 Dec 20;58(6):998-1014. Epub 2016 Sep 20.

Research Group on Governance, Participation and Sustainability, Leuphana University of Lüneburg, Scharnhorststrasse 1, 21335 , Lüneburg, Germany.

This article attempts to shed new light on prevailing puzzles of spatial scales in multi-level, participatory governance as regards the democratic legitimacy and environmental effectiveness of governance systems. We focus on the governance re-scaling by the European Water Framework Directive, which introduced new governance scales (mandated river basin management) and demands consultation of citizens and encourages 'active involvement' of stakeholders. This allows to examine whether and how re-scaling through deliberate governance interventions impacts on democratic legitimacy and effective environmental policy delivery. To guide the enquiry, this article organizes existing-partly contradictory-claims on the relation of scale, democratic legitimacy, and environmental effectiveness into three clusters of mechanisms, integrating insights from multi-level governance, social-ecological systems, and public participation. We empirically examine Water Framework Directive implementation in a comparative case study of multi-level systems in the light of the suggested mechanisms. We compare two planning areas in Germany: North Rhine Westphalia and Lower Saxony. Findings suggest that the Water Framework Directive did have some impact on institutionalizing hydrological scales and participation. Local participation appears generally both more effective and legitimate than on higher levels, pointing to the need for yet more tailored multi-level governance approaches, depending on whether environmental knowledge or advocacy is sought. We find mixed results regarding the potential of participation to bridge spatial 'misfits' between ecological and administrative scales of governance, depending on the historical institutionalization of governance on ecological scales. Polycentricity, finally, appeared somewhat favorable in effectiveness terms with some distinct differences regarding polycentricity in planning vs. polycentricity in implementation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00267-016-0753-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085982PMC
December 2016

Memory CD8(+) T cells colocalize with IL-7(+) stromal cells in bone marrow and rest in terms of proliferation and transcription.

Eur J Immunol 2015 Apr 27;45(4):975-87. Epub 2015 Feb 27.

Department of Cell Biology, German Rheumatism Research Center (DRFZ), a Leibniz Institute, Berlin, Germany.

It is believed that memory CD8(+) T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL-7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8(+) T cells individually colocalize with IL-7(+) reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4-1BB (CD137), IL-2, or IFN-γ, despite the expression of CD69 on about 30% of the cells. Ninety-five percent of the memory CD8(+) T cells in BM are in G0 phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G2 of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8(+) memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8(+) memory T cells. Taken together, the present results suggest that CD8(+) memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL-7 receptor signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201445295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415462PMC
April 2015

Rpb4 subunit functions mainly in mRNA synthesis by RNA polymerase II.

J Biol Chem 2014 Jun 5;289(25):17446-52. Epub 2014 May 5.

From the Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich and the Department of Molecular Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany

RNA polymerase II (Pol II) is the central enzyme that carries out eukaryotic mRNA transcription and consists of a 10-subunit catalytic core and a subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). Rpb4/7 has been proposed to dissociate from Pol II, enter the cytoplasm, and function there in mRNA translation and degradation. Here we provide evidence that Rpb4 mainly functions in nuclear mRNA synthesis by Pol II, as well as evidence arguing against an important cytoplasmic role in mRNA degradation. We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis to show that Rpb4 deletion in Saccharomyces cerevisiae causes a drastic defect in mRNA synthesis that is compensated by down-regulation of mRNA degradation, resulting in mRNA level buffering. Deletion of Rpb4 can be rescued by covalent fusion of Rpb4 to the Pol II core subunit Rpb2, which largely restores mRNA synthesis and degradation defects caused by Rpb4 deletion. Thus, Rpb4 is a bona fide Pol II core subunit that functions mainly in mRNA synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.568014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067182PMC
June 2014

Transcriptome surveillance by selective termination of noncoding RNA synthesis.

Cell 2013 Nov 7;155(5):1075-87. Epub 2013 Nov 7.

Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.

