TY - JOUR A1 - Sanyal, Anirban A1 - Wallaschek, Nina A1 - Glass, Mandy A1 - Flamand, Louis A1 - Wight, Darren J. A1 - Kaufer, Benedikt B. T1 - The ND10 Complex Represses Lytic Human Herpesvirus 6A Replication and Promotes Silencing of the Viral Genome JF - Viruses N2 - Human herpesvirus 6A (HHV-6A) replicates in peripheral blood mononuclear cells (PBMCs) and various T-cell lines in vitro. Intriguingly, the virus can also establish latency in these cells, but it remains unknown what influences the decision between lytic replication and the latency of the virus. Incoming virus genomes are confronted with the nuclear domain 10 (ND10) complex as part of an intrinsic antiviral response. Most herpesviruses can efficiently subvert ND10, but its role in HHV-6A infection remains poorly understood. In this study, we investigated if the ND10 complex affects HHV-6A replication and contributes to the silencing of the virus genome during latency. We could demonstrate that ND10 complex was not dissociated upon infection, while the number of ND10 bodies was reduced in lytically infected cells. Virus replication was significantly enhanced upon knock down of the ND10 complex using shRNAs against its major constituents promyelocytic leukemia protein (PML), hDaxx, and Sp100. In addition, we could demonstrate that viral genes are more efficiently silenced in the presence of a functional ND10 complex. Our data thereby provides the first evidence that the cellular ND10 complex plays an important role in suppressing HHV-6A lytic replication and the silencing of the virus genome in latently infected cells. KW - human herpesvirus 6 KW - ND10 complex KW - PML KW - lytic replication KW - latency KW - PML nuclear-bodies KW - gene-expression KW - virus-infection KW - in-vitro KW - DNA Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227337 VL - 10 IS - 8 ER - TY - JOUR A1 - Dotterweich, Julia A1 - Schlegelmilch, Katrin A1 - Keller, Alexander A1 - Geyer, Beate A1 - Schneider, Doris A1 - Zeck, Sabine A1 - Tower, Robert J. J. A1 - Ebert, Regina A1 - Jakob, Franz A1 - Schütze, Norbert T1 - Contact of myeloma cells induces a characteristic transcriptome signature in skeletal precursor cells-implications for myeloma bone disease JF - Bone N2 - Physical interaction of skeletal precursors with multiple myeloma cells has been shown to suppress their osteogenic potential while favoring their tumor-promoting features. Although several transcriptome analyses of myeloma patient-derived mesenchymal stem cells have displayed differences compared to their healthy counterparts, these analyses insufficiently reflect the signatures mediated by tumor cell contact, vary due to different methodologies, and lack results in lineage-committed precursors. To determine tumor cell contact-mediated changes on skeletal precursors, we performed transcriptome analyses of mesenchymal stem cells and osteogenic precursor cells cultured in contact with the myeloma cell line INA-6. Comparative analyses confirmed dysregulation of genes which code for known disease-relevant factors and additionally revealed upregulation of genes that are associated with plasma cell homing, adhesion, osteoclastogenesis, and angiogenesis. Osteoclast-derived coupling factors, a dysregulated adipogenic potential, and an imbalance in favor of anti-anabolic factors may play a role in the hampered osteoblast differentiation potential of mesenchymal stem cells. Angiopoietin-Like 4 (ANGPTL4) was selected from a list of differentially expressed genes as a myeloma cell contact-dependent target in skeletal precursor cells which warranted further functional analyses. Adhesion assays with full-length ANGPTL4-coated plates revealed a potential role of this protein in INA6 cell attachment. This study expands knowledge of the myeloma cell contact-induced signature in the stromal compartment of myelomatous bones and thus offers potential targets that may allow detection and treatment of myeloma bone disease at an early stage. KW - marrow stromal cells KW - Endothelial growth-factor KW - precedes multiple-myeloma KW - monoclonial gammopathy KW - in-vitro KW - mesenchymal stem-cells KW - undetermined significance KW - angiogenic cytokines KW - peripheral-blood KW - gene-expression KW - Multiple myeloma KW - Bone disease KW - Angiopoietin-like 4 KW - Gene expression profiling KW - Mesenchymal stem cells KW - Osteogenic precursor cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186688 VL - 93 ER - TY - JOUR A1 - Ahmad, Ruhel A1 - Wolber, Wanja A1 - Eckardt, Sigrid A1 - Koch, Philipp A1 - Schmitt, Jessica A1 - Semechkin, Ruslan A1 - Geis, Christian A1 - Heckmann, Manfred A1 - Brüstle, Oliver A1 - McLaughlin, John K. A1 - Sirén, Anna-Leena A1 - Müller, Albrecht M. T1 - Functional Neuronal Cells Generated by Human Parthenogenetic Stem Cells JF - PLoS One N2 - Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases. KW - methylation KW - derivation KW - blastocysts KW - pluripotent KW - differentiation KW - lines KW - brain development KW - in-vitro KW - mice KW - specification Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130268 VL - 7 IS - 8 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Josupeit, Rafael A1 - Rudolph, Stephan A1 - Ehrig, Klaas A1 - Donat, Ulrike A1 - Weibel, Stephanie A1 - Chen, Nanhai G. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Heisig, Martin A1 - Thamm, Douglas A1 - Stritzker, Jochen A1 - MacNeill, Amy A1 - Szalay, Aladar A. T1 - Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma JF - PLoS One N2 - Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS. KW - breast-tumors KW - animal-model KW - nude-mice KW - cell-line KW - in-vitro KW - glv-1h68 KW - cancer KW - virotherapy KW - dogs KW - neutrophils Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129998 VL - 7 IS - 5 ER - TY - JOUR A1 - Yan, Yan A1 - Hong, Ni A1 - Chen, Tiansheng A1 - Li, Mingyou A1 - Wang, Tiansu A1 - Guan, Guijun A1 - Qiao, Yongkang A1 - Chen, Songlin A1 - Schartl, Manfred A1 - Li, Chang-Ming A1 - Hong, Yunhan T1 - p53 Gene Targeting by Homologous Recombination in Fish ES Cells JF - PLoS One N2 - Background: Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes). Methodology and Principal Findings: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1 similar to MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by similar to 12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. Conclusions: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology. KW - mouse KW - in-vitro KW - drug selection KW - chimera formation KW - medakafish oryzias latipes KW - embryonic stem-cells KW - zebrafish KW - differentiation KW - cultures KW - pluripotency Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133416 VL - 8 IS - 3 ER - TY - JOUR A1 - Hohenauer, Tobias A1 - Berking, Carola A1 - Schmidt, Andreas A1 - Haferkamp, Sebastian A1 - Senft, Daniela A1 - Kammerbauer, Claudia A1 - Fraschka, Sabine A1 - Graf, Saskia Anna A1 - Irmler, Martin A1 - Beckers, Johannes A1 - Flaig, Michael A1 - Aigner, Achim A1 - Höbel, Sabrina A1 - Hoffmann, Franziska A1 - Hermeking, Heiko A1 - Rothenfusser, Simon A1 - Endres, Stefan A1 - Ruzicka, Thomas A1 - Besch, Robert T1 - The neural crest transcription factor Brn3a is expressed in melanoma and required for cell cycle progression and survival JF - EMBO Molecular Medicine N2 - Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit-Oct-Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi-mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double-strand breaks as evidenced by Mre11/Rad50-containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage-independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene-induced senescence in non-malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis. KW - oncogene-induced senescence KW - BRN-3A KW - DNA KW - DNA damage KW - tumourigenesis KW - P53 KW - in-vitro KW - neural crest factors KW - family KW - apoptosis KW - melanoma KW - BRAF mutations KW - domain Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122193 SN - 1757-4676 VL - 5 ER - TY - JOUR A1 - Ngwa, Che Julius A1 - Scheuermayer, Matthias A1 - Mair, Gunnar Rudolf A1 - Kern, Selina A1 - Brügl, Thomas A1 - Wirth, Christine Clara A1 - Aminake, Makoah Nigel A1 - Wiesner, Jochen A1 - Fischer, Rainer A1 - Vilcinskas, Andreas A1 - Pradel, Gabriele T1 - Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito JF - BMC Genomics N2 - Background: The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector. Results: To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy. Conclusions: The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector. KW - parasitophorous vacuole KW - sexual development KW - gametocyte KW - transcriptome KW - signal peptide peptidase KW - host cell interface KW - alpha-tubulin-II