Center for Medical Biotechnology

Regensburg, Germany

Center for Medical Biotechnology

Regensburg, Germany
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Gracia J.C.M.,Ghent University | Van Den Hoecke S.,Center for Medical Biotechnology | Van Den Hoecke S.,Ghent University | Saelens X.,Center for Medical Biotechnology | And 2 more authors.
PLoS ONE | Year: 2017

H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations. © 2017 Mancera Gracia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Jiang H.,Swedish University of Agricultural Sciences | Moreno-Romero J.,Swedish University of Agricultural Sciences | Santos-Gonzalez J.,Swedish University of Agricultural Sciences | De Jaeger G.,Ghent University | And 6 more authors.
Genes and Development | Year: 2017

Hybrid seed lethality as a consequence of interspecies or interploidy hybridizations is a major mechanism of reproductive isolation in plants. This mechanism is manifested in the endosperm, a dosage-sensitive tissue supporting embryo growth. Deregulated expression of imprinted genes such as ADMETOS (ADM) underpin the interploidy hybridization barrier in Arabidopsis thaliana; however, the mechanisms of their action remained unknown. In this study, we show that ADM interacts with the AT hook domain protein AHL10 and the SET domain-containing SU (VAR)3-9 homolog SUVH9 and ectopically recruits the heterochromatic mark H3K9me2 to AT-rich transposable elements (TEs), causing deregulated expression of neighboring genes. Several hybrid incompatibility genes identified in Drosophila encode for dosage-sensitive heterochromatin-interacting proteins, which has led to the suggestion that hybrid incompatibilities evolve as a consequence of interspecies divergence of selfish DNA elements and their regulation. Our data show that imbalance of dosage-sensitive chromatin regulators underpins the barrier to interploidy hybridization in Arabidopsis, suggesting that reproductive isolation as a consequence of epigenetic regulation of TEs is a conserved feature in animals and plants. © 2017 Jiang et al.


Leyh M.,University of Regensburg | Leyh M.,Center for Medical Biotechnology | Seitz A.,University of Ulm | Durselen L.,University of Ulm | And 6 more authors.
Stem Cell Research and Therapy | Year: 2014

Introduction. In the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a "tri-culture" model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants. Methods. Gene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures). Results: In general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young's and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only. Conclusions: Our results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1β and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs' chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies. © 2014 Leyh et al.; licensee BioMed Central Ltd.


Bedal K.B.,University of Regensburg | Bedal K.B.,Center for Medical Biotechnology | Grassel S.,University of Regensburg | Spanier G.,University of Regensburg | And 3 more authors.
Carcinogenesis | Year: 2015

Collagen XVI, a fibril-associated collagen with interrupted triple helix (FACIT) collagen, is involved in oral squamous cell carcinoma (OSCC) and glioblastoma progression. The NC11 domain of collagen XVI has been described previously with a strong implication in physiological processes. We detected the non-collagenous (NC) 11-domain in supernatants of OSCC cells after recombinant expression of full-length collagen XVI and in sera from OSCC patients and healthy individuals. Stable expression of NC11-green fluorescent protein (GFP) fusion protein in OSCC cells initiated proliferation control and block of anchorage-independent growth. Moreover, the NC11 domain triggered the generation of tubular-like net structures on laminin-rich matrix in contrast to mock-GFP control cells and cells expressing full-length collagen XVI. Taqman® quantitative PCR and diaminobenzidine staining in 2D- and 3D cell culture revealed a significantly increased gene and protein expression of VEGFR1, VEGFR2 and uPAR in recombinant NC11-GFP-expressing cells. Specific VEGF receptor inhibition with Axitinib or fetal calf serum heat inactivation prevented formation of tubular-like net structures. Accordantly, NC11-GFP coated culture slides led to an increase of focal adhesion contact formation and the upregulation of VEGFR1 and uPAR in three different non-transfected OSCC cell lines. In summary, we suggest that the NC11 domain of collagen XVI is a potential biomarker for OSCC and triggers vasculogenic mimicry via upregulation of endothelial receptors VEGFR1, VEGFR2 and uPAR in 2D- and 3D OSCC cell culture conditions. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.