Pervasive transcription of eukaryotic genomes stems to a large extent from bidirectional promoters that synthesize mRNA and divergent noncoding RNA (ncRNA). Here, we show that ncRNA transcription in the yeast S. cerevisiae is globally restricted by early termination that relies on the essential RNA-binding factor Nrd1. Depletion of Nrd1 from the nucleus results in 1,526 Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at most promoters and antisense NUTs in most 3' regions of genes. Nrd1 and its partner Nab3 preferentially bind RNA motifs that are depleted in mRNAs and enriched in ncRNAs and some mRNAs whose synthesis is controlled by transcription attenuation. These results define a global mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and to suppress antisense transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2013.10.024DOI Listing
November 2013

Decoding signaling and function of the orphan G protein-coupled receptor GPR17 with a small-molecule agonist.

Sci Signal 2013 Oct 22;6(298):ra93. Epub 2013 Oct 22.

1Molecular, Cellular, and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, 53115 Bonn, Germany.

Replacement of the lost myelin sheath is a therapeutic goal for treating demyelinating diseases of the central nervous system (CNS), such as multiple sclerosis (MS). The G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR17, which is phylogenetically closely related to receptors of the "purinergic cluster," has emerged as a modulator of CNS myelination. However, whether GPR17-mediated signaling positively or negatively regulates this critical process is unresolved. We identified a small-molecule agonist, MDL29,951, that selectively activated GPR17 even in a complex environment of endogenous purinergic receptors in primary oligodendrocytes. MDL29,951-stimulated GPR17 engaged the entire set of intracellular adaptor proteins for GPCRs: G proteins of the Gα(i), Gα(s), and Gα(q) subfamily, as well as β-arrestins. This was visualized as alterations in the concentrations of cyclic adenosine monophosphate and inositol phosphate, increased Ca²⁺ flux, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as multifeatured cell activation recorded with label-free dynamic mass redistribution and impedance biosensors. MDL29,951 inhibited the maturation of primary oligodendrocytes from heterozygous but not GPR17 knockout mice in culture, as well as in cerebellar slices from 4-day-old wild-type mice. Because GPCRs are attractive targets for therapeutic intervention, inhibiting GPR17 emerges as therapeutic strategy to relieve the oligodendrocyte maturation block and promote myelin repair in MS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.2004350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114018PMC
October 2013

Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation.

Genome Res 2012 Jul 30;22(7):1350-9. Epub 2012 Mar 30.

Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-Universität München, Munich, Germany.

To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.130161.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396375PMC
July 2012

Measurement of genome-wide RNA synthesis and decay rates with Dynamic Transcriptome Analysis (DTA).

Bioinformatics 2012 Mar 28;28(6):884-5. Epub 2012 Jan 28.

Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Standard transcriptomics measures total cellular RNA levels. Our understanding of gene regulation would be greatly improved if we could measure RNA synthesis and decay rates on a genome-wide level. To that end, the Dynamic Transcriptome Analysis (DTA) method has been developed. DTA combines metabolic RNA labeling with standard transcriptomics to measure RNA synthesis and decay rates in a precise and non-perturbing manner. Here, we present the open source R/Bioconductor software package DTA. It implements all required bioinformatics steps that allow the accurate absolute quantification and comparison of RNA turnover.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/bts052DOI Listing
March 2012

Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast.

Mol Syst Biol 2011 Jan;7:458

Gene Center and Department of Biochemistry, Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-Universität München, Munich, Germany.

To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA-derived mRNA synthesis rates identified 16 stress-specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/msb.2010.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049410PMC
January 2011

Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase.

Mol Cell 2009 Apr;34(2):212-22

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

Quality control pathways such as ER-associated degradation (ERAD) employ a small number of factors to specifically recognize a wide variety of protein substrates. Delineating the mechanisms of substrate selection is a principle goal in studying quality control. The Hrd1p ubiquitin ligase mediates ERAD of numerous misfolded proteins including soluble, lumenal ERAD-L and membrane-anchored ERAD-M substrates. We tested if the Hrd1p multispanning membrane domain was involved in ERAD-M specificity. In this work, we have identified site-directed membrane domain mutants of Hrd1p impaired only for ERAD-M and normal for ERAD-L. Furthermore, other Hrd1p variants were specifically deficient for degradation of individual ERAD-M substrates. Thus, the Hrd1p transmembrane region bears determinants of high specificity in the ERAD-M pathway. From in vitro and interaction studies, we suggest a model in which the Hrd1p membrane domain employs intramembrane residues to evaluate substrate misfolding, leading to selective ubiquitination of appropriate ERAD-M clients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2009.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710143PMC
April 2009