KW - life-cycle KW - protein kinases KW - in-vitro KW - erythroyte invation KW - blocking antibodies KW - malaria KW - plasmodium falciparum KW - gametogenesis KW - mosquito KW - transmission Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121905 SN - 1471-2164 VL - 14 IS - 256 ER - TY - JOUR A1 - Fernandez-Robredo, P. A1 - Sancho, A. A1 - Johnen, S. A1 - Recalde, S. A1 - Gama, N. A1 - Thumann, G. A1 - Groll, J. A1 - Garcia-Layana, A. T1 - Current Treatment Limitations in Age-Related Macular Degeneration and Future Approaches Based on Cell Therapy and Tissue Engineering JF - Journal of Ophtamology N2 - Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. With an ageing population, it is anticipated that the number of AMD cases will increase dramatically, making a solution to this debilitating disease an urgent requirement for the socioeconomic future of the European Union and worldwide. The present paper reviews the limitations of the current therapies as well as the socioeconomic impact of the AMD. There is currently no cure available for AMD, and even palliative treatments are rare. Treatment options show several side effects, are of high cost, and only treat the consequence, not the cause of the pathology. For that reason, many options involving cell therapy mainly based on retinal and iris pigment epithelium cells as well as stem cells are being tested. Moreover, tissue engineering strategies to design and manufacture scaffolds to mimic Bruch's membrane are very diverse and under investigation. Both alternative therapies are aimed to prevent and/or cure AMD and are reviewed herein. KW - endothelial growth factor KW - pluripotent stem cells KW - subfoveal choroidal neovascularization KW - human bruchs membrane KW - ciliary neurotrophic factor KW - randomized clinical trial KW - dystrophic RCS rats KW - subretinal space KW - in-vitro KW - retinal pigment epithelium Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118004 SN - 2090-0058 IS - 510285 ER - TY - JOUR A1 - Sander, Brigitta A1 - de Jong, Daphne A1 - Rosenwald, Andreas A1 - Xie, Wanling A1 - Balagué, Olga A1 - Calaminici, Maria A1 - Carreras, Joaquim A1 - Gaulard, Philippe A1 - Gribben, John A1 - Hagenbeek, Anton A1 - Kersten, Marie José A1 - Molina, Thierry Jo A1 - Lee, Abigail A1 - Montes-Moreno, Santiago A1 - Ott, German A1 - Raemaekers, John A1 - Salles, Gilles A1 - Sehn, Laurie A1 - Thorns, Christoph A1 - Wahlin, Bjorn E. A1 - Gascoyne, Randy D. A1 - Weller, Edie T1 - The reliability of immunohistochemical analysis of the tumor microenvironment in follicular lymphoma: a validation study from the Lunenburg Lymphoma Biomarker Consortium JF - Haematologica N2 - The cellular microenvironment in follicular lymphoma is of biological and clinical importance. Studies on the clinical significance of non-malignant cell populations have generated conflicting results, which may partly be influenced by poor reproducibility in immunohistochemical marker quantification. In this study, the reproducibility of manual scoring and automated microscopy based on a tissue microarray of 25 follicular lymphomas as compared to flow cytometry is evaluated. The agreement between manual scoring and flow cytometry was moderate for CD3, low for CD4, and moderate to high for CD8, with some laboratories scoring closer to the flow cytometry results. Agreement in manual quantification across the 7 laboratories was low to moderate for CD3, CD4, CD8 and FOXP3 frequencies, moderate for CD21, low for MIB1 and CD68, and high for CD10. Manual scoring of the architectural distribution resulted in moderate agreement for CD3, CD4 and CD8, and low agreement for FOXP3 and CD68. Comparing manual scoring to automated microscopy demonstrated that manual scoring increased the variability in the low and high frequency interval with some laboratories showing a better agreement with automated scores. Manual scoring reliably identified rare architectural patterns of T-cell infiltrates. Automated microscopy analyses for T-cell markers by two different instruments were highly reproducible and provided acceptable agreement with flow cytometry. These validation results provide explanations for the heterogeneous findings on the prognostic value of the microenvironment in follicular lymphoma. We recommend a more objective measurement, such as computer-assisted scoring, in future studies of the prognostic impact