Ratzinger S.,Center for Medical Biotechnology | Ratzinger S.,University of Regensburg | Grassel S.,University of Regensburg | Grassel S.,Center for Medical Biotechnology | And 4 more authors.
Matrix Biology | Year: 2011

Type XVI collagen belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). Recently, high affinity to integrin alpha1beta1 has been shown allowing cells expressing those integrins to attach and spread on recombinant type XVI collagen. Here, we show that type XVI collagen is overexpressed in dysplastic areas of mucosal epithelium from oral squamous cell carcinoma (OSCC) patients. Induction of its expression in OSCC cell lines (COLXVI cells) leads to an increased expression of Kindlin-1. Moreover, we demonstrate a significantly increased Kindlin-1/beta1-integrin interaction. Additionally, we detected a higher number of activated beta1-integrins in COLXVI cells and found a neo-expression of alpha1 integrin subunit on these cells. FACS analysis revealed a significantly higher amount of COLXVI cells in S-phase and G2/M-phase 6. h after synchronisation leading to a markedly higher proliferation activity. Blocking beta1-integrins with a specific antibody resulted in reduced proliferation of COLXVI cells. In summary, we demonstrate that overexpression of type XVI collagen in aberrant oral keratinocytes leads to Kindlin-1 induction, increased Kindlin-1/beta1-integrin interaction, integrin activation and subsequently to a proliferative cellular phenotype. © 2011 International Society of Matrix Biology.


Bedal K.B.,University of Regensburg | Bedal K.B.,Center for Medical Biotechnology | Grassel S.,University of Regensburg | Grassel S.,Center for Medical Biotechnology | And 5 more authors.
PLoS ONE | Year: 2014

Collagen XVI belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). It is overexpressed during the progression of oral squamous cell carcinoma (OSCC). The present data show a strong collagen XVI-dependent induction of MMP9 and an increase in OSCC cell invasion. We found activated integrin-linked kinase (ILK) in a complex with kindlin-1 and activation of protein kinase B (PKB/Akt) to be responsible for MMP9 induction. Inhibition of the formation of focal adhesions reduced MMP9 expression. Moreover, collagen XVI overexpressing OSCC cell clones (COLXVI cell clones) transfected with vectors containing different MMP9 promoter fragments adjacent to a luciferase reporter revealed an increase in luciferase signal dependent on AP-1 binding sites. Deletion of the AP-1 binding site 98 bp upstream of the reported transcription start site and inhibition of AP-1 with Tanshinone IIA resulted in decreased MMP9 expression. The AP-1 subunit JunB showed differential expression between COLXVI cell clones and mock control cells. Additionally, mass spectrometric analysis of immunoprecipitates revealed that c-Fos interacted strongly with dyskerin in COLXVI cell clones compared to mock controls. © 2014 Bedal et al.


Grassel S.,University of Regensburg | Grassel S.,Center for Medical Biotechnology | Beckmann J.,University of Regensburg | Rath B.,University of Regensburg | And 4 more authors.
International Journal of Molecular Medicine | Year: 2010

The pathogenetic mechanisms of osteonecrosis of the femoral head are unresolved to date. This study analyzed the matrix metalloprotease (MMP) and tissue inhibitor of matrix metalloprotease (TIMP) expression and activity which might add to the impaired bone matrix repair capacity affecting the balance between bone resorption and de novo bone formation in osteonecrosis of the femoral head (ONFH). Cancellous bone biopsies were taken at the femoral head and neck of patients with advanced ONFH and patients with primary osteoarthritis (OA) who were undergoing total hip arthroplasty. We assessed the gene expression levels of MMP-2 and -9, TIMP-1 and -2, IL-1β, IL-6 and TNF-α in both biopsies. These data were corroborated by MMP activity screening, collagen profiling and ELISAs for determination of MMP, TIMP, IL-1β, IL-6 and TNF-α concentrations. Gene expression rates of MMP-2 and TIMP-1 were higher in ONFH biopsies whereas TIMP-2 and MMP-9 gene expression was not significantly altered. MMP protein profile shifted towards increased biosynthesis of both, active and pro-MMP-2 in ONFH bone lysates and decreased pro-MMP-9 production. Expression profiles of pro-inflammatory cytokines and collagens in OA and ONFH bone lysates were highly similar. Increased biosynthesis and activation of MMP-2 in ONFH samples may add to shifting the bone matrix turnover balance towards resorption of bone macromolecules thereby counteracting new bone matrix production. OA- and ONFH-affected bone exhibits a similar pro-inflammatory cytokine and collagen expression profile suggesting a relationship on the molecular level of inflammation and collagenous matrix composition between both diseases.