of microenvironment in follicular lymphoma patients. KW - CD/metabolism KW - flow cytometry KW - antigens KW - regulatory T-cells KW - independent predictor KW - gene expression KW - high numbers KW - CD40 ligand KW - Riutximab KW - survival KW - marcophages KW - transformation KW - in-vitro Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116875 SN - 1592-8721 VL - 99 IS - 4 ER - TY - JOUR A1 - Oli, Swarna A1 - Abdelmohsen, Usama Ramadan A1 - Hentschel, Ute A1 - Schirmeister, Tanja T1 - Identification of Plakortide E from the Caribbean Sponge Plakortis halichondroides as a Trypanocidal Protease Inhibitor using Bioactivity-Guided Fractionation JF - MARINE DRUGS N2 - In this paper, we report new protease inhibitory activity of plakortide E towards cathepsins and cathepsin-like parasitic proteases. We further report on its anti-parasitic activity against Trypanosoma brucei with an IC50 value of 5 mu M and without cytotoxic effects against J774.1 macrophages at 100 mu M concentration. Plakortide E was isolated from the sponge Plakortis halichondroides using enzyme assay-guided fractionation and identified by NMR spectroscopy and mass spectrometry. Furthermore, enzyme kinetic studies confirmed plakortide E as a non-competitive, slowly-binding, reversible inhibitor of rhodesain. KW - plakortis halichondroides KW - plakortide E. KW - protease inhibitor KW - slowly-binding reversible inhibitor KW - cathepsin KW - trypanosoma brucei KW - cysteine protease KW - malaria parasites KW - cathepsin-L KW - in-vitro KW - rhodesain Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116536 SN - 1660-3397 VL - 12 IS - 5 ER - TY - JOUR A1 - Liedert, Astrid A1 - Röntgen, Viktoria A1 - Schinke, Thorsten A1 - Benisch, Peggy A1 - Ebert, Regina A1 - Jakob, Franz A1 - Klein-Hitpass, Ludger A1 - Lennerz, Jochen K. A1 - Amling, Michael A1 - Ignatius, Anita T1 - Osteoblast-Specific Krm2 Overexpression and Lrp5 Deficiency Have Different Effects on Fracture Healing in Mice JF - PLOS ONE N2 - The canonical Wnt/beta-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5(-/-)) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5(-/-) mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5(-/-) mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active beta-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis. KW - autosomal-dominant osteopetrosis KW - receptor related protein KW - high-bone-mass KW - WNT pathway KW - in-vitro KW - cells KW - gene KW - proliferation KW - osteoclasts KW - mutations Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115782 SN - 1932-6203 VL - 9 IS - 7 ER - TY - JOUR A1 - Kern, Selina A1 - Agarwal, Shruti A1 - Huber, Kilian A1 - Gehring, Andre P. A1 - Strödke, Benjamin A1 - Wirth, Christine C. A1 - Brügl, Thomas A1 - Abodo, Liane Onambele A1 - Dandekar, Thomas A1 - Doerig, Christian A1 - Fischer, Rainer A1 - Tobin, Andrew B. A1 - Alam, Mahmood M. A1 - Bracher, Franz A1 - Pradel, Gabriele T1 - Inhibition of the SR Protein-Phosphorylating CLK Kinases of Plasmodium falciparum Impairs Blood Stage Replication and Malaria Transmission JF - PLOS ONE N2 - Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-beta-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs. KW - parasite KW - expression KW - mosquito KW - splicing factors KW - lactate dehydrogenase KW - xanthurenic acid KW - in-vitro KW - RNA-SEQ KW - identification KW - culture Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115405 SN - 1932-6203 VL - 9 IS - 9 ER - TY - THES A1 - Hausmann, Michael Franz Toni T1 - Untersuchungen zum Differenzierungspotential humaner Monozyten / Makrophagen in vitro T1 - A study: The Potential of human monocyte / macrophage-differentiation in vitro N2 - Unter dem Einfluss von M-CSF und GM-CSF entwickeln sich CD14-positive periphere humane Blutmonozyten zu CD68-positiven M-CSF- bzw. GM-CSF-Makrophagen. M-CSF-Makrophagen lassen sich mit INFg und LPS zu klassisch aktivierten M1-Makrophagen, oder mit IL-4 und IL-10 zu alternativ aktivierten M2-Makrophagen differenzieren. Durch GM-CSF werden aus Monozyten GM-CSF-Makrophagen induziert. Im Gegensatz zu M1-Makrophagen sind GM1-Makrophagen bisher noch wenig untersucht. Mit INFg und LPS werden GM-CSF-Makrophagen zu GM1-Makrophagen aktivert. In der vorliegenden Arbeit wurde überprüft, wie groß die Übereinstimmung zwischen M-CSF- und M2-Makrophagen sowie zwischen GM-CSF- und M1-Makrophagen / GM1-Makrophagen ist. Im Gegensatz zu M-CSF- und GM-CSF stellt Laktat aber keinen Differenzierungsfaktor für Monozyten dar. Jedoch beeinflusst Laktat den Phänotyp von M2-Makrophagen und hemmt die Ausschüttung von IL-12 und NO durch M1- und GM1-Makrophagen. N2 - In the presence of M-CSF and GM-CSF CD14-positive human blood monocytes develop into CD68-pos M-CSF- or GM-CSF-macrophages. M-CSF-macrophages differentiate when incubated with INFg and LPS to classicly activated M1-marophages, when incubated with IL-4 and IL-10 zu alternativly activated M2-macrophages. Monocytes presented to GM-CSF eventually become GM-CSF-macrophages. There are only few examinations of GM-CSF-macrophages and classicly activated GM1-macrophages compared to M1-macrophages. In this study we focused on comparing the non-activated M-CSF-macrophages to alternativly activated M2-macrophages and non-activated GM-CSF-macrophages to classicly activated GM1-macrophages/M1-macrophages. Furthermore we point out that lactate is not a potential differentiation-signal für monocytes. But lactate does infact interfere with certain macrophage-phenotypes and the secretion of proinflammatory IL-12 and NO-molecules. KW - Differenzierung KW - Makrophage KW - Monozyt KW - Mensch KW - Zellkultur KW - differentiation KW - macrophage KW - monocyte KW - human KW - in-vitro Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-77801 ER - TY - THES A1 - Straßberger, Ulla T1 - Fragmentbefestigung bei Wurzelfrakturen - Eine In-vitro-Untersuchung zum Bruchverhalten verschiedener Dentinadhäsivsysteme T1 - Fragment-fixation with root-fractures - An in-vitro investigation about the failuremode of different dentine adhesive systems N2 - In einer vorangegangenen In-vitro-Untersuchung wurden die pallatinalen Wurzeln menschlicher Molaren im Scherversuch (10mm/Min) gebrochen. Zahnfragmente wurden mit Hilfe verschiedener Dentinhaftvermittler (OptiBond FL, Syntac, Adhese, Prompt L-Pop und OptiBond FL in Kombination mit Tetrac Flow) adhäsiv wiederbefestigt. Die so rekonstruierte Zahneinheit wurde erneut an der ursprünglichen Bruchfläche gebrochen und die Bruchfestigkeit ermittelt. In der vorliegenden In-vitro-Untersuchung wurden die Bruchflächen der erneut gebrochenen Zähne digital abfotografiert und lichtmikroskopisch untersucht, um den Frakturmodus zu ermitteln. Die Frakturmodi (Adhäsivfraktur, Kohäsivfraktur im Dentin oder Befestigungsmaterial) wurden absolut und in Relation zur gesamten Bruchfläche ermittelt. Hierzu wurde ein spezielles CAD-Programm verwendet. Mittels statistischer Tests wurden die Adhäsivsysteme auf signifikante Unterschiede hinsichtlich der aufgetretenen Frakturmodi untersucht. Ergebnisse: Keine signifikanten Unterschiede. Kohäsivfrakturen im Befestigungsmaterial traten unabhängig vom Dentinhaftvermittler am häufigsten auf. Vor allem konnte dieser Bruchmodus bei Verwendung von OptiBond FL beobachtet werden. Adhäsivfrakturen traten am zahlreichsten bei Gebrauch von Prompt L-Pop und Kohäsivfrakturen im Dentin bei Verwendung von Syntac auf. N2 - In a preceding in-vitro-investigation roots of human molars were broken in shear-test (10mm/min). Teeth were restored by fragment fixation with different adhesive systems (OptiBond FL, Syntac, Adhese, Prompt L-Pop and OptiBond FL in combination with Tetrac Flow) and shear test was repeated to investigate fracture-toughness. In this in-vitro investigation pictures of the surfaces of the broken roots were taken and they were analysed using light-microscope. Failure-modes (adhesive-fracture, cohesive-fracture in dentine or restaurative material) were calculated absolute and in relation to the whole surface by a special CAD-software. Statistical tests were done to find out significant difference in failure-mode between the tested adhesive systems. Results: No significant differences. In every adhesive system cohesive fracture in restaurative material was the most frequent fracture-mode. Most of them could be observed if OptiBond FL was used. Adhesive fractures especially appeared in the group of Prompt L-Pop, and cohesive fractures in dentine by using syntac. KW - Bruchverhalten KW - Mikroskop KW - In vitro KW - Frakturmodus KW - Adhäsivfraktur KW - Kohäsivfraktur KW - Wurzelfraktur KW - Dentinhaftvermittler KW - Microscope KW - in-vitro KW - adhesive fracture KW - cohesive fracture Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-26119 ER -