Ratzinger S.,University of Regensburg | Ratzinger S.,Center for Medical Biotechnology | Eble J.A.,Goethe University Frankfurt | Pasoldt A.,University of Regensburg | And 5 more authors.
Matrix Biology | Year: 2010

In Crohn's disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF) provided by intact tissue is disturbed due to inflammation and thus, cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original ECM leads ISEMF to exit this cycle. In contrast, during fibrosis, ISEMF persist. It is known that ISEMF produce and deposit collagen types I, III, IV and V; however synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed the distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell-matrix interactions in different phases of the inflammatory process and their effect on cell spreading, proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall where it co-localizes with fibrillin-1 and integrin α1. ISEMF reveal increasing gene and protein expression of collagen XVI concurrent to increasing inflammation. ISEMF reveal more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This involves recruitment of α1β1 integrin, which shows increased cell surface expression on ISEMF in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and, even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell-matrix interaction preferentially through recruitment of α1ß1 integrin into the tips of the focal adhesion contacts. © 2009 Elsevier B.V.


Grassel S.,University of Regensburg | Grassel S.,Center for Medical Biotechnology | Bauer R.J.,Center for Medical Biotechnology | Bauer R.J.,University of Regensburg
Matrix Biology | Year: 2013

Collagen XVI, by structural analogy a member of the FACIT- (fibril-associated collagens with interrupted triple helices) family of collagens, is described as a minor collagen component of connective tissues. Collagen XVI is expressed in various cells and tissues without known occurrence of splice variants or isoforms. For skin and cartilage tissues its suprastructure is known. Presumably, there it acts as an adaptor protein connecting and organizing large fibrillar networks and thus modulates integrity and stability of the extracellular matrix (ECM).Collagen XVI is produced by myofibroblasts in the normal intestine and its synthesis is increased in the inflamed bowel wall where myofibroblasts develop increased numbers of focal adhesion contacts on collagen XVI. Consequently, recruitment of α1 integrin into the focal adhesions at the tip of the cells is induced followed by increased cell spreading on collagen XVI. This presumably adds to the maintenance of myofibroblasts in the inflamed intestinal regions and thus promotes fibrotic responses of the tissue. Notably, α1/α2 integrins interact with collagen XVI through an α1/α2β1 integrin binding site located in the COL 1-3 domains.Collagen XVI may act as a substrate for adhesion and invasion of connective tissue tumor cells. In glioblastoma it induces tumor invasiveness by modification of the β1-integrin activation pattern. Thus, altering the cell-matrix interaction through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. In this line, in oral squamous cell carcinoma collagen XVI expression is induced which results in an upregulation of Kindlin-1 followed by an increased interaction with beta1-integrin. Consequently, collagen XVI induces a proliferative tumor phenotype by promoting an early S-phase entry.In summary, collagen XVI plays a decisive role in the interaction of connective tissue cells with their ECM, which is impaired in pathological situations. Alteration of tissue location and expression level of collagen XVI appears to promote tumorigenesis and to perpetuate inflammatory reactions. © 2012 International Society of Matrix Biology.


Grum D.,Center for Medical Biotechnology | van den Boom J.,Center for Medical Biotechnology | Neumann D.,Center for Medical Biotechnology | Matena A.,Center for Medical Biotechnology | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2010

3′-Phospho-adenosine-5′-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5′-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells. © 2010 Elsevier Inc. All rights reserved.

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